Extracellular Vesicles As A Drug Delivery System A Systematic Review of Preclinical Studies

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Extracellular vesicles as a drug delivery system:A systematic review of pre-

clinical studies
Pol Escudé Martinez de Castilla, Lingjun Tong, Chenyuan Huang,

Alexandros Marios Sofias, Giorgia Pastorin, Xiaoyuan Chen, Gert Storm,
Raymond M. Schiffelers, Jiong-Wei Wang
PII: S0169-409X(21)00175-7
DOI: https://doi.org/10.1016/j.addr.2021.05.011
Reference: ADR 13801
To appear in: Advanced Drug Delivery Reviews
Received Date: 18 February 2021

Revised Date: 10 May 2021
Accepted Date: 15 May 2021
Please cite this article as: P. Escudé Martinez de Castilla, L. Tong, C. Huang, A. Marios Sofias, G. Pastorin, X.
Chen, G. Storm, R.M. Schiffelers, J-W. Wang, Extracellular vesicles as a drug delivery system:A systematic
review of preclinical studies, Advanced Drug Delivery Reviews (2021), doi: https://doi.org/10.1016/j.addr.
2021.05.011
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Extracellular vesicles as a drug delivery system:
A systematic review of preclinical studies
Pol Escudé Martinez de Castilla a,1, Lingjun Tong b,c,1, Chenyuan Huang b,c,1, Alexandros Marios Sofias
d, Giorgia Pastorin e, Xiaoyuan Chen b,f,g,h,i, Gert Storm b,i,j,k, Raymond M. Schiffelers a,*, Jiong-Wei Wang
b,c,i,l,*
a CDL Research, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands
b Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 119228
Singapore, Singapore
c Cardiovascular Research Institute (CVRI), National University Heart Centre Singapore (NUHCS),
117599 Singapore, Singapore
d Institute for Experimental Molecular Imaging, Faculty of Medicine, RWTH Aachen University, 52074
Aachen, Germany
e Department of Pharmacy, Faculty of Science, National University of Singapore, 117543 Singapore,

Singapore
f Department of Diagnostic Radiology, Yong Loo Lin School of Medicine, National University of
Singapore, 119074 Singapore, Singapore
g Departments of Chemical and Biomolecular Engineering, and Biomedical Engineering, Faculty of

Engineering, National University of Singapore, 117575 Singapore, Singapore
h Clinical Imaging Research Centre, Centre for Translational Medicine, Yong Loo Lin School of
Medicine, National University of Singapore, 117599 Singapore, Singapore
i Nanomedicine Translational Research Programme, the Centre for NanoMedicine, Yong Loo Lin
School of Medicine, National University of Singapore, 117609 Singapore, Singapore
j Department of Pharmaceutics, Faculty of Science, Utrecht University, 3584 CG Utrecht, the

Netherlands
k Department of Biomaterials, Science and Technology, Faculty of Science and Technology, University
of Twente, 7522 NB Enschede, the Netherlands
l Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore 117593, Singapore
1These authors contributed equally
*Correspondence to: [email protected] (R.M.S.) and [email protected] (J.W.W.)
1
Abstract:
During the past decades, EVs have emerged as attractive drug delivery systems. Here, we assess their
pre-clinical applications, in the form of a systematic review. For each study published during the past
decade, disease models, animal species, EV donor cell types, active pharmaceutical ingredients (APIs),
EV surface modifications, API loading methods, EV size and charge, estimation of EV purity, presence
of biodistribution studies and administration routes were qualitatively analyzed in a defined and
reproducible way. We have interpreted the trends we observe over the past decade, to define the niches
where to apply EVs for drug delivery in the future and to provide a basis for regulatory guidelines.
Number of words: 108
Keywords:
Extracellular vesicles, liposomes, systematic review, drug delivery, preclinical animal models, drug
loading
Highlights:
 There are over 150 publications of EVs in drug delivery in preclinical models published during
the past decade.
 These preclinical studies primarily focus on murine cancer models and use intravenously
injected EVs. Major EV donor cell types are cancer cells, stem cells and HEK293 derived cells
while the majority of drug classes are nucleic acid therapeutics and small molecule drugs.
 The lack of regulatory guidelines in the EV field is pulling the brakes on clinical research.
2
3
Table of contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …. 5
1.1 Types of EVs: exosomes, microvesicles and apoptotic bodies . . . . . . . . . . . . . . . . …….. 5
1.2 Functions of EVs ...................................................... 5
2. Systematic review of EVs as drug delivery systems in preclinical studies . .............. 7
2.1 Introduction of the systematic review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …. 7
2.2 Approaches for Systematic Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …. 8
2.3 Inclusion and exclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ….. 9
3. Data analysis of systematic review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ……. 10
3.1. Categorization of selected publications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 10
3.2. Diving deeper into the tables: analytical interpretation. . . . . . . . . . . . . . . . . . . . . . . . . . ... 30
3.2.1. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …….. 30
3.2.2. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2.3. Donor cell types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2.4. Active pharmaceutical ingredients (API) associated with EVs . . . . . . . . . . . . . . . . . . . . . 32
3.2.5. API loading procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 33
3.2.6. Surface modification status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.7. Size and zeta potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.2.8. Estimation purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.2.9. Biodistribution studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2.10. Administration routes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3. Clinical trials of EVs as DDS: on the way to reach patients . . . . . . . . . . . . . . . . . . . . . . . . 38
4. Liposomes versus EVs for drug delivery: heads up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.1. Short overview of EVs as therapeutic delivery vehicles in comparison with liposomes . . . . . 40
4.2. Physical features, production and quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3. In vivo administration of EVs and liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4
4.3.1. Nanoparticles (EVs & liposomes) are rapidly cleared by the mononuclear phagocyte system
(MPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ……………… 41
4.3.2. Accelerated blood clearance (ABC) phenomenon upon multiple injections of nanoparticles42
4.3.3. Complement activation-related pseudoallergy (CARPA) upon nanoparticle injection . . . . .42
4.3.4. Biodistribution profiles: passive vs. active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . … 43
4.3.5. Pharmacokinetics and pharmacodynamics (PK/PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
5. Critical discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 44
5.1. Seeing the glass half empty: Missing gaps for establishing EVs as effective and safe drug delivery
systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ……………...45
5.1.1. Challenges in production, isolation, purity and characterization . . . . . . . . . . . . . . . . . . . . 45
5.1.2. Misleading information on EV biodistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
5.1.3. Complications in upscaling, loading strategies and storage . . . . . . . . . . . . . . . . . . . . . . . 46
5.1.4. Lack of comparison with liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47
5.2. Seeing the glass half full: accomplished landmarks of EVs for drug delivery . . . . . . . . . . . . 47
5.2.1. Active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.2.2. Improved circulation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.2.3. Upgraded loading efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.2.4. EVs in intracellular trafficking: Escaping the endosomal system . . . . . . . . . . . . . . . . . . . 48
5.2.5. Crossing the Blood-Brain Barrier (BBB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.2.6. Emerging EV platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
5.3. Defining the niches of EVs in comparison to liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.4. Reaching the clinic: Guidance on how to evaluate EVs as drug delivery systems . . . . . . . . 49
6. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …………. 55
5
1. Introduction
Extracellular vesicles (EVs) are particles released by all cells and mediate a conserved form of
intercellular communication [1]. Enclosed by one or more lipidic membranes, they consist of aqueous
compartments which carry a vast array of biomolecules from the parental cell, such as lipids, proteins,
various types of nucleic acids and soluble small molecules [2, 3]. EVs have been observed among all
kingdoms of life, from bacteria and archaea to mammals, highlighting their evolutionary importance [4].
In mammals, EVs are present in all biofluids such as blood, saliva, breast milk, urine, cerebrospinal fluid,
amniotic fluid, semen and ascites [5-7].
1.1. Types of EVs: exosomes, microvesicles and apoptotic bodies

The term “extracellular vesicles” encompasses all secreted membrane vesicles from the cell, yet these
vesicles appear particularly heterogeneous. As a matter of fact, during the past decade different subsets
of EVs have been called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic
bodies, and multiple other names [8]. To simplify nomenclature, classes are defined based on their
biogenesis. As a result, EVs have been divided into three main populations: exosomes, microvesicles
and apoptotic bodies [9, 10]. Exosomes are small vesicles (30-120 nm) formed and contained
intracellularly inside multivesicular bodies (MVBs). Exosomes are released to the extracellular space
through fusion of the MVB with the plasma membrane [10-12]. Microvesicles (50-1000 nm) are released
from the cells by direct budding from the plasma membrane [12]. The last population of EVs are the
apoptotic bodies (50-5000 nm), which are also released directly from the cell membrane but only by
cells undergoing apoptosis [10, 13, 14]. The heterogeneity between EV populations, with overlapping
sizes, and the lack of consensus on specific proteins that are unique to each EV subtype have greatly
hindered their characterization at the subtype level, as well as the modification of subclass-specific
properties and study of differences between subtype functions [15-18]. Nevertheless, in recent years
the term “exosomes” has become increasingly popular in publications to refer to what is likely a mixture
of different heterogeneous EV subtypes. In this review we will only use the term EVs.
1.2. Functions of EVs

EVs have a broad range of biological functions and participate in multiple physiological and pathological
processes [19]. Their ability to mediate intercellular communication by transferring a wide spectrum of
molecules between cells gives them an important role in complex biological processes like
tumorigenesis [20], preparation of metastatic niches [21], elimination of cytotoxic drugs such as cisplatin
[22], inflammation [23], immune response modulation [24], angiogenesis [25, 26], tissue repair [27],
apoptosis [28-30] and also in maintenance of homeostasis [31], amongst many others [32]. Since their
composition reflects the parental cell status at the time of production, this makes them very attractive
for the diagnostics field [2, 33]. In addition, they are stable in many biological fluids and are relatively
abundant, endowing EVs with plenty of potential as a reservoir of biomarkers. Liquid biopsies containing
circulating EVs could allow monitoring of prognosis, progression of the disease and response to therapy
in patients [2, 33-35].
6
Furthermore, EVs are able to modulate cell phenotypes, differentiation and recruitment in a paracrine
fashion [36]. As such, EVs possess similar therapeutic features as the parental cells such as stem cells.
However, EVs cannot self-replicate, hence potentially conferring a safer profile over stem cell
transplantation in regenerative medicine [36-42]. Interestingly, EVs derived from biological fluids, such
as plasma, also exert intrinsic bioactivities although the specific components are not always defined
[43]. Given the capacity of EVs to effectively carry a broad variety of biological molecules through
different biofluids with cellular specificity, EVs hold promise for drug delivery [3]. Taking it one step
forward, an EV-based theranostic delivery platform has been recently proposed by loading both imaging
tracers (for diagnosis) and therapeutic compounds (for delivery) into (or onto) EVs [2, 44]. Taken
together, EVs are emerging as a diagnostic toolbox, a new class of therapeutics, and a drug delivery
vehicle (Fig.1). All these potential applications are in the process of validation in many preclinical and
clinical studies. In this review, we focus on their application as drug delivery systems (DDS) by
systematically reviewing and analyzing the preclinical studies over the past decade (for earlier studies
readers are recommended to an elegant review by Johnsen et al. [45]).
Fig. 1. Potential applications of EVs. (A) Diagnostic (and prognostic) potential of EVs obtained from
various sources. EVs generated under pathological microenvironments are able to capture complex
intracellular molecular signatures that are unique for specific disease stages or injuries, therefore
becoming an attractive reservoir of biomarkers. (B) Therapeutic potential of EVs. EVs derived from
multiple cells can interact with the target cells via various pathways, including endocytosis, direct
7
binding, phagocytosis, and direct fusion, imparting specific therapeutic effects. (C) EVs as a potential
DDS. EVs can be loaded with therapeutics such as RNAs, proteins, and small-molecule drugs,
delivering these cargoes to target cells.
2. Systematic review of EVs as drug delivery systems in preclinical studies

2.1. Introduction of the systematic review
Having analyzed several aspects of EVs as DDS in comparison with the conventional DDS liposomes,
we realize the urgency of a systematic review that offers an overview of the development of EVs as
DDS, especially in the past decade when EVs have been extensively explored for drug delivery in
various disease models (Fig. 2). In fact, for a comprehensive evaluation of any therapeutic delivery
vehicle, it is crucial to test in detail the pharmacodynamics and the pharmacokinetics in preclinical
models that resemble the human condition. In consequence, the choice of animal models based on the
resemblance of their physiological features with the disease modus operandi, is of key importance to
determine the translatability of the results into human therapies. Up to now, only mice, rats and zebrafish
have been used in published preclinical studies of EVs as DDS. Pigs have been used for preclinical
testing of EVs as therapeutics per se but not as DSS [46, 47]. There are no published records to
investigate EVs, either as therapeutics per se or as DSS, in non-human primates, which are generally
used as a model in the final preclinical stages prior to human clinical trials. Another factor to take into
account for the development of DDS is the drug-encapsulation efficiency, which can be relatively high
for liposomal drug formulations [48]. For EVs, availability of drug loading strategies and efficiencies is
limited as they are biological products derived from cellular activity that offer less freedom to adapt
composition of lipid membranes and interior in comparison to liposomal delivery systems. There are two
main strategies for loading drugs into EVs: A) Before EV isolation, drugs are loaded by addition to and
manipulation of the EV donor cells. This strategy demands compatibility and suitability of the parental
cells with the drugs to encapsulate in EVs. B) Drugs are loaded after EV isolation. This strategy requires
preservation of the structure and functionality of the vesicles [49]. In addition, the routes of
administration, size, charge and surface modifications of EVs are influencing the pharmacokinetics and
pharmacodynamics of drug-loaded EVs. In Fig. 2, we provide an overview of the parameters, including
payloads, drug loading strategies, EV surface modifications, administration routes, animal models and
disease indications, that have been considered in the experimental design of preclinical testing of EVs
as DDS.
8
Fig. 2. Schematic overview of EVs as DDS in preclinical animal models. (A) General simplification of
EV contents, drug loading procedures, and surface ligand incorporation, before or after EV isolation. (B)
Various animal models and administration routes for preclinical testing of EVs for drug delivery. (C)
Examples of disease indications for drug delivery via EVs.
In this article, we will assess the performance of EVs as DDS in preclinical models, in the form of a
systematic review. For each study published in the past decade, disease, animal model, EV donor cell
type, active pharmaceutical ingredient (API) loaded, EV surface modifications, API loading procedure,
EV size and charge, estimation of EV purity, presence of biodistribution studies and administration route
were qualitatively analyzed in a defined and reproducible way. After analysis of the performance of EVs
as DDS in comparison with liposomes, we interpret the trends observed for the past decade and try to
define the niches where to apply EVs in the future.
2.2. Approaches for Systematic Review

The main goal of this systematic review is to comprehensively analyze and interpret all the literature
from the past decade on the preclinical testing of EVs as a DDS with a non-biased, precise and
reproducible approach, following the principles defined in the Preferred Reporting Items for Systematic
Reviews and Meta-analysis (PRISMA) statement [50] and the Cochrane Handbook for Systematic
Reviews of Interventions [51]. The search parameters and criteria employed (Table 1) were defined
based on a consensus between multiple investigators (P.E.M., L.T., C.H., G.S., R.S., and J.W.W.) using
exclusively PubMed as a database.
9
Table 1
Eligibility criteria for this systematic review.
Publication
Database PubMed
Language English
Time period 01/01/2010 - 01/01/2021
Publication type Journal Article
Species Other Animals
extracellular vesicles / exosomes / microvesicles / apoptotic
bodies / microparticles
drug / therapeutic / small molecule / antioxidant / anti-inflammatory
/ chemotherapeutic / silencing / siRNA / miRNA / mRNA / plasmid
Keywords
/ kinase inhibitor
[Title/Abstract]
animal / mice / mouse / murine / rats / rat / pig / zebrafish / primate
/ monkey / chimpanzee
in vivo / preclinical
2.3. Inclusion and exclusion criteria

As we intended to carry out a systematic review of the preclinical status of EVs as DDS during the past
decade, we only included journal articles published in English from the 1st of January 2010 until the 1st
of January 2021. In this manner we aimed to provide an overview of the recent development in the field.
We focus on original research articles, while excluding all other types of publications such as opinion
articles, case reports, and editorials. Moreover, as this review provides an overview of the preclinical
status of EVs as DDS, the PubMed option “Other animals” was applied to the species filter. EVs were
defined according to the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018)
guidelines [8], excluding synthetic nanoparticles and exosome-mimetic nanovesicles. Regarding the
selection of keywords for our search we set a criterion that the articles of potential interest should contain
in their titles or abstracts at least one term of each of the 4 categories that were defined in the Keywords
section of Table 1. Furthermore, “EVs as drug delivery systems” in this study were defined as EVs that
were purposely used for loading and delivery of therapeutic molecules. Enriched EV fractions containing
endogenous molecules which were not introduced for a delivery purpose were excluded from our
systematic review.
Articles that did not meet all the selection criteria were excluded for analysis. Additionally, to increase
the power and the sensitivity of our search, we performed a subsequent PubMed search with the same
search keywords format as described in Table 1 but opted for Reviews and Systematic Reviews instead
of Journal Articles and opted out Other Animals. The aim of this additional search was to screen the
resulting reviews for analytical tables or figures which referenced journal articles that fulfilled our criteria
10
but that we might have missed with our previous search parameters. Explicit search parameters are
provided in the Supplementary Materials Section A. Any studies that generated eligibility doubts were
brought to and resolved by JWW, RS and GS. The workflow for this Systematic Review is presented in
Fig. 3.
Fig. 3. Flowchart diagram of the systematic review according to the PRISMA statement.
3. Data analysis of systematic review

3.1. Categorization of selected publications
In this systematic review, we categorized the selected 157 publications, based on disease or pathogenic
conditions where EVs were investigated as DDS, into 5 groups: cancer (Table 2a), cardiovascular
disease (Table 2b), neurological disease (Table 2c), inflammatory disease (Table 2d) and other
diseases (Table 2e). All the tables were designed with ten categories (disease/condition tested, animal
model, EV donor cell type, API loaded, EV surface modifications, API loading method, EV size & charge,
EV purity analysis, biodistribution studies and administration route). EV isolation issues have been
exhaustedly reviewed in the literature [2, 8, 12, 52-55] and therefore were not included in the current
study. We carried out a systematic analysis of the approaches and trends in the field for the past decade
in a rigorous and unbiased manner. The detailed criteria employed for data analysis can be found in
Supplementary Materials Section B.
11
Table 2a
Preclinical studies of EVs as drug delivery systems for cancer treatment
Disease/condition Animal Donor cell Active pharmaceutical Surface API loading Size/charge* Purity Biodistribution Route of Year of Ref.
model type ingredient (API) modifications procedure estimat studies administration publication
ion
Brain cancer
Glioma Mouse BMSCs Indocyanine green & None After isolation EVs, 160 nm (DLS) / - No Yes I.V. 2020 [56]
Curcumin Physical 16 mV
(Electroporation)
Glioma Mouse MSCs, DCs & PTEN-mRNA None Before EVs isolation, 70-110 nm (DLS) No Yes I.V. 2020 [57]
HEK293T Transfection based
(Plasmid)
Glioma Mouse RAW264.7 Curcumin & Neuropilin-1- After isolation EVs, 122.7±6.5 nm No Yes I.V. 2018 [58]
Macrophage superparamagnetic iron targeted physical (Incubation) (NTA) / -24.1±2.2
oxide nanoparticles peptide mV
Glioma Mouse MSCs miRNA-124a None Before EVs isolation, 100-125 nm No No I.P. 2018 [59]
Transfection based (NTA)
(Lentivirus)
Glioblastoma Rat HEK293T Anti-miR-21 T7 peptide After isolation EVs, 15-50 nm (DLS) / No Yes I.V. 2020 [60]
Physical -10 to -3 mV
(Electroporation)
Glioblastoma Mouse Malignant cells CRISPR/Cas9 TNF-α After isolation EVs, NA No No I.V. 2019 [61]
Physical
(Electroporation)
Glioblastoma Mouse Embryonic stem Paclitaxel cRGD After isolation EVs, 125±27 nm No Yes I.V. 2019 [62]
cells Physical (Incubation) (NanoFCM)
Glioblastoma Rat HEK-293T miRNA-21-Sponge None Before EVs isolation, 66.65±39.88 nm, No No Local 2019 [63]
Transfection based PDI 0.317 (DLS)
(Plasmid)
Glioblastoma Mouse HEK293T CD-UPRT mRNA & None Before EVs isolation, 88-152 nm (NTA) No No Local 2017 [64]
protein Transfection based
(Plasmid)
Glioblastoma- Zebrafish bEND.3 cells siVEGF None After isolation EVs, NA No No I.V. 2017 [65]
astrocytoma Chemical
(Lipofetamine® 2000
transfection)
Glioblastoma- Zebrafish U-87 MG, Rhodamine 123, None After isolation EVs, 30-100 nm (DLS) No Yes I.V. 2015 [66]
astrocytoma bEND.3, PFSK- Paclitaxel & Doxorubicin Physical (Incubation)
12
1 & A-172
Glioblastoma Mouse L929 cells Methotrexate & KLA LDL & KLA Before EVs isolation, 318.3±15.5 nm No Yes I.V. 2018 [67]
multiforme peptide peptide Ultraviolet irradiation (DLS) / about -10
mV
Glioblastoma Rat MSCs miRNA-146b None Before EVs isolation, NA No No Local 2013 [68]
multiforme Transfection based
(Plasmid)
Breast cancer
Breast cancer Mouse HEK293T PH20 hyaluronidase & Folic acid & Before EVs isolation, About 100 nm No Yes Local 2021 [69]
Doxorubicin PH20 Transfection based (DLS)
hyaluronidase (Plasmid); After
isolation EVs,
Physical (Incubation &
electroporation)
Breast cancer Mouse HEK293T AntiCD3 & antiHer2 AntiCD3 & Before EVs isolation, 109 nm (NTA) Yes No I.V. 2020 [70]
(Expi293) antibody antiHer2 Transfection based
antibody (Plasmid)
Breast cancer Mouse RAW264.7 Paclitaxel & Doxorubicin None After isolation EVs, Dox-EV 162.1± No No I.V. 2020 [71]
Macrophage Physical (Incubation, 5.5 nm & PTX-
sonication & exclusion EV 129.4±2.3 nm
chromatography) (NTA)
Breast cancer Mouse 4T1 cells TK-NTR–encoding None Before EVs isolation, 136-160 nm No No Local 2019 [72]
minicircle DNA Transfection based (NTA)
(Plasmid)
Breast cancer Mouse BMSCs Doxorubicin DARPin After isolation EVs, 120 nm (DLS) No Yes I.V. 2019 [73]
Physical
(Electroporation)
Breast cancer Mouse Blood Chimeric peptide (ChiP) Chimeric After isolation EVs, 132.6 nm, PDI No Yes I.V. 2019 [74]
peptide (ChiP) Physical (Incubation 0.306 (DLS)
on ice)
Breast cancer Mouse MSCs Paclitaxel None Before EVs isolation, 204±93.1 nm No No I.V. 2019 [75]
Incubation (NTA) /
-43.08±1.58 mV
Breast cancer Mouse M1-polarized Paclitaxel None After isolation EVs, 172.8 nm No Yes I.V. 2019 [76]
macrophages Physical (Sonication) (DLS&NTA) /
-12 mV
Breast cancer Mouse 4T1 cells Sinoporphyrin sodium Sinoporphyrin After isolation EVs, 126.71±3.86, PDI No Yes I.V. 2019 [77]
sodium Physical (Incubation) 0.18±0.05
13
(DLS&NTA) /
-10.67±0.52 mV
Breast cancer Mouse H22 & Bel7402 Doxorubicin None Before EVs isolation, 260±15 nm, PDI No Yes I.V. 2019 [78]
cells Transfection based 0.145±0.032
(Plasmid) (DLS) /
-11.0±0.4 mV
Breast cancer Mouse MSCs miRNA-142-3p None After isolation EVs, 103 nm (DLS) No Yes I.V. 2018 [79]
Physical
(Electroporation)
Breast cancer Mouse HEK293T siSurvivin Folate, PSMA After isolation EVs, 103-120 nm Yes Yes I.V. 2018 [80]
RNA aptamer Chemical (ExoFect (NTA) /
& EGFR RNA Exosome transfection -15.6±27.9 mV
aptamer (All kit)
conjugated to
3WJ)
Breast cancer Mouse Dendritic cells Paclitaxel AS1411 After isolation EVs, 111 nm (NTA) / - No Yes I.V. 2018 [81]
aptamer Physical (Sonication) 25.6 mV
conjugated to
cholesterol-
PEG
Breast cancer Mouse HEK293 HchrR6 mRNA LS-ML39-C1– Before EVs isolation, 30-100 nm (NTA) No No I.P. 2018 [82]
C2-His (EVHB) Transfection based
(Plasmid)
Breast cancer Mouse HEK293 PH20 hyaluronidase & PH20 Before EVs isolation, 95 nm (DLS) No Yes Local 2018 [83]
Doxorubicin hyaluronidase Transfection based
(Plasmid); After
isolation EVs,
Physical (Incubation)
Breast cancer Mouse Human red Anti-miR-125b, Cas9 None After isolation EVs, About 140 nm, No Yes I.P. & Local 2018 [84]
blood cells mRNA, & guide RNAs Physical PDI 0.07
(Electroporation) (NTA&DLS) /
-11.5 mV
Breast cancer Mouse Dendritic cells miRNA let-7 & siRNA- AS1411 After isolation EVs, 77 nm (NTA) / No Yes I.V. 2017 [85]
VEGF aptamer Physical -16.4 mV
(Electroporation)
Breast cancer Mouse MDA-MB-231 Doxorubicin None After isolation EVs, 101 nm (NTA) No No I.P. 2016 [86]
Physical
& STOSE (Electroporation)
Breast cancer Mouse MDA-MB-231 & Doxorubicin None After isolation EVs, 176±53 nm, No Yes I.V. 2015 [87]
HCT-116 Physical 209±54 nm,
(Electroporation) respectively
(NTA)
14
Breast cancer Mouse MCF-7 cells Doxorubicin None Before EVs isolation, 40-100 nm (TEM) No No S.C. 2015 [88]
Incubation
Breast Cancer Mouse Immature Doxorubicin AlphaV After isolation EVs, 93 nm (NTA) No Yes I.V. 2014 [89]
dendritic cells integrin- Physical
specific iRGD (Electroporation)
peptide
Breast cancer Mouse HEK293 miRNA-let-7a Transmembran Before EVs isolation, NA No Yes I.V. 2013 [90]
e domain of Transfection based
platelet- (miRNA)
derived growth
factor receptor
fused to GE11
peptide
Breast cancer Mouse HEK293T Doxorubicin Lipidomimetic Before EVs isolation, 449.1±15.1 nm, No Yes I.V. 2019 [91]
multidrug resistance chains-grafted Ultraviolet irradiation PDI 0.29±0.02
hyaluronic acid (DLS)
Breast cancer with Mouse Murine Laurate functionalized Pt None After isolation EVs, 61.9±1.74 nm, No Yes I.V. 2019 [92]
Lung metastasis macrophage (IV) prodrug Physical (Incubation) PDI 0.168±0.021
(DLS) /
- 9.39±0.56 mV
Metastatic breast Mouse HUVECs & 4T1 siS100A4 None After isolation EVs, 263.71±24.84, No Yes I.V. 2020 [93]
cancer cells Physical (Incubation & PDI 0.32±0.01
extrusion) (DLS) /
-28.63±0.33 mV
Hypoxic breast Mouse MDA-MB-231 Olaparib SPIO After isolation EVs, 110-170 nm No Yes Local 2018 [94]
cancer tumors (superparamag Physical (NTA)
netic iron (Electroporation)
oxide)
nanoparticles
Triple-negative Mouse HEK293T PH20 hyaluronidase PH20 Before EVs isolation, About 100 nm No No Local 2019 [95]
breast cancer hyaluronidase Transfection based (DLS)
(Plasmid)
Triple-negative Mouse Macrophages Doxorubicin & Disintegrin and After isolation EVs, 94.1±104.4 nm No Yes I.V. 2019 [96]
breast cancer cholesterol-modified metalloprotein Chemical (Mixing with (empty# A15-
miRNA-159 ase 15 (A15) trimethylamine exosome, NTA) /
solution); After −14.67±1.53 mV
isolation EVs, (miRNA loaded)
Colorectal cancer
Colorectal cancer Mouse MSCs Doxorubicin MUC1 aptamer After isolation EVs, 120±12 nm, PDI No Yes I.V. 2020 [97]
15
Physical 0.5±0.02 (DLS) /
(Electroporation -80±12 mV
method
(DOX@exosome))
Colorectal cancer Mouse HEK293T si-ciRS-122 None Before EVs isolation, About 100 nm No No I.V. 2020 [98]
Transfection based (NTA)
(Plasmid)
Colorectal cancer Mouse HEK293T siSur-A647 & Folate Folic acid After isolation EVs, 136.5±3.5 (NTA) No No I.V. 2019 [99]
Chemical (ExoFect
exosomes transfection
kit); After isolation
EVs, Physical (Heat-
shock)
Colorectal cancer Mouse LIM1215 cells Doxorubicin A33Ab-US After isolation EVs, 187.83±6.76 nm No Yes I.V. 2018 [100]
Physical (Incubation) (DLS) /
−9.57±0.38 mV
Colorectal cancer Mouse HEK293T siSurvivin Folate, PSMA After isolation EVs, 103-120 nm Yes Yes I.V. 2018 [80]
aptamer (All kit)
conjugated to
3WJ)
.Colorectal cancer Mouse THLG-293T & 5-Fluorouracil (5-FU) & Her2 binding After isolation EVs, 110±11.3 nm No Yes I.V. 2020 [101]
LG-293T Anti-miR-21 affibody Physical (DLS) / -11±2.7
(Electroporation) mV
Colorectal cancer Mouse HEK-293T SIRPα protein SIRPα protein Before EVs isolation, About 100 nm No No Local 2018 [102]
Transfection based (DLS)
(Plasmid)
Colorectal cancer Mouse HEK293T SIRPα proteins SIRPα proteins Before EVs isolation, About 100 nm No Yes I.V. & Local 2017 [103]
Transfection based (DLS)
(Plasmid)
Colorectal cancer Mouse CT26-CIITA MHC class II molecule MHC class II Before EVs isolation, NA No No I.D. 2013 [104]
cells molecule Transfection based
(Retrovirus)
Colorectal cancer Mouse LL/2, MC-38, Oncolytic adenovirus None Before EVs isolation, 50-400 nm (NTA) No Yes I.V. 2019 [105]
A549 & human Ad5/3-CD40L Transfection based / About -40 mV
liver samples (Adenovirus)
Cervical cancer
Cervical cancer Mouse HeLa Paclitaxel None Before EVs isolation, 285.58±2.95 nm, No No I.V. 2020 [106]
Ultraviolet irradiation PDI 0.104±0.106
16
(DLS)
Cervical cancer Mouse THP-1 Doxorubicin RGD, After isolation EVs, 30-300 nm No Yes I.V. 2018 [107]
macrophages sulfhydryl Physical (empty#, DLS)
groups, AuNRs (Electroporation)
& Folic acid
Cervical cancer Rat Bovine milk Curcumin None After isolation EVs, 93±6 nm, PDI No No Oral 2017 [108]
Chemical (Mixing with 0.21±0.04 (DLS)
ethanol: acetonitrile)
Cervical cancer Mouse Macrophages Doxorubicin Biotin, After isolation EVs, 100-1000 nm No Yes I.V. 2017 [109]
streptavidin- Physical (DLS) / About -10
modified iron (Electroporation) mV
oxide
nanoparticles
SA-IONPs &
Folic acid
Cervical cancer Mouse THP-1 m-THPC photosensitizer None Before EVs isolation, 550±50 nm (DLS) No Yes Local 2013 [110]
macrophages Incubation
Digestive system cancer
Digestive system Mouse Bovine milk siBcl-2 None After isolation EVs, 68.06 nm (DLS) No No I.V. 2020 [111]
cancer Physical (Ultrasound)
Gastric cancer Mouse Urinary PMA/Au-BSA@Ce6 None After isolation EVs, 75±7.6 nm Yes Yes I.V. 2020 [112]
nanoparticles Physical (DLS&NTA) /
(Electroporation) -31.4±3.1 mV
Gastric cancer Mouse HEK293T Anti-miR-214 None Before EVs isolation, NA No No I.V. 2018 [113]
Transfection based
(Anti-miR)
Gastric cancer Mouse HEK293T miRNA-29a/c None Before EVs isolation, NA No No I.V. 2016 [114]
Transfection based
(miRNA)
Lung cancer
Lewis lung Mouse M1 Cisplatin None After isolation EVs, 100-300 nm No No I.V. 2020 [115]
carcinoma macrophages Physical (NTA)
(Electroporation)
Lewis lung Mouse LL/2, MC-38, Oncolytic adenovirus None Before EVs isolation, 50 -400 nm (NTA) No Yes I.V. 2019 [105]
carcinoma A549 & human Ad5/3-CD40L Transfection based / About -40 mV
liver samples (Adenovirus)
17
Lewis Lung Mouse LL/2 cells Immunogenic oncolytic None After isolation EVs, 50-400 nm (NTA) No Yes I.V. 2019 [116]
carcinoma adenovirus Ad5D24-CpG Physical (Incubation); / About -40 mV
Before EVs isolation,
& Paclitaxel Transfection based
(Adenovirus)
Lewis lung Mouse A549 cells Cisplatin & Methotrexate None Before EVs isolation, 300-800 nm No Yes I.P. & I.V. 2016 [117]
carcinoma & Doxorubicin & Ultraviolet irradiation (empty#, DLS)
Paclitaxel
Lung metastatic Mouse H22 & Bel7402 Doxorubicin None Before EVs isolation, 260±15 nm, PDI No Yes I.V. 2019 [78]
cancer cells Transfection based 0.145±0.032
(Plasmid) (DLS) /-11.0±0.4
mV
Lung cancer with Mouse Bovine milk siKRAS Folic acid After isolation EVs, NA No Yes I.V. 2019 [118]
mutated KRAS Physical
(Electroporation);
After isolation EVs,
Chemical (ExoFect
exosomes transfection
kit)
Lung cancer Mouse LLC, MC38, Methotrexate None Before EVs isolation, 30-930 nm, mean No Yes Local 2019 [119]
patients with B16-F10, A549 Ultraviolet irradiation at 264 nm (NTA)
malignant pleural & MCF-7
effusion
Lung cancer Mouse LL/2 cells Oncolytic adenovirus None Before isolation EVs, NA No Yes I.V. 2018 [120]
Ad5D24 Transfection based
(Adenovirus)
Lung cancer Mouse Malignant cells CRISPR/Cas9 TNF-α After isolation EVs, NA No No I.V. 2019 [61]
Physical
(Electroporation)
Lung cancer Mouse A549 cells Oncolytic Ad5D24-CpG None Before EVs isolation, 50-1000 nm, with No Yes I.V. 2018 [121]
& Paclitaxel Transfection based major peak
(Adenovirus); After around 100 nm
isolation EVs, (NTA) / About -40
Physical (Incubation) mV
Lung cancer Mouse Bovine milk Paclitaxel None After isolation EVs, 108.1±1.5, PDI No No Oral 2017 [122]
Chemical (Mixing with 0.190±0.006
acetonitrile: ethanol) (DLS) / -7.4±0.7
mV
Lung cancer Mouse Bovine milk Berry anthocyanidins None After isolation EVs, 83±1.7 nm, PDI No No Oral 2017 [123]
Chemical (Mixture of 0.23 (DLS)
acetonitrile: ethanol)
18
Lung cancer Mouse Bovine milk Paclitaxel & Docetaxel & Folic acid After isolation EVs, 40-100 nm (NTA), No Yes I.V. & I.P. 2016 [124]
Curcumin & Chemical (Mixture PDI 0.22±0.06
with ethanol & (DLS)
acetonitrile)
Lung cancer Mouse Bovine milk Celastrol (CEL) None After isolation EVs, 106±9 nm, PDI No No Oral 2016 [125]
Chemical (Dissolved 0.15 (DLS)
in ethanol)
Lung Cancer Mouse RAW264.7 Paclitaxel None After isolation EVs, 159-217.9 nm No No I.N. 2016 [126]
Macrophages Physical (Sonication, (NTA&DLS) /
electroporation & -14.07 to -9.33
incubation) mV
Non-small cell lung Mouse MDA-MB-231 miRNA-126 None After isolation EVs, 30-120 nm (DLS) No Yes I.V. 2020 [127]
cancer cells Chemical (ExoFectin®
kit transfection)
Non-small cell lung Mouse Human plasma Imperialine Integrin After isolation EVs, 169.9±50.7 nm Yes Yes I.V. 2019 [128]
cancer Physical (Incubation & (NTA), PDI 0.192
α3β1-binding ultrasonic) / -17.3±4.32 mV
octapeptide
cNGQGEQc
Non-small cell lung Mouse RAW264.7 Paclitaxel Aminoethylanis After isolation EVs, 280.8±3.1 No No I.V. 2018 [129]
cancer Macrophage amide-PEG) Physical (Sonication & (NTA&DLS) /
incubation) -4.4±0.1 mV
Small cell lung Mouse HEK293 Soluble fms-like tyrosine None Before EVs isolation, About 100 nm No No Local 2019 [130]
cancer kinase-1 Transfection based (NTA)
(Lentivirus)
Liver cancer
Hepatocellular Mouse MSCs miRNA-199a None Before EVs isolation, 80±1.9 nm (NTA) No No I.V. 2020 [131]
carcinoma Transfection based
(Lentivirus)
Hepatocellular Mouse H22 tumor cell Bi2Se3 nanodots & None Before EVs isolation, 366.71±20.41 nm No Yes I.V. 2020 [132]
carcinoma doxorubicin Electroporation & (DLS) /
ultraviolet irradiation -12.02±0.59 mV
Hepatocellular Mouse H22 & Bel7402 Doxorubicin None Before EVs isolation, 260±15 nm, PDI No Yes I.V. 2019 [78]
carcinoma cells Transfection based 0.145±0.032
(Plasmid) (DLS) / -11.0±0.4
mV
Hepatocellular Mouse Blood Doxorubicin Superparamag After isolation EVs, Peak around 100 No Yes I.V. 2016 [133]
carcinoma netic magnetite Physical (Incubation) nm (DLS)
colloidal
nanocrystal
19
clusters
Hepatocellular Mouse H22 & A2780 Methotrexate None Before EVs isolation, 100-1000 nm No No I.P. 2012 [134]
carcinoma Ultraviolet irradiation (TEM)
Hepatocellular Mouse BM dendritic Doxorubicin Tumor derived Before EVs isolation, About 400 nm No Yes I.V. 2017 [135]
carcinoma ascites cells antigens Ultraviolet irradiation (empty#, DLS)
Hepatocellular Mouse A549 cells Cisplatin & Methotrexate None Before EVs isolation, 300-800 nm No Yes I.P. & I.V. 2016 [117]
carcinoma ascites & Doxorubicin & Ultraviolet irradiation (empty#, DLS)
Paclitaxel
Lymphoma
Lymphoma Mouse K562 cells TRAIL protein TRAIL protein Before EVs isolation, 140 nm (NTA) No Yes I.V. & Local 2016 [136]
Transfection based
(Lentivirus)
T-cell lymphoma Mouse EL4 cells Doxorubicin None After isolation EVs, 37 nm (DLS) No Yes I.V. 2018 [137]
Melanoma
Melanoma Mouse MSCs TNF-α Superparamag Before EVs isolation, 40-80 nm (DLS) No Yes I.V. 2020 [138]
netic iron oxide Transfection based
(Plasmid)
nanoparticles
(SPION)
Melanoma Mouse HEK293T PH20 hyaluronidase PH20 Before EVs isolation, About 100 nm No No Local 2019 [95]
hyaluronidase Transfection based (DLS)
(Plasmid)
Melanoma Mouse MSCs TRAIL protein TRAIL protein Before EVs isolation, 71.9 nm (empty#, No Yes I.V. 2018 [139]
Transfection based DLS)
(Plasmid)
Melanoma Mouse B16BL6 cells Immunostimulatory CpG Streptavidin- After isolation EVs, 109±10 nm No No I.D. 2016 [140]
DNA lactadherin Physical (Incubation) (qNano-TRPS) / -
32±1.6 mV
Melanoma Mouse Human siVEGF Streptavidin- After isolation EVs, 629.2 nm, PDI No Yes Local 2015 [141]
umbilical vein conjugated Physical 0.296 (empty#,
endothelial cell quantum dots (Electroporation) DLS)
Ovarian cancer
Ovarian cancer Mouse SKOV3 cells Triptolide None After isolation EVs, 145 nm (NTA) No Yes I.P. 2019 [142]
Physical (Sonication)
20
Ovarian cancer Mouse HEK293 & CRISPR/Cas9-NLS & None After isolation EVs, 50-150 nm No Yes I.V. & Local 2017 [143]
SKOV3 cells PARP-1 sgRNA Physical (empty#, DLS)
(Electroporation)
Ovarian cancer Mouse Bovine milk Berry anthocyanidins & None After isolation EVs, Not reported No No Oral 2017 [144]
Paclitaxel Chemical (Mixing & (NTA and DLS)
dissolving with
ethanol)
Ovarian cancer Mouse MDA-MB-231 Doxorubicin None After isolation EVs, 101 nm (NTA) No No I.P. 2016 [86]
Physical
& STOSE (Electroporation)
Ovarian cancer Mouse H22 & A2780 Methotrexate None Before EVs isolation, 100-1000 nm No No I.P. 2012 [134]
Ultraviolet irradiation (TEM)
Pancreatic cancer
Pancreatic cancer Mouse PANC-1 siPAK4 None After isolation EVs, 97.1±1.7 nm Yes No Local 2021 [145]
Physical (empty#, NTA) /
(Electroporation) -15 to -10 mV
Pancreatic cancer Mouse PANC-1 Gemcitabine None After isolation EVs, About 100-150 No Yes I.V. 2020 [146]
Physical (Incubation & nm (NTA)
sonication)
Pancreatic cancer Mouse MSCs siKRASG12D None After isolation EVs, 179 nm (empty#, Yes Yes I.P. 2018 [147]
Physical NTA)
(Electroporation)
Pancreatic cancer Mouse MSCs siKRASG12D & pLKO.1- CD47 After isolation EVs, Peak around 150 Yes Yes I.P. 2017 [148]
shKRASG12D Physical nm (empty#, NTA)
(Electroporation)
Pancreatic ductal Mouse hucMSCs miRNA-145-5p None After isolation EVs, 52.5-185.5 nm No No Local 2019 [149]
adenocarcinoma Chemical (Exo-Fect™ (empty#, NTA)
Exosome Transfection
Reagent)
Pancreatic ductal Mouse K989 cells & Anti-miR-365 None Before EVs isolation, 135 nm (empty#, No No I.P. 2018 [150]
adenocarcinoma tumor- Chemical Transfection NTA)
associated based (Plasmid)
macrophages
Prostate cancer
Prostate cancer Mouse HEK293T siSurvivin Folate, PSMA After isolation EVs, 103-120 nm Yes Yes I.V. 2018 [80]
aptamer (All kit)
21
conjugated to
3WJ)
Prostate cancer Mouse HEK293 PH20 hyaluronidase & PH20 Before EVs isolation, 95 nm (DLS) No Yes Local 2018 [83]
Doxorubicin hyaluronidase Transfection based
(Plasmid); After
isolation EVs,
Sarcoma
Fibrosarcoma Mouse HT1080 & HeLa Doxil® None After isolation EVs, 87.7±2.4 (NTA) Yes Yes I.V. 2020 [151]
Physical (Incubation
followed by extrusion)
Sarcoma Mouse Fibroblast L929 siTGF-β1 None Before EVs isolation, 60-100 nm No Yes I.V. 2014 [152]
Transfection based (empty#, TEM)
(siRNA)
Sarcoma Mouse THP-1 & 293T Anti-miR-150 None Before EVs isolation, NA No No I.V. 2013 [153]
Chemical Transfection
based (Anti-miR)
Other cancer
Carcinoma (KB Mouse Ginger root siSurvivin Folic acid After isolation EVs, About 100 nm Yes No I.V. 2018 [154]
xenograft) Chemical (ExoFect (NTA)
Exosome transfection
kit)
Chronic Mouse HEK293T Imatinib & siBCR-ABL IL3 Before EVs isolation, 30-60 (empty#, No Yes I.P. 2017 [155]
myelogenous Transfection based DLS)
leukemia (siRNA) & Incubation
Nasopharyngeal Mouse HUVECs Anti-miR-BART10-5p & iRGD Before EVs isolation, About 100 nm No Yes I.V. 2020 [156]
cancer Anti-miR-18a Transfection based (empty#, DLS)
(Plasmid)
Neuroendocrine Mouse HEK293 Verrucarin A & Anti-SSTR2 After isolation EVs, 125±6 nm No Yes I.V. 2020 [157]
cancer romidepsin mAb Physical (Incubation) (empty#, NTA)
Renal cell Mouse RCC cells miRNA-31-5p None Before EVs isolation, 53.3-57.8 nm No No I.V. 2020 [158]
carcinoma Transfection based (empty#, NTA)
(miRNA mimetics)
Schwannoma Mouse HEK-293T CD-UPRT mRNA & None Before EVs isolation, 159 nm (empty#, No No Local 2013 [159]
protein Transfection based NTA)
(Plasmid)
22
Thyroid cancer Mouse Malignant cells CRISPR/Cas9 TNF-α After isolation EVs, NA No No I.V. 2019 [61]
Physical
(Electroporation)
Table 2b
Preclinical studies of EVs as drug delivery systems for cardiovascular disease treatment
Disease/condition Animal EV donor Active pharmaceutical EV surface API loading EV size/charge* EV Biodistribution Route of Year of Ref.
model cell type ingredient (API) modifications procedure purity studies administration publication
stated
Myocardial infarction
Acute myocardial Rat MSCs lncRNA-H19 None Before EV isolation, About 100 nm No No Local 2019 [160]
infarction Stimulated by (NTA)
atorvastatin
Acute myocardial Rat Adipose- miRNA-126 None Before EVs isolation, 50-100 nm (NTA) No No I.V. 2018 [161]
infarction stem cells Transfection based
(miRNA)
Acute myocardial Rat MSCs Akt None Before EVs isolation, About 100 nm No No I.V. 2017 [162]
infarction Transfection based (NTA)
(Adenovirus)
Acute Myocardial Rat MSCs TIMP2 protein None Before EVs isolation, 40-90 nm (TEM) No No Local 2019 [163]
infarction Transfection based
(Lentivirus)
Acute Myocardial Mouse HEK293T miRNA-21 None Before EVs isolation, 30-150 nm (NTA) No No Local 2019 [164]
(Plasmid)
Acute Myocardial Mouse Human miRNA-21 None After isolation EVs, About 104 nm No No Local 2019 [165]
infarction peripheral Chemical (Exo-Fect™ (empty#, NTA)
blood Exosome
Transfection kit)
Acute Myocardial Mouse MSCs Stromal‐derived factor 1 None Before EVs isolation, 112.2±19.6 nm No No Local 2019 [166]
(empty#, NTA)
(Plasmid)
Myocardial ischemia Rat BMSCs miRNA‑125b None Before EVs isolation, 60–100 nm No No Local 2020 [167]
reperfusion injury Transfection based (empty#, TEM)
(miRNA)
23
Stroke
Cerebral ischemia Rat MSCs miRNA-223-3p None Before EVs isolation, 30-150 nm (TEM) No No I.V. 2020 [168]
Transfection based
(Lentivirus)
Cerebral ischemia Mouse BMSCs Curcumin c(RGDyK) After isolation EVs, About 107 nm No Yes I.V. 2018 [169]
peptide physical (Incubation) (NTA) / -21.6 mV
(empty#)
Cerebral ischemia Mouse HEK293T Bioactive nerve growth RVG peptide Before EVs isolation, 20-500 nm (NTA) No Yes I.V. 2020 [170]
factor Transfection based
(Plasmid)
Cerebral ischemia Mouse BM-MSCs miRNA-124 RVG peptide After isolation EVs, NA No Yes I.V. 2017 [171]
Physical
(Electroporation)
Cerebral ischemia- Rat RAW264.7 Curcumin None Before EVs isolation, 110.1±8.1 nm No No I.V. 2020 [172]
reperfusion injury macrophage Incubation (DLS)
Cerebral ischemia- Rat Adipose- Pigment epithelium- None Before EVs isolation, About 100nm No No Local 2018 [173]
reperfusion injury derived stem derived factor Chemical (TEM)
cells Transfection based
(Plasmid)
Cerebral ischemia- Mouse MSCs Curcumin None After isolation EVs, About 118 nm No No I.N. 2016 [174]
reperfusion injury Physical (Incubation (empty#, NTA)
followed by freeze-
thaw cycle)
Other cardiovascular disease
Aging-induced Mouse UMSCs miRNA-675 None Before EVs isolation, NA No No Local 2019 [175]
vascular dysfunction Transfection based
(miRNA)
Atherosclerosis Mouse THP-1 & Anti-miR-150 None Before EVs isolation, NA No No I.V. 2013 [153]
293T Chemical
Transfection based
(Anti-miR)
Cardiotoxicity Mouse Blood miRNA-21 None After isolation EVs, 40-400 nm No No I.V. 2020 [176]
Physical (empty#, NTA)
(Electroporation)
Diabetic Mouse Hsp20-TG Hsp20 None Before EVs isolation, About 100 nm No No I.V. 2016 [177]
cardiomyopathy Cardiomyocy Transfection based (DLS)
tes (Adenovirus)
24
Transthyretin Mouse Human siTTR with pre-miR-45 None Before EVs isolation, About 100 nm No Yes I.V. 2020 [178]
amyloidosis primary backbone Transfection based (NTA)
neonatal (Lentivirus)
fibroblasts &
HEK293T
Table 2c
Preclinical studies of EVs as drug delivery systems for neurological disease treatment
Disease/condition Animal EV donor cell Active pharmaceutical EV surface API loading EV EV Biodistribution Route of Year of Ref.
model type ingredient (API) modifications procedure size/charge* purity studies administration publication
stated
Alzheimer’s disease
Alzheimer's disease Mouse RAW264.7 Curcumin None Before EVs isolation, 117.4±10.5 nm No Yes I.P. 2019 [179]
Macrophages Incubation −4.9 mV (DLS)
Alzheimer’s disease Mouse Dendritic cells siBACE1 RVG peptide After isolation EVs, About 88 nm No No I.V. 2011 [180]
Physical (empty#, NTA)
(Electroporation)
Parkinson’s disease
Parkinson’s disease Mouse Primary DCs Anti-alpha-synuclein RVG peptide After isolation EVs, About 100 nm No No I.V. 2019 [181]
shRNA-minicircle Physical (NTA)
(Electroporation)
Parkinson's disease Mouse HEK293T DNA Aptamer F5R2 α- RVG peptide After isolation EVs, About 100 nm No No I.P. 2019 [182]
Synuclein Physical (Incubation) (empty#, TEM)
Parkinson’s disease Mouse HEK293T Catalase mRNA CD63-L7Ae, Before EVs isolation, About 100 nm No Yes S.C. 2018 [183]
RVG peptide Transfection based (NTA)
& Cx43 S368A (Plasmid)
Parkinson's disease Mouse Blood Dopamine None After isolation EVs, 70-100 nm Yes Yes I.V. 2018 [184]
Physical (Incubation) (TRPS)
Parkinson's disease Mouse RAW264.7 Catalase None After isolation EVs, 100.5-183.7 No No I.N. 2015 [185]
Macrophages Physical (Sonication, nm, PDI 0.2-
incubation or 0.48 (DLS) &
extrusion); After 99.5-162.4 nm
isolation EVs, (NTA)
Chemical (Incubation
25
with saponin)
Parkinson’s disease Mouse Murine siRNA α-Syn RVG peptide After isolation EVs, About 100 nm No No I.V. 2014 [186]
dendritic cells Physical (empty#, NTA)
(Electroporation)
Other neurological disease
Huntington’s Mouse U87 cells hsiHtt None After isolation EVs, About 140 nm No No Local 2016 [187]
disease Chemical (Incubation) (NTA) / -32 mV
Multiple sclerosis Mouse MSCs LJM-3064 aptamer LJM-3064 After isolation EVs, 133±15nm No No I.V. 2019 [188]
aptamer Chemical (Click (DLS) / −40.8
chemistry) mV
Table 2d
Preclinical studies of EVs as drug delivery systems for inflammatory disease treatment
Disease/condition Animal EV donor Active pharmaceutical EV surface API loading EV size/charge* EV Biodistribution Route of Year of Ref.
model cell type ingredient (API) modifications procedure purity studies administration publication
stated
Arthritis
Osteoarthritis Rat SMSCs miRNA-140-5p None Before EVs 95.01±35.91 nm No No Local 2017 [189]
isolation,
(DLS)
Transfection based
(Lentivirus)
Osteoarthritis Rat Dendritic miRNA-140 Chondrocyte- After isolation EVs, 40-200 nm No No Local 2020 [190]
cells affinity peptide Physical (empty#, NTA)
(Electroporation)
Rheumatoid arthritis Mouse MSCs miRNA-150-5p None Before EVs About 100 nm No No I.P. 2018 [191]
isolation, (DLS)
Transfection based
(Plasmid)
Other inflammatory disease
Allergic cutaneous Mouse T cells miRNA-150 Ab LC After isolation EVs, About 130 nm No No I.V. 2013 [192]
contact dermatitis Physical (empty#, NTA)
(Incubation)
26
Brain inflammatory Mouse 3T3L1, 4T1, Curcumin & JSI124 None After isolation EVs, NA No Yes I.N. 2011 [193]
diseases CT26, A20 & (cucurbitacin I) Physical
EL4 (Incubation)
Central nervous Rat Embryonic PsiRNA-ASC None After EVs isolation, NA No No I.V. 2016 [194]
system injury cortical Physical
neuroinflammation neuronal (Electroporation)
culture
Intestinal fibrosis Rat BMSCs miRNA-200b None Before EVs About 500 nm No No I.V. 2017 [195]
isolation, (TEM)
Transfection based
(Lentivirus)
Inflammation-related Mouse EL4 Curcumin None After isolation EVs, NA No Yes I.P. 2010 [196]
diseases &Macrophag Physical
es (Incubation)
Inflammatory bowel Rat Dendritic IL-10 IL-10 Before EVs NA No No I.P. 2010 [197]
disease cells isolation,
Incubation
Table 2e
Preclinical studies of EVs as drug delivery systems for other disease treatment
Disease/condition Animal EV donor Active pharmaceutical EV surface API loading EV EV Biodistribution Route of Year of Ref.
model cell type ingredient (API) modifications procedure size/charge* purity studies administration publication
stated
Obesity and Diabetes
Diabetes Mouse THP-1 & Anti-miR-150 None Before EVs NA No No I.V. 2013 [153]
293T isolation,
Chemical
Transfection
based (Anti-
miR)
Type-1 diabetes Mouse hBMSCs siFas & Anti-miR-375 None Before EVs NA No Yes I.V. 2016 [198]
isolation,
Transfection
based (Plasmid)
Obesity Mouse HEK293T miRNA-148a-responsive None Before EVs 50-300 nm No Yes I.V. 2020 [199]
PGC1a mRNA isolation, (NTA)
Transfection
based (miRNA)
27
Infectious disease
HIV-1 Mouse Dendritic HIV-1-Gp120 None Before EVs NA No No I.V. 2012 [200]
cells isolation,
Transfection
based
(Adenovirus)
Tuberculosis Mouse Bone Mycobacterium RNA None Before EVs About 170 nm No No I.T. 2019 [201]
marrow isolation, (NTA)
macrophag Transfection
e based (Bacteria)
Kidney disease
Acute kidney injuries Mouse MSCs miRNA-10a-5p & None Before EVs 100-300 nm No No I.V. 2019 [202]
miRNA-29a-3p & isolation, (NTA)
miRNA-127-3p & Transfection
miRNA-486-5p based (miRNA)
Chronic kidney Mouse Primary miRNA-29 RVG peptide Before EVs 91 ± 1.9 nm No Yes Local 2019 [203]
disease mouse isolation, (NTA)
satellite Transfection
cells based
(Adenovirus)
Chronic kidney Mouse BM-MSCs Erythropoietin Erythropoietin Before EVs NA No No I.V. 2015 [204]
disease isolation,
Incubation
Liver disease
Acute liver failure Mouse hUCMSCs TNF-α None Before EVs 30-150 nm No No I.V. 2020 [205]
isolation, (NTA)
Incubation
Acute liver injury Mouse HEK293 dCas9- VPR/sgRNA None Before EVs About 50 and Yes Yes I.V. 2018 [206]
HGF isolation, 1000 nm peaks
Transfection (DLS) / About
based (Plasmid) 140nm
(exosome
fraction, NTA)
Acute liver injury Mouse HEK293T miRNA-155 & C/ebpα CD9-HuR Before EVs 100 nm No Yes I.V. 2019 [207]
gRNA CRISPR/dCas9 fusion protein isolation, (empty#, DLS)
Transfection
based
(Lentivirus)
28
Hepatitis C Mouse Huh7 shCD81 None Before EVs NA No No I.V. 2012 [208]
isolation,
Transfection
based
(Lentivirus)
Muscular disease
Muscular dystrophy Mouse Human CD63-propeptide CD63- Before EVs About 110.5 No Yes I.V. 2020 [209]
293FT propeptide isolation, nm (NTA)
cells Transfection
based
(Lentivirus)
Muscular dystrophy Mouse C2C12 Phosphorodiamidate CPO5 peptide After isolation About 117.1 No Yes I.V. 2018 [210]
cells morpholino oligomer EVs, Physical nm (NTA)
(Incubation)
Others
Cutaneous wound Rat MSCs miRNA-126-3p None Before EVs 64.33±28.88 No No I.V. 2017 [211]
isolation,
nm (DLS)
Transfection
based
(Lentivirus)
Immunomodulation Mouse THP1 & FasL protein AS1411 After isolation 88.0±0.09 to No No I.P. 2019 [212]
J774A.1 aptamer EVs, physical
(Incubation) 88.9±1.4 nm
(DLS) / -10.0±
1.2 to -20.4±
2.05 mV
I.V.: Intravenous administration; I.P.: Intraperitoneal injection; I.N.: Intranasal; S.C.: Subcutaneous injection; I.D.: Intradermal injection; I.T.: Intratracheal
administration; MSCs: Mesenchymal stem cells; RVG: Rabies virus glycoprotein. *Transmission electron microscopy (TEM) was commonly used to observe EV
morphology and frequently measure particle size as a secondary readout. If the authors used nanoparticle tracking analysis (NTA), dynamic light scattering (DLS) or
tunable resistive pulse sensing (TRPS) as their main readout for particle size we did not include the size estimated by TEM (following the MISEV2018 guidelines [7])
in the tables. # “empty” means the size of EVs was determined prior to the loading of APIs.
29
3.2. Diving deeper into the tables: analytical interpretation
3.2.1. Diseases
The main tables of this systematic review were grouped by disease categories of the study (Tables 2
a,b,c,d,e) and then the table contents were organized by specific diseases/conditions, to facilitate the
search of study parameters by readers who are interested in one particular disease or disease category.
As shown in Fig. 4a and Table 2a, the majority (66.2%) of publications on EVs for drug delivery during
the past decade were focused on cancer treatment. 70.6% of total studies in the year of 2020 were
cancer related (Fig. 4b), of which 27% were on breast cancer, 18% on lung cancer and 12% on brain
cancer. The extensive studies of EVs in cancer-related preclinical models may be partly attributable to
the relatively easy adoption and readily availability of tumor animal models. Studies on cardiovascular
disease (12.7%), the second most investigated disease type, were essentially represented by myocardial
infarction and stroke (Table 2b, Fig. 4a). Neurological and inflammatory disease studies constitute 6.4%
and 5.7% of the total, respectively, (Fig. 4a) where the majority of publications were on Parkinson’s and
Alzheimer’s disease (in the neurological class) and arthritis (for the inflammatory diseases category)
(Table 2c, Table 2d). In the “other” disease group (10.2%), we categorized a variety of liver, kidney,
muscular and infectious diseases together with diabetes, obesity, immunomodulation and wound healing
studies.
A B
Diseases/conditions Trends in diseases/conditions
40
Cancer Cancer
35
Cardiovascular 30
Cardiovascular
No of articles
Neurological Neurological
25
Inflammatory Inflammatory
20
Other disease
15 Other
10 Total publications
5
0
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
Fig. 4. Diseases tested in preclinical studies of EVs as drug delivery systems. Proportion (A) and trend
(B) analysis of diseases/conditions investigated.
3.2.2. Animal models

With regard to the animal models used (Fig. 5a), a vast majority of publications used mice (87.3%),
followed by rats (11.5%) and zebrafish (1.3%). The overwhelming majority of studies using mice has
been increasing over time (Fig. 5b). The extensive usage of mice as experimental animal model is mainly
because of historic preferences and available data from related studies as well as cost-effectiveness.
These percentages though, are different between disease groups (Table 2a). Rats represent 55% and
40% of animal studies for inflammatory and cardiovascular diseases, respectively. In the case of
cardiovascular diseases rats have been the main animal model for decades, where detailed and effective
experimental procedures for multiple conditions, like myocardial infarction and stroke are available as
well as rat strains that spontaneously develop these diseases [213]. A similar argument applies to
inflammatory diseases [214]. The two publications using zebrafish, were focused on treating brain cancer
30
(Table 2a) as zebrafish represents a model that might gain popularity due to its cost-effective
maintenance and the opportunity to dynamically visualize tumor growth in vivo [215].
A B
Animal models Trends in animal species
40
Mouse Mouse
Rat Rat
30
Zebrafish
No of articles
Zebrafish
20 Total publications
10
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
Fig. 5. Animal models in preclinical studies of EVs as drug delivery systems. Proportion (A) and trend
(B) analysis animal models used.
3.2.3. Donor cell types

Based on the origin of EVs, we grouped the EV donor cell types into eight different categories (Fig. 6a,b).
Among all the analyzed preclinical studies, EVs derived from cancer cell lines (23.6%), stem cells
(22.9%), and HEK293 cells (21.7%) were most commonly used. Interestingly, in the past five years, a
few studies explored the drug delivery potential of EVs derived from blood (plasma or serum), milk or
plant although the cellular origin of those EVs were mostly less defined (Fig. 6b). For studies using
cancer cell-derived EVs, the vast majority (87%) were focused on the treatment of cancer (Fig. 6c), likely
due to the assumed tropism of cancer cell-derived EVs towards tumors [105, 216]. Other frequently used
donor cells in cancer related studies were HEK293 cells (24%) and stem cells (14.4%) (Fig. 6d). In
studies of cardiovascular diseases (Fig. 6e), stem cell-derived EVs were commonly used (60%).
31
A B
EV donor cell types Trends in EV donor cell types
12
Cancer cell line
Cancer cell line
Stem cell derived Stem cell derived
9
No of articles
HEK293 derived HEK293 derived
Macrophage
6 Macrophage
Dendritic cell
Milk derived Dendritic cell
Blood derived Milk derived
3
Other sources
Blood derived
0 Other sources
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
C D E
Cancer cell lines use by disease Cancer studies EV donor cell types Cardiovascular disease EV donor cells
1.0 0.4 0.8
Proportion of publications
0.8 0.3 0.6
0.6
0.2
0.4
0.4
0.1
0.2
0.2
0.0
0.0 0.0
s
em d
es
dr ved
de e
ro ells
ll
so d
ce
n
ce
St ive
ve
M hag
lli
s
M de ed
O r c es
B 3 d lls
so ne
ry
e
m r
ur
c
th eri
ov i ca
la
ce
lo tic
ce
D der
r
C cro ive
as
el
eu ato
g
ce
od iv
er lli
th scu
d
In Can
ur
rc
ce ha
se
g
l o er
th el
ilk
3
od
lo
EK tem
er
ce
29
en
an p
di
ac
a
ro
m
an
EK
M
er
S
B
fla
29
di
a
H
N
ar
O
C
H
Fig. 6. EV donor cell types in preclinical studies of EVs as drug delivery systems. Proportion (A) and
trend (B) analysis of EV donor cell types used. (C) EVs of cancer cell origin used in studies of various
diseases. (D) Proportion of EV donor cell types used in cancer-related publications. (E) Proportion of EV
donor cell types used in cardiovascular disease publications.
3.2.4. Active pharmaceutical ingredients (API) associated with EVs

Among all the APIs loaded into or onto EVs in the analyzed preclinical studies (Fig. 7a,b), nucleic acids
(46.5%) and small molecule drugs (39.5%) were the most popular, followed by proteins/peptides (17.8%),
oncolytic viruses (2.5%) and nanoparticles (1.9%). Of note, three nanoparticles, Bi2Se3 nanodots/DOX
[123], PMA/Au-BSA@Ce6 nanoparticles [103] and superparamagnetic iron oxide
nanoparticles/Curcumin [49] were considered as APIs for this analysis since they were encapsulated into
EVs as therapeutics. As the most popular API, the studied nucleic acids (Fig. 7c,d) were mainly miRNA
(39.7%), siRNA/shRNA (30.1%), antagomiR (11%), mRNA (9.6%), and CRISPR/CAS9 (6.8%). The other
nucleic acids (9.6%) encompassed plasmid DNA, DNA aptamer, lncRNA, CpG DNA and minicircle DNA.
Interestingly, miRNA was particularly used for inflammatory disease and cardiovascular disease,
constituting 55.6% and 40% of the respective publications (Supplementary Fig. S1a). siRNA/shRNA,
however, was frequently used as an API in the studies (40%) of neurological disease (Supplementary
Fig. S1a). Of notice, siRNA/shRNA, antagomiR, mRNA and CRISPR/CAS9 used as APIs were mainly
reported in cancer studies, whereas miRNA as an API was used more often for other types of diseases
(Supplementary Fig. S1b).
32
A B
Active pharmaceutical ingredients (APIs) Trends in active pharmaceutical ingredients (APIs)
40
Nucleic acids Nucleic acids
Small molecule drugs 35
Small molecule
Protein/Peptides 30
No of articles
drugs
Oncolytic virus 25
Nanoparticles Protein/Peptides
20
Oncolytic virus
15
Nanoparticles
10
Total publications
5
0
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
C D
API: Nucleic acids Trends in nucleic acids loaded
20
miRNA miRNA
siRNA/shRNA siRNA/shRNA
AntagomiR 15
No of articles
mRNA AntagomiR
CRISPR/CAS9 10 mRNA
Other nucleic acids
CRISPR/CAS9
5 Other nucleic acids
Total nucleic acids
0
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
Fig. 7. Active pharmaceutical ingredients (APIs) in preclinical studies of EVs as drug delivery systems.
Proportion (A) and trend (B) analysis of APIs used. Proportion (C) and trend (D) analysis of the subtypes
of nucleic acids used as APIs.
3.2.5. API loading procedures

APIs can be associated with EVs either before or after EV isolation. Before EV isolation, APIs were
loaded via incubation, transfection-based methods or ultraviolet irradiation (Fig. 8a), whereas after EV
isolation (Fig. 8b) APIs were loaded by either physical (electroporation, plain incubation, sonication, etc.)
or chemical procedures (transfection kit, mix with organic solvent, etc.). Interestingly, we found no
obvious preference of API loading before versus after EV isolation in the past decade (Supplementary
Fig. S2). API loading before EV isolation was mainly achieved through transfection-based methods
(77.9%), including viral transduction, plasmid, miRNA, and antagomiR transfection (Fig. 8a). The
frequency of API loading before EV isolation using plain incubation (11.7%) and incubation post UV
irradiation of the cells (10.4%) is similar. As shown in Fig. 8b, API loading after EV isolation was mainly
performed with physical procedures like electroporation (39%), plain incubation (29.3%) and sonication
(12.2%); and less than 20% studies employed chemical procedures such as transfection (9.8%) and
mixing with organic solvents (7.6%). For the past decade, the use of electroporation has been increasing
and reached 58.8% in the year 2020 among all studies involving API loading after EV isolation (Fig. 8b).
Surprisingly, only nucleic acids (59.4%) and small molecule drugs (40.6%) were the APIs loaded by
electroporation (Supplementary Fig. S3).
33
A
API loading procedures before EV isolation Trends in API loading procedures before EV isolation
20
Transfection based Transfection based
Incubation
Ultraviolet irradiation Incubation
15
No of articles
Ultraviolet irradiation
Total publications API loading
10
before EV isolation
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Total=77
Year
B
API loading procedures after EV isolation Trends in API loading procedures after EV isolation
20
Physical: Electroporation Physical: Electroporation
Physical: Plain incubation
Physical: Sonication Physical: Plain incubation
15
No of articles
Chemical: Transfection kit Physical: Sonication
Chemical: Mix with organic solvent Chemical: Transfection kit
Other 10
Chemical: Mix with organic solvent
5 Other
Total publications API loading
after EV isolation
0
10
11
12
13
14
15
16
17
18
19
20
Total=82
20
20
20
20
20
20
20
20
20
20
20
Year
Fig. 8. API loading procedures for preclinical studies of EVs as DDS. (A) Proportion and trend analysis
of API loading procedures before EV isolation. Total: 77 publications. (B) Proportion and trend analysis
of API loading procedures after EV isolation. Total: 82 publications.
3.2.6. Surface modification status

Surface modification of EVs, and nanoparticles in general, with targeting ligands and other molecules
represents a well-known and widespread method for targeting cells in organs and tissues of interest.
Digging deeper into the targeting ligands used in EVs up to date, they were extensively discussed in a
recent review by Gudbergsson et al., i.e., the most common type of targeting ligands used in EVs as
DDS are small peptides (38%), transmembrane proteins (34%) and antibody fragments (25%) [217]. For
EVs as DDS in preclinical models, similar numbers of studies used surface-modified and surface-
unmodified EVs although more studies intended to use EVs without surface modification in recent years
(Fig. 9a,b). Despite PEGylation is commonly used for liposomes and other non-cell derived DDS to
increase the circulation time, we found that only two recent studies (1.3%) using EVs as DDS in
preclinical models incorporated PEG to modify EV surface [81, 129]. We further investigated the
application of EV surface modifications in different disease studies (Fig. 9c). We found that 85% of
cardiovascular disease studies used EVs without surface modifications, perhaps due to the difficulties in
manipulating MSCs, representing 60% of EV donor cells in cardiovascular disease, and the extensive
use of local administration (40%) in cardiovascular disease studies (Supplementary Fig. S4). In
contrast, 60% of the neurological disease studies used EVs with surface modifications, mainly featuring
the addition of the RVG peptide (83.3%) that presumably facilitates transcytosis of EVs across the blood
brain barrier (BBB) via targeting the alpha-7-subunit of the nicotinic acetylcholine receptor found in the
brain [180, 218].
34
A B
EV surface modifications Trends in surface modifications
40
Unmodified Total
Modified
Unmodified
30
No of articles
Modified
20
10
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
C
Proportion of surface modifications in EVs by disease
1.0
0.9 Cancer
0.8 Cardiovascular
0.7 Neurological
0.6
0.5 Inflammatory
0.4 Other disease
0.3
0.2
0.1
0.0
ed
ed
ed
ed
ed
ed
ed
ed
ed
ed
ifi
ifi
ifi
ifi
ifi
ifi
ifi
ifi
ifi
ifi
od
od
od
od
od
od
od
od
od
od
M
M
nm
nm
nm
nm
nm
U
Fig. 9. Surface modification status in preclinical studies of EVs as drug delivery systems. Proportion (A)
and trend (B) analysis of surface modifications of EVs as DDS in preclinical studies. (C) EVs with or
without surface modifications used as DDS in preclinical studies of various diseases.
3.2.7. Size and zeta potential

Measuring the size and size distribution of nanoparticles is crucial to assessing the stability and drug
delivery efficiency of a DDS. EVs are notorious for their high heterogeneity in size, which is influenced
by multiple factors, including cell sources, cell culture conditions or EV producing microenvironment, and
EV isolation procedures [219]. In addition, most of the current methodologies have various technical
limitations and are not able to detect the smallest EVs (bellow 30 nm), making it particularly difficult to
compare EV parameters such as concentrations, size and size distribution [220]. In our systematic
analysis (Fig. 10a,b) we observed that the majority of publications used nanoparticle tracking analysis
(NTA) and dynamic light scattering (DLS) to determine EV size, representing 47.1% and 38.9% of total
publications, respectively. Importantly, the utilization of these two methods keeps increasing in the recent
years. DLS is broadly used to measure nanoparticles of a wide range of sizes; however, it suffers from
severe limitations of accurately measuring the size or size distribution of polydisperse samples that
contain mixed particle populations. In contrast, NTA, obtains size information based on the Brownian
motion of individual particles. Given the high heterogeneity of isolated EVs, NTA outperforms DLS and
therefore is recommend for measuring EV size by the International Society for EVs [8, 221-223]. 12.7%
of the publications, to our surprise, did not measure EV size or size distribution. Furthermore, although
most studies used transmission electron microscopy (TEM) to confirm EV size (as a secondary readout
to DLS or NTA) while examining EV morphology, 5.7% of total studies only used TEM to estimate EV
35
size. However, the vacuum pressure in TEM and sample preparations including fixation and dehydration
probably affect the size of EVs [220]. Other EV size measure methods, namely Tunable Resistive Pulse
Sensing (TRPS) and flow cytometry acquainted for 1.3% and 0.6% of the publications, respectively.
Zeta potential, determined by DLS, which measures the surface charge of nanoparticles, is another
important parameter affecting the stability, potential of aggregation and drug delivery effectiveness of
EVs used as DDS. However, only 21.7% of the publications reported zeta potential of EVs (Fig. 10c).
This means that nearly half of the studies using DLS for EV size measurement did not determine zeta
potential at the same time. Nevertheless, among those studies that determined the zeta potential of EVs
(Fig. 10d), 23.5% reported a neutral surface charge (between 0 and -10mV) and 76.5% reported a
negative surface charge (zeta potential below -10mV).
A B
EV size measurement Trends in EV size measurement
40
NTA NTA
DLS
No measurement DLS
30
No of articles
TEM No measurement
Other EV size
20 TEM
measurements
Other EV size
measurements
10
Total publications
0
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
C D
EV zeta potential EV zeta potential
Present Neutral
Absent (-) charged
Fig. 10. EV size and zeta potential measurement in preclinical studies of EVs as DDS. Proportion (A)
and trend (B) analysis of EV size measurement methods. Proportion (C) of studies reporting EV zeta
potential and qualitative analysis of the reported zeta potential (D).
3.2.8. Estimation of purity

Purity has become a critical issue to develop EVs as DDS due to the complex biological origin of EVs
and the existing challenges in isolation procedures. As a drug delivery platform, low purity of EVs may
compromise the drug loading efficiency and cause unexpected toxicity [224]. However, purity
assessment is difficult. For instance, NTA cannot distinguish EVs from other particles such as LDL and
protein complexes [225]. TEM allows the distinction of EVs from other non-EV particles [225, 226],
however, it is not able to quantify soluble contaminants in the sample [227]. Another informative strategy
is using the western blotting to semi-quantitatively examine the presence of EV proteins and known non-
EV markers (e.g., calnexin) [227]. In this systematic review, we identified 8 studies that assessed EV
purity according to the MISEV2018 guidelines, i.e., reporting quantitative ratios of protein:particle,
36
lipid:particle or lipid:protein as an indicator of the purity of EVs [8], representing only 5.1% of the reviewed
publications (Fig. 11a). In fact, all those studies reported the ratio of protein:particle rather than ratios of
lipid:particle or lipid:protein. Excitingly, we observed an increasing trend in the number of publications
that evaluated EV purity by reporting protein:particle ratio over recent years (Fig. 11b), especially after
the publication of the latest MISEV guidelines in 2018. Still, the applicability of this methodology is an
issue and impurities in the form of protein aggregates [226] or non-EV particles of a similar size (e.g.,
LDL and VLDL) [228] cannot be discriminated. To address those issues, other methodologies such as
flow cytometry detecting EV-specific markers (e.g., CD63, CD81, CD9) [148, 151, 229, 230] may be
combined with the current ones. All in all, our findings point out the lack of consensus of a “gold standard”
to determine EV purity.
Fig. 11. Purity analysis of EVs as DDS in preclinical studies. (A) Proportion and (B) trend analysis of
studies reporting EV purity with protein:particle ratio.
3.2.9. Biodistribution studies

Monitoring biodistribution is an essential step in preclinical studies to elucidate the final destination of the
administered EVs, thus providing valuable information on their targeting efficiency, pharmacokinetics and
potential toxicity. Among all preclinical studies of EVs as DDS included in our systematic analysis, 49.7%
performed biodistribution experiments (Fig. 12a). Despite the number of studies reporting EV
biodistribution has been increasing over the past decade, the change in the percentage of such studies
is not impressive (Fig. 12b). The higher proportion of studies reporting EV biodistribution in 2018 (66.7%)
and 2020 (55.9%) suggests that this issue is gaining attention in the field. Furthermore, we noticed that
two methodologies were commonly used to investigate biodistribution EVs as DDS: tracing EVs labeled
with fluorescence probes via in vivo optical imaging [80, 184]; or quantitative profiling the delivered
cargoes in various animal organs ex vivo using analytical chemistry methods (e.g. HPLC or LC-MS) [75].
Given the possible disassociation between the vector and the cargo, combination of the two
methodologies to monitor the biodistribution of both EVs (DDS) and delivered cargoes is recommended
for future studies.
37
A B
EV biodistribution studies Trends in EV biodistribution studies
40
Present Present
Absent
Absent
30
No of articles
Total publications
20
10
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
Fig. 12. EV biodistribution in preclinical studies of EVs as DDS. Proportion (A) and trend analysis (B) of
studies reported EV biodistribution.
3.2.10. Administration routes

The administration routes of EVs as DDS in preclinical studies are important as they play a critical role
in the pharmacokinetics and pharmacodynamics of drug-loaded EVs. Some studies have given evidence
that tissue distribution and clearance rate of EVs might be influenced by the route of administration [227].
For this purpose, we evaluated the different administration routes of EVs that were used in the selected
publications. Intravenous administration has been the dominant route of administration over the past
decade (63.1%), gaining particular popularity in recent years (Fig. 13a,b). In 2020, 88.2% of all the
publications used intravenous administration to apply EVs for drug delivery studies (Fig. 13b). Examining
the routes of administration for each of the disease groups (Supplementary Fig. S3) shows that the
main administration routes for cancer studies are intravenous (64.4%), local (19.2%) and intraperitoneal
(11.5%) administration, while for cardiovascular diseases they are intravenous (55%) and local (40%)
administration.
A B
Administration routes Trends in administration routes
40 Intravenous administration
Intravenous administration 35 Local administration
Local administration
Intraperitoneal injection 30 Intraperitoneal injection
No of articles
Oral administration 25 Oral administration

Intranasal administration 20
Subcutaneous injection
Intranasal administration
Intradermal injection 15 Other administration forms
Intratracheal administration 10 Total
5
0
10
11
12
13
14
15
16
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
Year
Fig. 13. Administration routes in preclinical studies of EVs as drug delivery systems. Proportion (A) and
trend (B) analysis of administration routes of EVs as DDS in preclinical studies.
3.3. Clinical trials of EVs as DDS: on the way to reach patients

A clear reflection of the promising therapeutic results accomplished with EVs in preclinical studies is the
appearance of multiple clinical studies involving drug delivery through EVs in patients (Table 3).
38
Table 3
Clinical trials of EVs as DDS.
Disease EV donor cell Compound Phase, Status NCT NO.

type
Enrollment
Acute Mesenchymal MiR-124 Phase I/II, Recruiting (Estimated NCT03384433
ischemic stem cells Study Completion
stroke 5* Date: March 17,
2021)
Pancreatic Mesenchymal siRNA Phase I, Not yet recruiting NCT03608631
cancer stem cells KrasG12D (Estimated Study
28* Completion Date:
March 2022)
Colon Plant cells Curcumin Phase I, Active, not recruiting NCT01294072
cancer (Estimated Study
7 Completion Date:
December 2022)
Malignant Tumor cells Chemothera- Phase II, Unknown1 (Estimated NCT01854866
ascites & peutics Study Completion
Pleural 30* Date: March 2014)
effusion
Malignant Autologous Methotrexate Phase II, Recruiting (Estimated NCT02657460
pleural tumor cells Study Completion
effusion 90* Date: December
2019)
1 Study has passed its completion date and status has not been verified since September 2013.
* Estimated enrollment.
Already five clinical trials have been using EVs as drug delivery systems in patients. Two of those clinical
trials are in Phase I, one is in Phase I/II and the other two are in Phase II. In line with what can be seen
from preclinical studies of the past decade, where 66.2% of the articles correspond to cancer studies,
80% of the clinical trials (4/5) are focused on cancer treatment. The remaining clinical trial aims to treat
patients with acute ischemic stroke. Also, in preclinical studies cardiovascular diseases were second
during the past decade (12.7%). Cancer cells and stem cells represent the two main EV donor cell types,
similar to that in preclinical studies, in clinical studies (40% of the totality each). Moreover, the types of
APIs loaded in EVs, including small molecule drugs, miRNA and siRNA represent 60%, 20% and 20%,
respectively, of the five clinical trials, also similar to the trends observed in preclinical studies (39.5%,
18.5% and 14%, respectively). Although it is difficult to reach a statistically meaningful conclusion with
only five clinical trials, the interesting similarities observed between clinical and preclinical studies as
discussed above may not be coincidental.
Along with the recent increase in pre-clinical and clinical trials using EVs as drug delivery systems, up to
date there are 20 other clinical trials based on the intrinsic therapeutic function of unmodified EVs
(Supplementary Table S2).
We consider that it is of vital importance to establish a multifactorial comparison between the current
gold standard of nanomedicine, the liposomes, and EVs, to achieve a meaningful assessment of the
possible niches where to potentially use EVs as DDS.
39
4. Liposomes versus EVs for drug delivery: heads up
4.1. Short overview of EVs as therapeutic delivery vehicles in comparison with liposomes
The main challenges in delivery of therapeutics to the site of action are off-target toxicity, rapid clearance,
and low accumulation and bioavailability in target tissue, cell or organelle [229]. To circumvent these
challenges, a broad range of synthetic delivery vehicles (liposomes, lipid nanoparticles, polymeric
micelles, inorganic nanoparticles, dendrimers, etc.) have been developed in the last few decades with
some of them already clinically approved. The main concept of a drug delivery vehicle is that the tissue
distribution and the body clearance are governed by the characteristics of the vehicle instead of those of
the drug itself [230-232]. Out of the available spectrum of all nanoparticles, the most successful delivery
vehicles up to date, with the highest number of clinical approvals on the market, are the liposomes. Given
their similarity, a side-by-side comparison of EVs with liposomes, regarding their physicochemical
properties and their drug delivery capacity, is presented next (Fig. 14).
Fig. 14. Liposomes versus EVs. (A) PEGylated liposomes split in quadrants: lipophilic drugs loaded in
the bilayer membrane; ligands can be incorporated to increase tissue targeting specificity; hydrophilic
drugs can be loaded in the lumen of liposomes; Onpattro, the first U.S. Food and Drug Administration
(FDA) approved siRNA loaded lipid nanoparticles, is made of ionizable lipids, cholesterol, PEGylated
lipids, and helper lipids. (B) EVs as a drug delivery system (DDS): proteins, hydrophilic drugs and nucleic
acids (miRNA, siRNAs, mRNAs, etc.) can be loaded in the lumen of vesicles whereas targeting ligands,
membrane proteins and lipophilic drugs can be incorporated in the membrane.
4.2. Physical features, production and quality control

Liposomes are structurally similar to EVs as they are composed of a lipid bilayer around an aqueous
compartment [233]. Similarly, as a drug delivery system (DDS), EVs can carry hydrophobic drugs within
the lipid membrane bilayer and hydrophilic drugs in the aqueous core [234]. In addition, the dimensions
40
of the clinically approved liposomes are around 100 nm [235-239], similar to exosomes and microvesicles
[8, 53]. This size is compatible with avoidance of premature clearance by macrophages, which increases
with increasing particle size, and renal clearance, which occurs to particles with hydrodynamic radius
lower than 5-6 nm [240, 241]. Moreover, the size of these liposomes allows extravasation at certain body
sites after intravenous administration and uptake by cells [242, 243]. Despite the similarities they share,
a number of differences exist between liposomes and EVs as drug delivery vehicles (Fig. 14). This can
be illustrated by comparing EVs with the pioneer liposomal formulation of Doxil®/Caelyx®, PEGylated
liposomes loaded with the chemotherapeutic doxorubicin [244]. Being approved by the FDA in 1995 and
the European Medicines Agency (EMA) in 1996, it was the first nanoparticular drug brought to the market.
Compared to EVs, clinically used liposomes like Doxil® are composed of a limited number of defined
lipids but no cellular components such as proteins and genetic materials [245], and, therefore, they are
relatively easy to handle in pharmaceutical quality control and large-scale manufacture processes. While
the initial Doxil® contained naturally sourced phospholipids, like hydrogenated soy phosphatidylcholine,
the current formulation only uses synthetic phospholipids. A variety of high throughput methods exist to
manufacture liposomes, including extrusion, ethanol injection and microfluidic mixing. Because of the
self-assembly of liposomes from all the individual components, full control exists over the composition,
including the constitution of the aqueous phase. This is exploited in the Doxil® formulation by loading a
high concentration of ammonium sulphate in the liposome interior. The gradient of this salt over the
membrane serves as the driving force for the subsequent remote loading of doxorubicin. Similarly, in
Vyxeos®, the exact proportion of two co-encapsulated drugs (daunorubicin and cytarabine) can be
controlled during production to obtain the optimal ratio for synergistic action.
EVs, however, contain the full repertoire of cellular lipids and are particularly enriched in lipid raft
components like sphingomyelin, cholesterol and lysophospholipids. It is noticeable that a higher degree
of complexity can be achieved with EVs in comparison with mixing individual components in liposomes.
Furthermore, due to the presence of biomolecules in the membrane and core, additional binding pockets
may be present in EVs for drug loading. However, loading into pre-formed EVs is extremely challenging
[49]. This presents a challenge for manufacture and quality control as the particular EV composition is a
snapshot of the cell at the time of production which may vary from cell to cell, from culture condition to
culture condition and over time. In terms of production and harvesting of EVs, so far scale-up remains
highly challenging.
4.3. In vivo administration of EVs and liposomes

4.3.1. Nanoparticles (EVs & liposomes) are rapidly cleared by the mononuclear phagocyte system
(MPS)
Liposomes represent biodegradable and biocompatible DDS with exceptionally versatile high‐throughput
preparation and drug encapsulation efficiencies, allowing lyophilization and surface modifications [246].
However, there are still some obstacles that need to be overcome to obtain improved drug efficacy,
diminished drug toxicity and reduced off-target effects. One of the biggest hurdles to obtain optimal drug
delivery to the target tissue is the rapid systemic clearance of liposomes from the blood through the
41
mononuclear phagocyte system (MPS) mediated by the opsonization of the liposomes (surface
adsorption of serum proteins) and the subsequent phagocytosis by macrophages, mainly those in the
liver and spleen [247]. Apart from macrophages, other cells of the reticuloendothelial system (RES), such
as liver sinusoidal endothelial cells, have also been reported to take up and clear liposomes [248, 249].
In a similar fashion, EVs are rapidly cleared by the MPS after systemic administration in vivo,
notwithstanding their composition [250, 251]. Likewise, after intravenous injection of EVs in vivo, the
biodistribution profile of EVs resembles that of non-pegylated liposomes of the same size and surface
charge [252]. In order to reduce immunogenicity and to avoid rapid blood clearance of liposomes,
polyethylene glycol (PEG) surface coating is widely used, thereby enabling more accumulation in the
target tissue [253]. The decoration of EVs with PEG or PEG-coupled targeting ligands has been proposed
as a promising strategy to enhance EV drug delivery capacities [81, 129, 254]. Another interesting
strategy is the selection of subsets of EVs that contain specific surface proteins like CD47. This protein
acts as a “don’t eat me signal” in EVs and may confer them the ability to circumvent the MPS and exhibit
longer circulation time [148, 255]. Furthermore, transiently blocking the RES through systemic
administration of empty liposomes or other substances like dextran sulfate has been explored to obtain
a substantial increase in functional drug delivery and tumor accumulation for both drug-loaded liposomes
and EVs [256-258].
4.3.2. Accelerated blood clearance (ABC) phenomenon upon multiple injections of nanoparticles
ABC was first reported by Dams et al. by showing that PEGylated liposomes exhibited enhanced
clearance and loss of effectiveness upon repeated administration [259]. This is due to their opsonization
by anti-PEG IgM antibodies and subsequent complement activation response [260, 261], which
accelerates the recognition and clearance of successive injections of PEGylated liposomes particularly
by liver macrophages. Strategies that have been reported to possibly circumvent the ABC phenomenon
include increasing the lipid dose for the first injection or administering a small pre-dose before the
second liposomal injection [262]. Similarly, PEG chain modification of the EV surface is a common
method used to prolong the circulation time of EVs [254], and it is reasonable to assume that the ABC
effect also applies to PEG-coated EVs and subsequently affects the effectiveness of EVs, since the
occurrence of the ABC effect in case of other PEGylated non-liposomal nanoparticles has been reported
as well [263, 264]. Different strategies could be applied in order to circumvent potential ABC effect in
EVs by; 1) using substitutions of PEG chains [260, 265]; 2) administering immune suppressive agents
[260] or 3) applying modified anti-PEG Ab to compete with the natural anti-PEG Ab [266].
4.3.3. Complement activation-related pseudoallergy (CARPA) upon nanoparticle injection

CARPA is an acute allergic reaction with symptoms including cardiopulmonary, hemodynamic, and an
array of other pathophysiological changes [267]. It has been reported that lipidic nanoparticles could
trigger adverse immunological reactions, and result in hypersensitivity CARPA, hindering their clinical
use in hypersensitive patients [268]. The severity of CARPA after systemic liposomal administration in
patients is influenced by morphological properties, size, surface charge, PEGylation, and cholesterol
42
content, amongst other characteristics of liposomes [268]. A representative example is Doxil®, which has
been reported to elicit rare but serious acute infusion reactions in cancer patients [269]. Therefore,
CARPA has been recognized as a safety issue. Although a number of studies have confirmed the
administration of liposomes could result in adverse CARPA reactions, its exact mechanism is still
unknown. At the same time this provides an opportunity for investigating the application of EVs as DDS,
but potentially without CARPA triggering. EVs may have the potential to overcome CARPA effects as
some studies claimed that EVs consist of a natural cocktail of biomolecules that do not cause adverse
reactions linked to liposomal particle infusion [270, 271]. But still, regarding possible CARPA adverse
reactions after administration of EVs, their occurrence remains uncertain. A Phase I clinical trial indicated
that dendritic cell-derived EVs caused mild inflammatory reactions after subcutaneous administration in
half of the patients [272, 273]. However, in some studies using pig models, CARPA-related adverse
effects were not observed after intracardiac administration of human MSC-derived EVs, while low doses
of liposomes could induce shock-like symptoms [46, 47]. Regarding the CARPA adverse reactions after
administration of EVs, these remain uncertain and demand extensive research as the current
experimental evidence is too limited [46, 47, 270, 273]. Of note, it is generally accepted that the choice
of species for preclinical validation is important to assess possible adverse reactions after infusion of
EVs and liposomes [3]. CARPA adverse reactions are not that common in rodents as they are in humans
and other species such as human-like pigs [3]. Therefore, the latter might be more suitable for these
types of studies.
4.3.4. Biodistribution profiles: passive vs. active targeting

All the approved liposomal drugs on the market rely on passive targeting, and only a small proportion of
the actively targeted formulations have reached clinical stages. This is due to the fact that even with
surface ligand-targeting a specific receptor on target cells, the accumulation of liposomes is still
considered to be dictated by passive extravasation processes referred to as the enhanced permeability
and retention (EPR) effect [274]. Regarding drug delivery in some types of cancer, liposomes take
advantage of the EPR effect caused by the leakiness of the tumor vessels to achieve higher tumor
accumulation [275]. Via the EPR effect, liposomes with longer circulation time are prone to accumulate
in the tumor [234] or injured myocardium [276]. Interestingly, liposomes have also been suggested in a
recent study to enter tumors through an process mediated by endothelial cells that line tumor blood
vessels [277]. A better understanding of those pathways will help the development of more effective
nanomedicine strategies.
In the case of EVs, studies have shown that EVs with a size below 100 nm are also capable of achieving
enhanced tumor targeting through the EPR effect in some tumors [278, 279]. Still, EVs naturally and
inevitably exhibit an interactive surface which makes them natural actively targeted carriers. However,
they display, in general, short circulation time which limits their opportunity to take advantage of passive
targeting. Enrichment of “don’t-eat-me” surface molecules such as CD47 that likely enable EVs to escape
from the MPS clearance system [148] might help to engineer EVs for passive targeting. Of note though,
the EPR effect is rather diversified being only present in some types of tumors and varies among
43
individuals and tumor types and should not be regarded as a general characteristic of solid malignancies,
as evidenced in clinical canine cancer patients [262]. Furthermore, the preclinical models used for cancer
studies may not necessarily represent the EPR features of human tumors as they often have a different
microenvironment and growth kinetics [280]. For preclinical testing of EVs as a DDS, these potential
challenges need to be addressed and the data should be extrapolated to the human situation with caution
as the EPR effect is less significant in the clinic than in rodents (especially xenograft models) [281].
Certain EV subsets display organotropic or tumor-targeting capacities [282], and the targeting properties
of EVs can be modified by genetic engineering of the donor cells [255]. From a biodistribution point of
view, the majority of administered liposomes end up in the liver and spleen due to removal by the MPS.
In a similar fashion, despites their interactive surfaces, passive biodistribution of EVs is a big challenge
for drug delivery applications as the predominant uptake organs for EVs in the reported studies so far
are also the liver and spleen [3]. Nonetheless, integrin expression profiles on EVs have been reported to
directly relate to tissue organotropism to the lung, liver and brain [282]. Moreover, each tumor-derived
EV population exhibits organotropism towards specific organs where EV parental cell lines originate
[282]. These results reinforce the concept that selection of EV sources might be key to achieve specific
tissue targeting [245, 278].
4.3.5. Pharmacokinetics and pharmacodynamics (PK/PD)

PK/PD as a simulation system based on the drug’s physiological and pharmacological effects can provide
valuable information on the treatment efficacy of drugs [283]. Compared with free forms of drugs,
encapsulation of drugs in liposomes prevents rapid clearance and remarkably changes the PK profile of
drugs [284]. For example, systemic clearance of doxorubicin in humans is reduced from 45 L/h to 0.1 L/h
and accumulation in targeted tumors is increased by 4 to 16-fold upon encapsulation into liposomes, i.e.
Doxil® [285]. Liposomal amikacin, Arikayce®, is another example of a marketed liposome suspension.
Compared to intravenous administration of free amikacin in rats, inhalation of Arikayce® increases the
concentrations of amikacin in the lung, airways, and macrophages by 42-, 69-, and 274-fold, respectively
[286]. Compared with liposomes, EVs might have potential to reduce MPS-mediated clearance due to
the presence of surface CD47 [148] but more evidence is demanded. EVs derived from five different cell
types showed comparable physicochemical (particle size and zeta potential) and pharmacokinetic
properties (biodistribution and EV time course of the serum) after injection into mice [287]. Unfortunately,
very little information is available regarding PK/PD properties of EVs, which is likely due to the challenge
of large-scale EV production and the presence of endogenous EVs. A comprehensive understanding of
the PK/PD properties of EVs as DDS is still missing but crucial for EVs to reach the clinic.
5. Critical discussion
Through the detailed, systematic analysis of the selected publications we obtained a global picture of the
field of EVs as DDS and its future trends. These studies tested EVs as DDS primarily in murine cancer
models after intravenous administration. The increasing number of publications using EVs as DDS in
cancer studies relates to the potential that EVs harbor for tumor targeting, cancer vaccination/antigen
presentation and their capacity to carry a vast array of therapeutic molecules such as nucleic acids and
44
small molecule drugs. The majority of the EV donor cell types used in those publications were cancer
cells, stem cells and HEK293 cells, while the majority of APIs loaded into EVs were nucleic acid
therapeutics and small molecule drugs. Regarding nucleic acids as APIs, these mainly corresponded to
miRNA and siRNA/shRNA. This is in line with the general increase in their therapeutic use and suitability
for EV loading demonstrated during the recent years. We found no obvious preference of API loading
before versus after EV isolation and the loading of isolated EVs was done by electroporation in about
one third of the studies. Moreover, nearly half of the studies used EVs subjected to surface modifications,
leaving plenty of room for debate on whether such modifications are of significance for increasing the
therapeutic efficacy of APIs but sacrificing simplicity and reproducibility. From our results we can also
infer that the usage of NTA and DLS for measuring EV size and size distribution has been increasing in
the recent years, whereas EV zeta potential is unexpectedly neglected in most studies. Furthermore,
approximal half of the studies performed EV biodistribution analysis. Finally, but disappointingly, reports
on the purity of tested EVs for drug delivery remain scarce.
5.1. Seeing the glass half empty: Missing gaps for establishing EVs as effective and safe drug delivery
systems
After analyzing the preclinical studies on EVs as drug delivery systems, we have to conclude that
essential information for a proper understanding of the behavior and effects of EVs used is missing in
many of the publications. Information such as EV heterogeneity, size and charge range, characterization
of EV intrinsic content, safety, robust biodistribution studies, calculations of final API loaded
concentration, storage conditions or comparison with liposomal carriers is often not or only poorly
reported.
5.1.1. Challenges in production, isolation, purity and characterization

Major challenges are production, isolation, purity and characterization. Biological production methods
are by definition difficult to standardize. It requires full control over cell culture conditions, genetic stability
of the culture and the presence of cellular subtypes or contaminants. Production of EVs is higher when
cells are in suspension but not all cell types can be grown under such circumstances. The development
of efficient bioreactors for EV production might offer a promising way of generating large amounts of EVs
with the possibility to scale up the process for clinical purposes. Even then, therapy with EVs will remain
a costly drug delivery strategy. Well-established isolation methods like ultracentrifugation represent a
hassle for large-scale production of undamaged EVs; other methods such as tangential flow filtration
(TFF) might be more suitable for this purpose [234]. EV samples are still a mixture of bilayered vesicles
and diverse lipoprotein particles in the vast majority of the studies we systematically reviewed, with little
or no indication given on the level of EV purity. The lack of EV purity in the isolated fractions is of big
concern and hampers their clinical translation. In addition, EV heterogeneity has hindered their
characterization at the subtype level, as well as subclass-specific modification of properties and biological
activity [15-18]. The lack of characterization, in turn, poses challenges in the interpretation of
biodistribution and intracellular delivery results [288]. Moreover, due to this lack of characterization of
EVs intrinsic content, additional attention is needed regarding safety. A clear risk example would be the
45
use of EVs derived from cancer cell lines as drug delivery systems, even though such EVs have been
reported to play a key role in malignant processes like the formation of pre-metastatic niches.
5.1.2 Misleading information on EV biodistribution

Another important challenge is the lack of standardized methods to study EV biodistribution. The use of
fluorescently labeled lipids, or lipid conjugated dyes presents two fundamental problems: 1) imaging of
a fluorophore or a tracer dye is not necessarily imaging of the EV itself as the labeling probes may drop
off from the EVs. This phenomenon has been recently reported for liposomes [289]. Other labeling
approaches (please refer to the comprehensive review by Gangadaran et al. [290]), including magnetic
nanoparticles, radiolabeling, or indirect labeling via genetic modification of the parental cells to pack
bioluminescent reporter proteins into or onto EVs, in combination with more sophisticated (but usually
expensive) imaging modalities like magnetic resonance imaging (MRI), may confer more accurate
biodistribution profiles of EVs. Similar to non-cell derived nanoparticles such as liposomes, majority of
injected EVs accumulate in the liver, spleen and lungs regardless of their origin, likely due to recognition
by the recipient’s MPS [291]. However, a recent study that genetically labeled cardiomyocyte-derived
EVs using a CD63NanoLuc reporter in transgenic mice demonstrated that endogenous EVs were mainly
taken up by the thymus, testis, lung and kidney instead of the liver [292]. These discrepancies in
biodistribution of endogenous and exogenous EVs indicate that EV isolation, storage, labeling strategy,
or allogeneic-induced recognition of EVs by the MPS alter the biological behavior of EVs in vivo.
Examining EV biodistribution has been considered as monitoring EV cellular uptake and therefore EV
delivery of cargoes, however, 2) cellular uptake of EVs might not correspond to the ultimate delivery of
the cargo as the loaded cargo may escape or be leaked from the EVs. A possible strategy to solve this
disconnection between cellular uptake of EVs and functional delivery of their cargoes would be the use
of two labeling methodologies simultaneously (for cargo and EV membrane, respectively) to study the
colocalization of two physiochemically distinct labels. Even that, cellular internalization of both EVs and
their cargoes may not guarantee the successful delivery. This was recently reported by Reshke et al.
that cellular uptake of GFP siRNA loaded EVs failed to knock down GFP in Kupffer cells in mice [178].
Thus, EV biodistribution data should be interpreted with caution.
5.1.3. Complications in upscaling, loading strategies and storage

In line with the previously mentioned issues regarding isolation and characterization of EVs comes the
challenge of efficient upscaling of EVs for clinical use. Reproducibility remains an issue with batch by
batch production [293] and this matter is also of great concern for academic research, which would
benefit enormously from reproducible and reliable experimental conditions. To use EVs in drug delivery,
the lack of effective loading strategies that do not compromise the structure and functions of EVs is
problematic. Exogenous loading methods have been reported to give variable outcomes and loading
efficiencies whereas for genetic engineering of the parental cells, loading capacities remain generally
low [293-295]. Better understanding of the intracellular formation, release and trafficking processes,
might improve API loading. Another aspect, which is often not mentioned in preclinical studies, is the
storage condition for the EVs used. It is essential that storage conditions that assure EV functionality are
46
used, in order to guarantee current Good Manufacturing Practices (cGMP) [18]. There is a general
agreement by the scientific community to support storage of EVs at −80 °C [52]. Cryoprotectants such
as trehalose may be necessary to protect damage and aggregation of EVs induced by freezing/thawing
during storage and recovery [296]. Notably, the study of EV stability under different conditions could be
crucial to determine their feasibility for clinical use as not all the countries have the resources to
guarantee -80 °C storage at clinical centers.
5.1.4. Lack of comparison with liposomes

Surprisingly, we found that as of today there are only very few publications that have done a head-to-
head comparison in delivery efficiency of EVs versus liposomes [148, 178, 297] and from these some
are comparing EVs to liposomes without giving sufficient information on the type of liposomes used [148].
Given the similarity in size, shape and basic membrane composition (phospholipids), to examine the
possible advantages of EVs as a new class of DDS, it may be recommendable to include liposomal
formulations as a control DDS in future studies. Liposomal systems could be used as a reference in order
to investigate EV cell targeting, routes of cellular uptake and intracellular trafficking in recipient cells.
5.2. Seeing the glass half full: accomplished landmarks of EVs for drug delivery
As it can be inferred from our study (Table 2), a variety of approaches and biologic materials have been
used in order to successfully use EVs as DDS in animal models. The constant increase in the number
of publications involving EVs as DDS during the past decade is a promising augury for the future of the
field. From the publications we systematically reviewed, some important landmarks have been achieved:
5.2.1. Active targeting

When it comes to active targeting, which is one of the main aims of using EVs as DDS, considerable
progress has been achieved already with EVs. Intravenously injected tumor-derived EVs tend to target
parental tumor tissues. Compared to the gold standard nanocarrier, Doxil®, drug-loaded EVs showed
increased therapeutic retention in tumor tissues and more pronounced tumor suppression in nude mice
[151]. Multiple strategies exist to introduce active targeting in EVs, such as the generation of EVs
decorated with rabies virus protein enabling targeting of EVs to the brain [180]. Similarly, EVs have been
coated with nanobodies, bispecific antibodies or peptides to improve tissue tropism, for example to target
regenerative exosomes to myocardial infarctions using a cardiac homing peptide [298]. Furthermore, a
recent publication demonstrated that small EVs exhibit targeted delivery of siRNA to specific cell
populations and tissues in mice [178].
5.2.2. Improved circulation time

As stated before, the short circulation time of EVs is a limiting factor for their drug delivery capacities and
therefore several studies focused on increasing the circulation time of EVs, a strategy reminiscent of the
liposome field. Addition of CD47 or PEG was shown to reduce clearance by macrophages of the RES
[299]. Whereas unmodified EVs were rapidly cleared within 10 min upon systemic administration, EVs
post-inserted with nanobody-PEG-lipids could be detected in plasma for over 60 min post injection and
47
exhibited improved cell specificity [254]. However, this circulation time is still very short in comparison
with PEGylated liposomes. Being optimistic, there is some evidence that the satisfactory performance of
EVs with respect to plasma stability might upgrade their therapeutic potency [217].
5.2.3. Upgraded loading efficiency

Drug loading remains one of the biggest hurdles to develop EV-based DDS. Interestingly, EVs seem to
be suitable for loading and delivery of RNA-based therapeutics. In a recent study, siRNA was integrated
into a pre-miRNA backbone in order to load it more efficiently into EVs [178]. Notably, those EVs achieved
similar gene knockdown efficiency with over ten-fold less siRNA than the typically required dose for lipid
nanoparticles [178]. Another recent study from our own group, which used a CRISPR/Cas9-based RNA
transfer reporter system [300], suggests that there is a difference of several orders of magnitude
regarding more potent delivery of sgRNA compared to liposomal systems [297]. An emerging strategy to
improve drug loading efficiency while maintaining the unique features of EVs is to generate hybrid EV
systems by incorporating components such as liposomes into EVs [301] or EV mimetics [302]. While
developing methodologies for improving drug loading efficiency into EVs, EV mimetics or hybrid EV
systems, as discussed in a recent article [55], heterogeneity of EVs in molecular composition, surface
feature, and subpopulation (in terms of cell origin, size, surface charge, etc.) may also impact EV drug
loading and delivery.
5.2.4. EVs in intracellular trafficking: Escaping the endosomal system

The delivery of RNA through nanoparticles is well known to be mediated through the endolysosomal
system at the subcellular level. This might represent a bottleneck for efficient RNA delivery, as only 1-
2% of the cargo escapes into the cytosol [303]. In this sense, there might be differences between
intracellular routing of EVs and other nanoparticles, in light of emerging evidence suggesting that EVs
are capable of escaping endosomal degradative pathways [178, 255, 297, 304, 305]. Elucidating the
details of the EV cargo delivery process might be key to upgrade the delivery of therapeutic molecules
through EVs when compared to other nanoparticulate systems [255, 297].
5.2.5. Crossing the Blood-Brain Barrier (BBB)

It has been shown that 98% of small molecule drugs, and ~100% of biologic drugs cannot cross the
blood-brain barrier (BBB) [302]. Therefore, it is understandable that to date there is not a single
nanoparticle formulation approved by the U.S. FDA that manages successful drug delivery across the
BBB. Of notice, important progress has been made regarding the discovery of EVs which are capable of
crossing specific biological barriers, such as the BBB in preclinical models. To illustrate this, exosomes
derived from human neuronal stem cells and a brain endothelial cell line were able to cross the BBB and
accumulate in injured sites [306] and brain cancer [66], respectively. Furthermore, EVs administered
intranasally were shown effective for the delivery of curcumin to the brain, suggesting an alternative
route for the treatment of brain inflammatory diseases [193].
5.2.6. Emerging EV platforms
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Recent theranostic platforms that allow imaging of therapeutic EVs harbor great potential to get a more
precise understanding of their ways of action. Magnetic EVs, through loading of iron oxide nanoparticles,
represent a sensitive and specific way of isolating EVs, enhancing tumor targeting under a magnetic
field, and tracking systemically administered therapeutic EVs through magnetic particle imaging [109,
138, 307]. Another emerging strategy is the direct transfer of mitochondria [308-310] through EVs, which
was recently reported to improve post-infarct cardiac function in vivo by restoring intracellular
bioenergetics and mitochondrial biogenesis in the recipient cardiomyocytes [310]. It is yet to be proven
whether EVs are capable of efficiently transferring other organelles of therapeutic relevance. Additionally,
improving other drug delivery nanoparticles by encapsulating or hybridizing them with EVs may also be
attractive to those nanoparticles of therapeutic potential but with a fast metabolism, limited bioavailability
and rapid clearance after systemic administration [196].
5.3. Defining the niches of EVs in comparison to liposomes

These important landmarks point to certain appealing characteristics of EVs as advanced delivery
systems. It may be argued that their structural and functional complexity together with their innate
therapeutic activity make EVs especially suited for multifactorial diseases/conditions (Table 4). Still, from
a realistic perspective, EVs have a long way to go to improve many aspects such as drug loading,
retention of drug, pharmaceutical stability during storage, PK, biodistribution and GMP manufacturing of
clinical batches.
Table 4
Defining the niches for EVs based on their attributes as DDS when compared to liposomes.
Liposomes EVs
Starting materials – limited number of chemically Crossing barriers – ability to cross biological
defined molecules barriers such as the BBB
Reproducibility – high control over manufacture, Trafficking – cell-specific interaction and
loading and physicochemical characteristics internalization routes through rich biomolecular
surface
Scalability – industrial scale production methods Complex cargo – Can carry a mix of bioactive
have been developed molecules (e.g. cell surface receptors, RNA
species & post-translationally modified proteins)
simultaneously
GMP – entire production process can be GMP Multifactorial modulation – can affect diseases at
grade multiple nodes of intervention
Storage – Long shelf live and pharmaceutical Flexibility – opportunities for extensive
stability bioengineering
5.4. Reaching the clinic: guidance on how to evaluate EVs as drug delivery systems
The lack of standardization of reproducible procedures for EVs as DDS in the field is putting serious
brakes on the clinical development of EVs (Fig. 15a,b). As a result, regulatory agencies like the FDA and
EMA are considering risks and release-criteria for EV products [288], influenced to a certain extent by
the guidance on liposomal products. Liposomes are generally composed of a handful of chemically
defined molecules and produced under specified conditions, and there is a high level of control over the
49
characteristics of the formulation. The FDA/EMA guidance on liposomal products, for example, provides
clear specifications of the data that should be reported in the dossier on liposomal products (Table 5).
Fig. 15. Standardization of EV-based delivery systems towards clinical trials. (A) Standardization of
procedures for EV drug loading. Cellular origin, isolation and purification, drug loading methods and
storage conditions need to be considered for the standardization. (B) EVs as DDS towards clinical trials.
Essential aspects need to be considered for developing EV-based DDS at all stages, including in vitro
screening, preclinical testing and clinical trials. Liposomes may be considered as a reference DDS.
Table 5
FDA/EMA guidelines point to a number of liposome characteristics that should be reported that are
deemed important for liposomal product performance.
Composition  Components of the liposome
 Quantities of the active substance and each lipid
 Molar ratio or percentage by weight of the lipid (including functional lipid)
to the active substance
Characterization  Particle size distribution
 Morphology and/or structure of the liposome
 Surface charge (zeta potential)
 Thermodynamic properties of the membrane
 Osmolality
50
 pH
 Aggregation
 Loading efficiency of the active substances
 Impurities
 Physical state of the encapsulated active substance
 Conformational structure, modification efficiency, and binding
capability
In vitro release  Release profile of the active substance from the liposome
 For internal triggered release liposomes, release profile under conditions
that reflect the physiological environment.
 For external triggered release liposome, release profile with external
stimulation
Manufacturing  Process of formation of liposomes
process & process  Encapsulation process of the active substance in liposomes
controls of  Sizing process
liposome drug  Process for surface modification
products  Sterilization process
 Stability
 Endotoxin content
 Biological assay for efficacy
It is clear that the same level of specifications cannot be met by EVs as they are complex vesicles that
come from living cells. As a result, the guidance for Advanced Therapy Medicinal Products (ATMP)
seems to be a better fit, although the requirements in this guidance are less well defined due to the large
variety of products and applications in this group of therapeutics. The ATMP guidelines are based on a
risk stratification approach that consists of 4 steps:
1. To identify risks associated with the clinical use of the ATMP

2. To identify product specific risk factors contributing to each identified risk
3. To map the relevant data for each identified risk factor against each of the identified risk
4. To conclude on the risk factor – risk relationships
These analyses are used to fill a risk-risk factor table in which these are described. Table 6 provides a
hypothetical analysis for an EV product.
Table 6
Hypothetical risk factor analysis of an EV product.
Unwanted Treatment failure Disease Tumor formation Toxicity
immunogenicity transmission
Cell starting Possible HLA Information on Oncogenic
material mismatching cell origin not transformation
complete because of cell
source
Culture Possible immune Composition Potential for Culture with
conditions reaction to compromises EV disease growth factors or
animal derived activity - insufficient transmission hormones may
materials or cells production of from cell source, induce tumor
bioactive animal derived formation
compounds materials
Cell population, Subpopulation Subpopulation Subpopulation
heterogeneity & formation of cells formation produces formation of cells
differentiation produces EVs
51
potential causes EVs with unfit with higher
immunogenicity properties potential of
oncogenic
transformation
Genetic stability Genetic Genetic instability
of source cells instability may may result in
increase potential loss of
immunogenicity secreted bioactive
substances
Isolation and Increased Potential impact of Potential impact of
Purification immunogenicity IaP procedures on IaP procedures on
(IaP) through IaP biological activity toxicity
procedures
Storage EVs lose their Preservatives or
conditions intrinsic activity and cryoprotective
vesicle stability reagents may
(e.g. drug leaking) confer cytotoxicity
Biodistribution Distribution to Potential loss of Tumor formation Delivery of
different activity due to in different organs bioactive
organs/cells limited arrival at substances
may increase risk target site of EVs in unintended
of microenvironments
immunogenicity may cause toxicity
Relevance of the Available animal Age, dosing,
animal model model is not immuno-
reflecting human competence
disease and duration of
animal study not
appropriate for
detection of tumor
formation.
Tumorigenicity.
Patient-related Risk for Risk for treatment Risk for unwanted
unwanted failure due to tissue formation
immunogenicity patient history due to
due to patient (age, suboptimal microenvironment
history microenvironment
and insufficient
dose finding data)
Disease-related Risk for suboptimal
EV performance
due to target tissue
microenvironment.
Medical Unwanted Risk for infection Hypertrophic
procedure- immune reaction due to growth due to
related & allergy to application application
concomitant procedure procedure.
substances at
site of application
6. Conclusions and future perspectives

EVs have been studied for over half a century and the field has been evolving quickly over the recent
years, from the discovery to the applications in diagnostics, from preclinical studies to clinical trials on
EVs as therapeutics and DDS, and to the recent standardization (i.e. guidelines) for EV research (Fig
16).
52
Fig. 16. Accomplished milestones in the EV field. EVs were discovered in 1967 as “platelet-dust” [311].
The terms “extracellular vesicle” [312] and “exosome” [313] were coined in 1971 and 1981, respectively.
In 1983, the first biological function of EVs was reported: transferrin carriers [314, 315]. In 1998, the fist
preclinical study of dendritic cell-derived EVs as a cancer vaccine in mice [316]. In 2004, urinary EVs
were explored for biomarker discovery [317]. In 2005, two Phase I clinical trials were initiated to test EVs
as therapeutics for patients with melanoma and advanced non-small cell lung cancer [272, 273]. In 2010,
the first preclinical study of EVs as DDS for small molecule drugs [196]. In 2011, the first study was
published on EVs as DDS for exogenous siRNA in a mouse model of Alzheimer’s disease [180]. In 2013,
the first clinical trial was launched to test EVs as DDS for small molecule (chemotherapeutic) drugs
(NCT01854866). In the same year, the Nobel Prize in Physiology or Medicine was awarded for the
discoveries of machinery regulating intracellular vesicle traffic. The guidelines for EV research, Minimal
Experimental Requirements for definition of EVs (MISEVs), were proposed in 2014 and revised in 2018
[8, 53].
The convergence of standardized procedures and regulations, together with the advent of new
discoveries in more adequate EV donor cell types and drug loading procedures that do not compromise
essential structural features of EVs, could possibly lead the field to future successes in the clinic.
Furthermore, it is important to compare EVs with other delivery systems such as liposomes, and possibly
to cellular systems such as therapeutic stem cells, to determine the ‘fit-for-purpose’. In cancer treatment,
for example, the primary objective is to deliver a maximal dose of chemotherapeutics to the tumor, which
seems to be a closer fit with DDS like liposomes. On the other hand, EVs hold promise as DDS for their
“homing” property, beyond the EPR effect, to specific tumors, their potential to cross certain biological
barriers such as the BBB, their unique capacity to encapsulate certain biomolecules such as membrane
receptors that are difficult to load into other DDS, and their plasticity for bioengineering. In fields like
regenerative medicine that involve multifactorial biological processes, a complex natural delivery system
53
like EVs can offer a competitive advantage. From this systematic review we conclude that there is an
increasing use of EVs for drug delivery in cancer treatment, along with an extensive application of nucleic
acids and small molecule drugs as APIs. We also notice an increasing tendency in the use of intravenous
administration of EVs, accompanied with coating EVs with agents that result in increased target capacity
(e.g. tumor antigens) and/or circulation time (e.g. PEG, CD47). We envision that the niches of EVs as
DDS in the near future may be dominated by incorporation of membrane proteins and perhaps delivery
of RNAs when the loading hurdles are solved, as well as delivery of proteins/peptides, for regenerative
medicine and development of vaccines. There is a vast array of fields that may benefit from the use of
EVs as DDS, but identification of the proper niches will come along with the maturation of the EV drug
delivery field and a deeper comprehension of the intracellular mechanisms underlying EV uptake by and
endosomal escape of the loaded compounds in target cells.
Acknowledgments/Funding
The authors would like to thank the funding support from the Singapore Ministry of Health’s National
Medical Research Council (NMRC/OFYIRG/0081/2018 to J.W.W., G.P. and G.S.), NUHS
(NUHSRO/2018/095/RO5+5/Seed-Nov/05 to J.W.W.), NUS ODPRT Cross-Faculty Research Fund
(CFGFY20P14 to J.W.W.), the National University of Singapore NanoNASH Program
(NUHSRO/2020/002/NanoNash/LOA to G.S., G.P., and J.W.W.), the National University of Singapore
start-up fund (NUHSRO/2020/133/Startup/08 to X.C.; NUHSRO/2019/077/STARTUP/03-ODPRT and
NUHSRO/2019/077/STARTUP/03-NUSMed to G.S.), the National University of Singapore (R-148-000-
296-114 and R-148-000-284-114 to G.P.) and the RIE2020 Advanced Manufacturing and Engineering
(AME) Industry Alignment Fund – Pre Positioning (IAF-PP) grant (A20G1a0046 and R-148-000-307-305
to G.P.), and the European Union’s Horizon 2020 research and innovation programme under the Marie
Skłodowska-Curie grant agreement (proEVLifeCycle, grant No 860303; and the B-SMART project, grant
No 721058; to R.M.S). Figures 1,2, 14,15 and 16 were created with BioRender.com.
54
References
[1] O.P.B. Wiklander, M. Brennan, J. Lötvall, X.O. Breakefield, S.E.L. Andaloussi, Advances in
therapeutic applications of extracellular vesicles, Sci. Transl. Med. 11 (2019) 1-16,
http://doi.org/10.1126/scitranslmed.aav8521.
[2] S.Y. Chong, C.K. Lee, C. Huang, Y.H. Ou, C.J. Charles, A.M. Richards, Y.R. Neupane, M.V. Pavon,
O. Zharkova, G. Pastorin, J.W. Wang, Extracellular Vesicles in Cardiovascular Diseases: Alternative
Biomarker Sources, Therapeutic Agents, and Drug Delivery Carriers, Int. J. Mol. Sci. 20 (2019),
http://doi.org/10.3390/ijms20133272.
[3] O.G. de Jong, S.A.A. Kooijmans, D.E. Murphy, L. Jiang, M.J.W. Evers, J.P.G. Sluijter, P. Vader, R.M.
Schiffelers, Drug Delivery with Extracellular Vesicles: From Imagination to Innovation, Acc. Chem. Res.
52 (2019) 1761-1770, http://doi.org/10.1021/acs.accounts.9b00109.
[4] E. Woith, G. Fuhrmann, M.F. Melzig, Extracellular Vesicles—Connecting Kingdoms, Int. J. Mol. Sci.
20 (2019) 5695-5695, http://doi.org/10.3390/ijms20225695.
[5] R. Kalluri, V.S. LeBleu, The biology, function, and biomedical applications of exosomes, Science 367
(2020), http://doi.org/10.1126/science.aau6977.
[6] M.A. Mori, R.G. Ludwig, R. Garcia-Martin, B.B. Brandão, C.R. Kahn, Extracellular miRNAs: From
Biomarkers to Mediators of Physiology and Disease, Cell Metab. 30 (2019) 656-673,
http://doi.org/10.1016/j.cmet.2019.07.011.
[7] L. Tong, H. Hao, X. Zhang, Z. Zhang, Y. Lv, L. Zhang, H. Yi, Oral Administration of Bovine Milk-
Derived Extracellular Vesicles Alters the Gut Microbiota and Enhances Intestinal Immunity in Mice, Mol.
Nutr. Food Res. 64 (2020) e1901251, http://doi.org/10.1002/mnfr.201901251.
[8] C. Thery, K.W. Witwer, E. Aikawa, M.J. Alcaraz, J.D. Anderson, R. Andriantsitohaina, A. Antoniou, T.
Arab, F. Archer, G.K. Atkin-Smith, D.C. Ayre, J.M. Bach, D. Bachurski, H. Baharvand, L. Balaj, S.
Baldacchino, N.N. Bauer, A.A. Baxter, M. Bebawy, C. Beckham, A. Bedina Zavec, A. Benmoussa, A.C.
Berardi, P. Bergese, E. Bielska, C. Blenkiron, S. Bobis-Wozowicz, E. Boilard, W. Boireau, A.
Bongiovanni, F.E. Borras, S. Bosch, C.M. Boulanger, X. Breakefield, A.M. Breglio, M.A. Brennan, D.R.
Brigstock, A. Brisson, M.L. Broekman, J.F. Bromberg, P. Bryl-Gorecka, S. Buch, A.H. Buck, D. Burger,
S. Busatto, D. Buschmann, B. Bussolati, E.I. Buzas, J.B. Byrd, G. Camussi, D.R. Carter, S. Caruso, L.W.
Chamley, Y.T. Chang, C. Chen, S. Chen, L. Cheng, A.R. Chin, A. Clayton, S.P. Clerici, A. Cocks, E.
Cocucci, R.J. Coffey, A. Cordeiro-da-Silva, Y. Couch, F.A. Coumans, B. Coyle, R. Crescitelli, M.F.
Criado, C. D'Souza-Schorey, S. Das, A. Datta Chaudhuri, P. de Candia, E.F. De Santana, O. De Wever,
H.A. Del Portillo, T. Demaret, S. Deville, A. Devitt, B. Dhondt, D. Di Vizio, L.C. Dieterich, V. Dolo, A.P.
Dominguez Rubio, M. Dominici, M.R. Dourado, T.A. Driedonks, F.V. Duarte, H.M. Duncan, R.M.
Eichenberger, K. Ekstrom, S. El Andaloussi, C. Elie-Caille, U. Erdbrugger, J.M. Falcon-Perez, F. Fatima,
J.E. Fish, M. Flores-Bellver, A. Forsonits, A. Frelet-Barrand, F. Fricke, G. Fuhrmann, S. Gabrielsson, A.
Gamez-Valero, C. Gardiner, K. Gartner, R. Gaudin, Y.S. Gho, B. Giebel, C. Gilbert, M. Gimona, I. Giusti,
D.C. Goberdhan, A. Gorgens, S.M. Gorski, D.W. Greening, J.C. Gross, A. Gualerzi, G.N. Gupta, D.
Gustafson, A. Handberg, R.A. Haraszti, P. Harrison, H. Hegyesi, A. Hendrix, A.F. Hill, F.H. Hochberg,
K.F. Hoffmann, B. Holder, H. Holthofer, B. Hosseinkhani, G. Hu, Y. Huang, V. Huber, S. Hunt, A.G.
Ibrahim, T. Ikezu, J.M. Inal, M. Isin, A. Ivanova, H.K. Jackson, S. Jacobsen, S.M. Jay, M. Jayachandran,
G. Jenster, L. Jiang, S.M. Johnson, J.C. Jones, A. Jong, T. Jovanovic-Talisman, S. Jung, R. Kalluri, S.I.
Kano, S. Kaur, Y. Kawamura, E.T. Keller, D. Khamari, E. Khomyakova, A. Khvorova, P. Kierulf, K.P. Kim,
T. Kislinger, M. Klingeborn, D.J. Klinke, 2nd, M. Kornek, M.M. Kosanovic, A.F. Kovacs, E.M. Kramer-
Albers, S. Krasemann, M. Krause, I.V. Kurochkin, G.D. Kusuma, S. Kuypers, S. Laitinen, S.M. Langevin,
L.R. Languino, J. Lannigan, C. Lasser, L.C. Laurent, G. Lavieu, E. Lazaro-Ibanez, S. Le Lay, M.S. Lee,
Y.X.F. Lee, D.S. Lemos, M. Lenassi, A. Leszczynska, I.T. Li, K. Liao, S.F. Libregts, E. Ligeti, R. Lim, S.K.
Lim, A. Line, K. Linnemannstons, A. Llorente, C.A. Lombard, M.J. Lorenowicz, A.M. Lorincz, J. Lotvall,
J. Lovett, M.C. Lowry, X. Loyer, Q. Lu, B. Lukomska, T.R. Lunavat, S.L. Maas, H. Malhi, A. Marcilla, J.
Mariani, J. Mariscal, E.S. Martens-Uzunova, L. Martin-Jaular, M.C. Martinez, V.R. Martins, M. Mathieu,
S. Mathivanan, M. Maugeri, L.K. McGinnis, M.J. McVey, D.G. Meckes, Jr., K.L. Meehan, I. Mertens, V.R.
Minciacchi, A. Moller, M. Moller Jorgensen, A. Morales-Kastresana, J. Morhayim, F. Mullier, M. Muraca,
L. Musante, V. Mussack, D.C. Muth, K.H. Myburgh, T. Najrana, M. Nawaz, I. Nazarenko, P. Nejsum, C.
Neri, T. Neri, R. Nieuwland, L. Nimrichter, J.P. Nolan, E.N. Nolte-'t Hoen, N. Noren Hooten, L. O'Driscoll,
T. O'Grady, A. O'Loghlen, T. Ochiya, M. Olivier, A. Ortiz, L.A. Ortiz, X. Osteikoetxea, O. Ostergaard, M.
Ostrowski, J. Park, D.M. Pegtel, H. Peinado, F. Perut, M.W. Pfaffl, D.G. Phinney, B.C. Pieters, R.C. Pink,
D.S. Pisetsky, E. Pogge von Strandmann, I. Polakovicova, I.K. Poon, B.H. Powell, I. Prada, L. Pulliam,
P. Quesenberry, A. Radeghieri, R.L. Raffai, S. Raimondo, J. Rak, M.I. Ramirez, G. Raposo, M.S.
Rayyan, N. Regev-Rudzki, F.L. Ricklefs, P.D. Robbins, D.D. Roberts, S.C. Rodrigues, E. Rohde, S.
Rome, K.M. Rouschop, A. Rughetti, A.E. Russell, P. Saa, S. Sahoo, E. Salas-Huenuleo, C. Sanchez,
J.A. Saugstad, M.J. Saul, R.M. Schiffelers, R. Schneider, T.H. Schoyen, A. Scott, E. Shahaj, S. Sharma,
55
O. Shatnyeva, F. Shekari, G.V. Shelke, A.K. Shetty, K. Shiba, P.R. Siljander, A.M. Silva, A. Skowronek,
O.L. Snyder, 2nd, R.P. Soares, B.W. Sodar, C. Soekmadji, J. Sotillo, P.D. Stahl, W. Stoorvogel, S.L.
Stott, E.F. Strasser, S. Swift, H. Tahara, M. Tewari, K. Timms, S. Tiwari, R. Tixeira, M. Tkach, W.S. Toh,
R. Tomasini, A.C. Torrecilhas, J.P. Tosar, V. Toxavidis, L. Urbanelli, P. Vader, B.W. van Balkom, S.G.
van der Grein, J. Van Deun, M.J. van Herwijnen, K. Van Keuren-Jensen, G. van Niel, M.E. van Royen,
A.J. van Wijnen, M.H. Vasconcelos, I.J. Vechetti, Jr., T.D. Veit, L.J. Vella, E. Velot, F.J. Verweij, B.
Vestad, J.L. Vinas, T. Visnovitz, K.V. Vukman, J. Wahlgren, D.C. Watson, M.H. Wauben, A. Weaver,
J.P. Webber, V. Weber, A.M. Wehman, D.J. Weiss, J.A. Welsh, S. Wendt, A.M. Wheelock, Z. Wiener, L.
Witte, J. Wolfram, A. Xagorari, P. Xander, J. Xu, X. Yan, M. Yanez-Mo, H. Yin, Y. Yuana, V. Zappulli, J.
Zarubova, V. Zekas, J.Y. Zhang, Z. Zhao, L. Zheng, A.R. Zheutlin, A.M. Zickler, P. Zimmermann, A.M.
Zivkovic, D. Zocco, E.K. Zuba-Surma, Minimal information for studies of extracellular vesicles 2018
(MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of
the MISEV2014 guidelines, J. Extracell. Vesicles. 7 (2018) 1535750,
http://doi.org/10.1080/20013078.2018.1535750.
[9] B. Gyorgy, T.G. Szabo, M. Pasztoi, Z. Pal, P. Misjak, B. Aradi, V. Laszlo, E. Pallinger, E. Pap, A.
Kittel, G. Nagy, A. Falus, E.I. Buzas, Membrane vesicles, current state-of-the-art: emerging role of
extracellular vesicles, Cell Mol. Life Sci. 68 (2011) 2667-2688, http://doi.org/10.1007/s00018-011-0689-
3.
[10] S. El Andaloussi, I. Mäger, X.O. Breakefield, M.J.A. Wood, Extracellular vesicles: Biology and
emerging therapeutic opportunities, Nat. Rev. Drug Discov. 12 (2013) 347-357,
http://doi.org/10.1038/nrd3978.
[11] E. Cocucci, J. Meldolesi, Ectosomes and exosomes: shedding the confusion between extracellular
vesicles, Trends. Cell. Biol. 25 (2015) 364-372, http://doi.org/10.1016/j.tcb.2015.01.004.
[12] E. Willms, C. Cabañas, I. Mäger, M.J.A. Wood, P. Vader, Extracellular vesicle heterogeneity:
Subpopulations, isolation techniques, and diverse functions in cancer progression, Front. Immunol. 9
(2018), http://doi.org/10.3389/fimmu.2018.00738.
[13] L. Console, M. Scalise, C. Indiveri, Exosomes in inflammation and role as biomarkers, Clin. Chim.
Acta. 488 (2019) 165-171, http://doi.org/10.1016/j.cca.2018.11.009.
[14] F.M. Barros, F. Carneiro, J.C. Machado, S.A. Melo, Exosomes and Immune Response in Cancer:
Friends or Foes?, Front. Immunol. 9 (2018), http://doi.org/10.3389/fimmu.2018.00730.
[15] J. Kowal, G. Arras, M. Colombo, M. Jouve, J.P. Morath, B. Primdal-Bengtson, F. Dingli, D. Loew, M.
Tkach, C. Théry, Proteomic comparison defines novel markers to characterize heterogeneous
populations of extracellular vesicle subtypes, Proc. Natl. Acad. Sci. U S A. 113 (2016) E968-E977,
http://doi.org/10.1073/pnas.1521230113.
[16] S.J. Gould, G. Raposo, As we wait: coping with an imperfect nomenclature for extracellular vesicles,
J. Extracell. Vesicles. 2 (2013), http://doi.org/10.3402/jev.v2i0.20389.
[17] G. van Niel, G. D'Angelo, G. Raposo, Shedding light on the cell biology of extracellular vesicles, Nat.
Rev. Mol. Cell. Biol. 19 (2018) 213-228, http://doi.org/10.1038/nrm.2017.125.
[18] O.M. Elsharkasy, J.Z. Nordin, D.W. Hagey, O.G. de Jong, R.M. Schiffelers, S.E.L. Andaloussi, P.
Vader, Extracellular vesicles as drug delivery systems: Why and how?, Adv. Drug. Deliv. Rev. 159 (2020)
332-343, http://doi.org/10.1016/j.addr.2020.04.004.
[19] Y. Yuana, A. Sturk, R. Nieuwland, Extracellular vesicles in physiological and pathological conditions,
Blood Rev. 27 (2013) 31-39, http://doi.org/10.1016/j.blre.2012.12.002.
[20] E. D’Asti, S. Chennakrishnaiah, T.H. Lee, J. Rak, Extracellular Vesicles in Brain Tumor Progression,
Cell Mol. Neurobiol. 36 (2016) 383-407, http://doi.org/10.1007/s10571-015-0296-1.
[21] J. Kong, H. Tian, F. Zhang, Z. Zhang, J. Li, X. Liu, X. Li, J. Liu, X. Li, D. Jin, X. Yang, B. Sun, T. Guo,
Y. Luo, Y. Lu, B. Lin, T. Liu, Extracellular vesicles of carcinoma-associated fibroblasts creates a pre-
metastatic niche in the lung through activating fibroblasts, Mol. Cancer. 18 (2019) 175-175,
http://doi.org/10.1186/s12943-019-1101-4.
[22] C. Federici, F. Petrucci, S. Caimi, A. Cesolini, M. Logozzi, M. Borghi, S. D'Ilio, L. Lugini, N. Violante,
T. Azzarito, C. Majorani, D. Brambilla, S. Fais, Exosome release and low pH belong to a framework of
resistance of human melanoma cells to cisplatin, PLoS One. 9 (2014) e88193,
http://doi.org/10.1371/journal.pone.0088193.
[23] A.T.A. Halim, N.A.F.M. Ariffin, M. Azlan, Review: the Multiple Roles of Monocytic Microparticles,
Inflammation. 39 (2016) 1277-1284, http://doi.org/10.1007/s10753-016-0381-8.
[24] M. Yanez-Mo, P.R. Siljander, Z. Andreu, A.B. Zavec, F.E. Borras, E.I. Buzas, K. Buzas, E. Casal, F.
Cappello, J. Carvalho, E. Colas, A. Cordeiro-da Silva, S. Fais, J.M. Falcon-Perez, I.M. Ghobrial, B.
Giebel, M. Gimona, M. Graner, I. Gursel, M. Gursel, N.H. Heegaard, A. Hendrix, P. Kierulf, K. Kokubun,
M. Kosanovic, V. Kralj-Iglic, E.M. Kramer-Albers, S. Laitinen, C. Lasser, T. Lener, E. Ligeti, A. Line, G.
Lipps, A. Llorente, J. Lotvall, M. Mancek-Keber, A. Marcilla, M. Mittelbrunn, I. Nazarenko, E.N. Nolte-'t
56
Hoen, T.A. Nyman, L. O'Driscoll, M. Olivan, C. Oliveira, E. Pallinger, H.A. Del Portillo, J. Reventos, M.
Rigau, E. Rohde, M. Sammar, F. Sanchez-Madrid, N. Santarem, K. Schallmoser, M.S. Ostenfeld, W.
Stoorvogel, R. Stukelj, S.G. Van der Grein, M.H. Vasconcelos, M.H. Wauben, O. De Wever, Biological
properties of extracellular vesicles and their physiological functions, J. Extracell. Vesicles. 4 (2015)
27066, http://doi.org/10.3402/jev.v4.27066.
[25] X. Wang, W. Huang, G. Liu, W. Cai, R.W. Millard, Y. Wang, J. Chang, T. Peng, G.-C. Fan,
Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-
320 into endothelial cells, J. Mol. Cell. Cardiol. 74 (2014) 139-150,
http://doi.org/10.1016/j.yjmcc.2014.05.001.
[26] G. Lombardo, P. Dentelli, G. Togliatto, A. Rosso, M. Gili, S. Gallo, M.C. Deregibus, G. Camussi,
M.F. Brizzi, Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3
Pro-angiogenic Paracrine Action, Sci. Rep. 6 (2016) 1-14, http://doi.org/10.1038/srep25689.
[27] M.T. Roefs, J.P.G. Sluijter, P. Vader, Extracellular Vesicle-Associated Proteins in Tissue Repair,
Trends Cell Biol. 30 (2020) 990-1013, http://doi.org/10.1016/j.tcb.2020.09.009.
[28] C. Hromada, S. Mühleder, J. Grillari, H. Redl, W. Holnthoner, Endothelial Extracellular Vesicles—
Promises and Challenges, Front. Physiol. 8 (2017), http://doi.org/10.3389/fphys.2017.00275.
[29] M.L. Liu, M.P. Reilly, P. Casasanto, S.E. McKenzie, K.J. Williams, Cholesterol enrichment of human
monocyte/macrophages induces surface exposure of phosphatidylserine and the release of biologically-
active tissue factor-positive microvesicles, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 430-435,
http://doi.org/10.1161/01.ATV.0000254674.47693.e8.
[30] L.C.P. Azevedo, M. Janiszewski, V. Pontieri, M.d.A. Pedro, E. Bassi, P.J.F. Tucci, F.R.M. Laurindo,
Platelet-derived exosomes from septic shock patients induce myocardial dysfunction, Crit. Care. 11
(2007) 1-10, http://doi.org/10.1186/cc6176.
[31] J.D. Hutcheson, E. Aikawa, Extracellular vesicles in cardiovascular homeostasis and disease, Curr.
Opin. Cardiol. 33 (2018) 290-297, http://doi.org/10.1097/HCO.0000000000000510.
[32] A. Hafiane, S.S. Daskalopoulou, Extracellular vesicles characteristics and emerging roles in
atherosclerotic cardiovascular disease, Metabolism. 85 (2018) 213-222,
http://doi.org/10.1016/j.metabol.2018.04.008.
[33] X. Zhou, F. Xie, L. Wang, L. Zhang, S. Zhang, M. Fang, F. Zhou, The function and clinical application
of extracellular vesicles in innate immune regulation, Cell Mol. Immunol. 17 (2020) 323-334,
[34] R.E. Lane, D. Korbie, M.M. Hill, M. Trau, Extracellular vesicles as circulating cancer biomarkers:
opportunities and challenges, Clin. Transl. Med. 7 (2018), http://doi.org/10.1186/s40169-018-0192-7.
[35] S. Pan, Y. Zhang, A. Natalia, C.Z.J. Lim, N.R.Y. Ho, B. Chowbay, T.P. Loh, J.K.C. Tam, H. Shao,
Extracellular vesicle drug occupancy enables real-time monitoring of targeted cancer therapy, Nat.
Nanotechnol. (2021), http://doi.org/10.1038/s41565-021-00872-w.
[36] O.G. De Jong, B.W.M. Van Balkom, R.M. Schiffelers, C.V.C. Bouten, M.C. Verhaar, Extracellular
Vesicles: Potential Roles in Regenerative Medicine, Front. Immunol. 5 (2014),
http://doi.org/10.3389/fimmu.2014.00608.
[37] M. Romano, A. Zendrini, L. Paolini, S. Busatto, A.C. Berardi, P. Bergese, A. Radeghieri, Extracellular
vesicles in regenerative medicine, Elsevier2020, pp. 29-58, http://doi.org/10.1016/B978-0-12-817838-
6.00002-4
[38] N.S. Roy, C. Cleren, S.K. Singh, L. Yang, M.F. Beal, S.A. Goldman, Functional engraftment of
human ES cell–derived dopaminergic neurons enriched by coculture with telomerase-immortalized
midbrain astrocytes, Nat. Med. 12 (2006) 1259-1268, http://doi.org/10.1038/nm1495.
[39] H. Ning, F. Yang, M. Jiang, L. Hu, K. Feng, J. Zhang, Z. Yu, B. Li, C. Xu, Y. Li, J. Wang, J. Hu, X.
Lou, H. Chen, The correlation between cotransplantation of mesenchymal stem cells and higher
recurrence rate in hematologic malignancy patients: outcome of a pilot clinical study, Leukemia. 22
(2008) 593-599, http://doi.org/10.1038/sj.leu.2405090.
[40] J.-O. Jeong, J.W. Han, J.-M. Kim, H.-J. Cho, C. Park, N. Lee, D.-W. Kim, Y.-S. Yoon, Malignant
Tumor Formation After Transplantation of Short-Term Cultured Bone Marrow Mesenchymal Stem Cells
in Experimental Myocardial Infarction and Diabetic Neuropathy, Circ. Res. 108 (2011) 1340-1347,
http://doi.org/10.1161/CIRCRESAHA.110.239848.
[41] F. Erdö, C. Bührle, J. Blunk, M. Hoehn, Y. Xia, B. Fleischmann, M. Föcking, E. Küstermann, E.
Kolossov, J. Hescheler, K.-A. Hossmann, T. Trapp, Host-Dependent Tumorigenesis of Embryonic Stem
Cell Transplantation in Experimental Stroke, J. Cereb. Blood Flow Metab. 23 (2003) 780-785,
http://doi.org/10.1097/01.WCB.0000071886.63724.FB.
[42] B.J. Dlouhy, O. Awe, R.C. Rao, P.A. Kirby, P.W. Hitchon, Autograft-derived spinal cord mass
following olfactory mucosal cell transplantation in a spinal cord injury patient, J. Neurosurg. Spine 21
(2014) 618-622, http://doi.org/10.3171/2014.5.SPINE13992.
57
[43] J.M. Vicencio, D.M. Yellon, V. Sivaraman, D. Das, C. Boi-Doku, S. Arjun, Y. Zheng, J.A. Riquelme,
J. Kearney, V. Sharma, G. Multhoff, A.R. Hall, S.M. Davidson, Plasma exosomes protect the myocardium
from ischemia-reperfusion injury, J. Am. Coll. Cardiol. 65 (2015) 1525-1536,
http://doi.org/10.1016/j.jacc.2015.02.026.
[44] K. Kusuzaki, T. Matsubara, H. Murata, M. Logozzi, E. Iessi, R. Di Raimo, F. Carta, C.T. Supuran, S.
Fais, Natural extracellular nanovesicles and photodynamic molecules: is there a future for drug delivery?,
J. Enzyme Inhib. Med. Chem. 32 (2017) 908-916, http://doi.org/10.1080/14756366.2017.1335310.
[45] K.B. Johnsen, J. Mar, M. Najbjerg, L. Pilgaard, T. Moos, M. Duroux, Biochimica et Biophysica Acta
A comprehensive overview of exosomes as drug delivery vehicles — Endogenous nanocarriers for
targeted cancer therapy, BBA. Rev. Cancer 1846 (2014) 75-87,
http://doi.org/10.1016/j.bbcan.2014.04.005.
[46] B.A. Potz, L.A. Scrimgeour, V.I. Pavlov, N.R. Sodha, M. Ruhul Abid, F.W. Sellke, M.R. Abid, F.W.
Sellke, M. Ruhul Abid, F.W. Sellke, Extracellular Vesicle Injection Improves Myocardial Function and
Increases Angiogenesis in a Swine Model of Chronic Ischemia, J. Am. Heart Assoc. 7 (2018),
http://doi.org/10.1161/JAHA.117.008344.
[47] S. Montaner-Tarbes, E. Novell, V. Tarancón, F.E. Borrás, M. Montoya, L. Fraile, H.A. del Portillo,
Targeted-pig trial on safety and immunogenicity of serum-derived extracellular vesicles enriched
fractions obtained from Porcine Respiratory and Reproductive virus infections, Sci. Rep. 8 (2018) 17487-
17487, http://doi.org/10.1038/s41598-018-36141-5.
[48] R. Vakili-Ghartavol, S.M. Rezayat, R. Faridi-Majidi, K. Sadri, M.R. Jaafari, Optimization of Docetaxel
Loading Conditions in Liposomes: proposing potential products for metastatic breast carcinoma
chemotherapy, Sci. Rep. 10 (2020) 1-14, http://doi.org/10.1038/s41598-020-62501-1.
[49] D.S. Sutaria, M. Badawi, M.A. Phelps, T.D. Schmittgen, Achieving the Promise of Therapeutic
Extracellular Vesicles: The Devil is in Details of Therapeutic Loading, Pharm. Res. 34 (2017) 1053-1066,
[50] D. Moher, A. Liberati, J. Tetzlaff, D.G. Altman, Preferred reporting items for systematic reviews and
meta-analyses: The PRISMA statement, Int. J. Surg. 8 (2010) 336-341,
http://doi.org/10.1016/j.ijsu.2010.02.007.
[51] T.J.C.J.C.M.L.T.P.M.J.W.V.A. Higgins Jpt, Cochrane Handbook for Systematic Reviews of
Interventions, Cochrane. (2020), http://www.training.cochrane.org/handbook.
[52] K.W. Witwer, E.I. Buzás, L.T. Bemis, A. Bora, C. Lässer, J. Lötvall, E.N. Nolte-‘t Hoen, M.G. Piper,
S. Sivaraman, J. Skog, C. Théry, M.H. Wauben, F. Hochberg, Standardization of sample collection,
isolation and analysis methods in extracellular vesicle research, J. Extracell. Vesicles. 2 (2013) 20360-
20360, http://doi.org/10.3402/jev.v2i0.20360.
[53] J. Lotvall, A.F. Hill, F. Hochberg, E.I. Buzas, D. Di Vizio, C. Gardiner, Y.S. Gho, I.V. Kurochkin, S.
Mathivanan, P. Quesenberry, S. Sahoo, H. Tahara, M.H. Wauben, K.W. Witwer, C. Thery, Minimal
experimental requirements for definition of extracellular vesicles and their functions: a position statement
from the International Society for Extracellular Vesicles, J. Extracell. Vesicles. 3 (2014) 26913,
http://doi.org/10.3402/jev.v3.26913.
[54] C. Huang, Y.R. Neupane, X.C. Lim, R. Shekhani, B. Czarny, M.G. Wacker, G. Pastorin, J.W. Wang,
Extracellular vesicles in cardiovascular disease, Elsevier Inc. , Adv. Clin. Chem. 2020, in press,
http://dx.doi.org/10.1016/bs.acc.2020.08.006.
[55] S.W. Ferguson, J. Nguyen, Exosomes as therapeutics: The implications of molecular composition
and exosomal heterogeneity, J. Control Release. 228 (2016) 179-190,
http://doi.org/10.1016/j.jconrel.2016.02.037.
[56] G. Li, J. Wang, M. Xu, H. Zhang, C. Tu, J. Yang, X. Chen, Q. Yao, P. Lan, M. Xie, Engineered
exosome for NIR-triggered drug delivery and superior synergistic chemo-phototherapy in a glioma model,
Appl. Mater. Today. 20 (2020), http://doi.org/10.1016/j.apmt.2020.100723.
[57] Z. Yang, J. Shi, J. Xie, Y. Wang, J. Sun, T. Liu, Y. Zhao, X. Zhao, X. Wang, Y. Ma, V. Malkoc, C.
Chiang, W. Deng, Y. Chen, Y. Fu, K.J. Kwak, Y. Fan, C. Kang, C. Yin, J. Rhee, P. Bertani, J. Otero, W.
Lu, K. Yun, A.S. Lee, W. Jiang, L. Teng, B.Y.S. Kim, L.J. Lee, Large-scale generation of functional
mRNA-encapsulating exosomes via cellular nanoporation, Nat. Biomed. Eng. 4 (2020) 69-83,
[58] G. Jia, Y. Han, Y. An, Y. Ding, C. He, X. Wang, Q. Tang, NRP-1 targeted and cargo-loaded
exosomes facilitate simultaneous imaging and therapy of glioma in vitro and in vivo, Biomaterials. 178
(2018) 302-316, http://doi.org/10.1016/j.biomaterials.2018.06.029.
[59] F.M.F. Lang, A. Hossain, J. Gumin, E.N. Momin, Y. Shimizu, D. Ledbetter, T. Shahar, S. Yamashita,
B. Parker Kerrigan, J. Fueyo, R. Sawaya, F.M.F. Lang, Mesenchymal stem cells as natural biofactories
for exosomes carrying miR-124a in the treatment of gliomas, Neuro. Oncol. 20 (2018) 380-390,
http://doi.org/10.1093/neuonc/nox152.
58
[60] G. Kim, M. Kim, Y. Lee, J. Woo, D. Won, M. Lee, Systemic delivery of microRNA-21 antisense
oligonucleotides to the brain using T7-peptide decorated exosomes, J. Control Release. 317 (2020) 273-
281, http://doi.org/10.1016/j.jconrel.2019.11.009.
[61] D. Gulei, I. Berindan-Neagoe, Activation of Necroptosis by Engineered Self Tumor-Derived
Exosomes Loaded with CRISPR/Cas9, Mol. Ther. Nucleic Acids. 17 (2019) 448-451,
http://doi.org/10.1016/j.omtn.2019.05.032.
[62] Q. Zhu, X. Ling, Y. Yang, J. Zhang, Q. Li, X. Niu, G. Hu, B. Chen, H. Li, Y. Wang, Z. Deng, Embryonic
Stem Cells‐Derived Exosomes Endowed with Targeting Properties as Chemotherapeutics Delivery
Vehicles for Glioblastoma Therapy, Adv. Sci. 6 (2019) 1801899-1801899,
http://doi.org/10.1002/advs.201801899.
[63] H. Monfared, Y. Jahangard, M. Nikkhah, J. Mirnajafi-Zadeh, S.J. Mowla, Potential Therapeutic
Effects of Exosomes Packed With a miR-21-Sponge Construct in a Rat Model of Glioblastoma, Front.
Oncol. 9 (2019) 1-11, http://doi.org/10.3389/fonc.2019.00782.
[64] E.P. Erkan, D. Senfter, S. Madlener, G. Jungwirth, T. Ströbel, N. Saydam, O. Saydam, Extracellular
vesicle-mediated suicide mRNA/protein delivery inhibits glioblastoma tumor growth in vivo, Cancer Gene
Ther. 24 (2017) 38-44, http://doi.org/10.1038/cgt.2016.78.
[65] T. Yang, B. Fogarty, B. LaForge, S. Aziz, T. Pham, L. Lai, S. Bai, Delivery of Small Interfering RNA
to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted
Exosome Nanovesicles for the Treatment of Brain Cancer, AAPS. J. 19 (2017) 475-486,
http://doi.org/10.1208/s12248-016-0015-y.
[66] T. Yang, P. Martin, B. Fogarty, A. Brown, K. Schurman, R. Phipps, V.P. Yin, P. Lockman, S. Bai,
Exosome Delivered Anticancer Drugs Across the Blood-Brain Barrier for Brain Cancer Therapy in Danio
Rerio, Pharm. Res. 32 (2015) 2003-2014, http://doi.org/10.1007/s11095-014-1593-y.
[67] Z. Ye, T. Zhang, W. He, H. Jin, C. Liu, Z. Yang, J. Ren, Methotrexate-Loaded Extracellular Vesicles
Functionalized with Therapeutic and Targeted Peptides for the Treatment of Glioblastoma Multiforme,
ACS. Appl. Mater. Interfaces. 10 (2018) 12341-12350, http://doi.org/10.1021/acsami.7b18135.
[68] M. Katakowski, B. Buller, X. Zheng, Y. Lu, T. Rogers, O. Osobamiro, W. Shu, F. Jiang, M. Chopp,
Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth, Cancer Lett. 335
(2013) 201-204, http://doi.org/10.1016/j.canlet.2013.02.019.
[69] C. Feng, Z. Xiong, C. Wang, W. Xiao, H. Xiao, K. Xie, K. Chen, H. Liang, X. Zhang, H. Yang, Folic
acid-modified Exosome-PH20 enhances the efficiency of therapy via modulation of the tumor
microenvironment and directly inhibits tumor cell metastasis, Bioact. Mater. 6 (2021) 963-974,
http://doi.org/10.1016/j.bioactmat.2020.09.014.
[70] X. Shi, Q. Cheng, T. Hou, M. Han, G. Smbatyan, J.E. Lang, A.L. Epstein, H.J. Lenz, Y. Zhang,
Genetically Engineered Cell-Derived Nanoparticles for Targeted Breast Cancer Immunotherapy, Mol.
Ther. 28 (2020) 536-547, http://doi.org/10.1016/j.ymthe.2019.11.020.
[71] M.J. Haney, Y. Zhao, Y.S. Jin, S.M. Li, J.R. Bago, N.L. Klyachko, A.V. Kabanov, E.V. Batrakova,
Macrophage-Derived Extracellular Vesicles as Drug Delivery Systems for Triple Negative Breast Cancer
(TNBC) Therapy, J. Neuroimmune Pharmacol. 15 (2020) 487-500, http://doi.org/10.1007/s11481-019-
09884-9.
[72] D. Hao, Y. Li, G. Zhao, M. Zhang, Microvesicle-mediated delivery of minicircle DNA results in
effective gene-directed enzyme prodrug cancer therapy, Thorac. Cancer. 10 (2019) 1962-1972,
http://doi.org/10.1111/1759-7714.13175.
[73] H. Gomari, M.F. Moghadam, M. Soleimani, M. Ghavami, S. Khodashenas, Targeted delivery of
doxorubicin to HER2 positive tumor models, Int. J. Nanomedicine. 14 (2019) 5679-5690,
http://doi.org/10.2147/IJN.S210731.
[74] H. Cheng, J.H. Fan, L.P. Zhao, G.L. Fan, R.R. Zheng, X.Z. Qiu, X.Y. Yu, S.Y. Li, X.Z. Zhang,
Chimeric peptide engineered exosomes for dual-stage light guided plasma membrane and nucleus
targeted photodynamic therapy, Biomaterials. 211 (2019) 14-24,
http://doi.org/10.1016/j.biomaterials.2019.05.004.
[75] C. Melzer, V. Rehn, Y. Yang, H. Bähre, J. von der Ohe, R. Hass, Taxol-loaded MSC-Derived
exosomes provide a therapeutic vehicle to target metastatic breast cancer and other carcinoma cells,
Cancers. 11 (2019) 1-20, http://doi.org/10.3390/cancers11060798.
[76] P. Wang, H. Wang, Q. Huang, C. Peng, L. Yao, H. Chen, Z. Qiu, Y. Wu, L. Wang, W. Chen,
Exosomes from M1-Polarized Macrophages Enhance Paclitaxel Antitumor Activity by Activating
Macrophages-Mediated Inflammation, Theranostics. 9 (2019) 1714-1727,
http://doi.org/10.7150/thno.30716.
[77] Y. Liu, L. Bai, K. Guo, Y. Jia, K. Zhang, Q. Liu, P. Wang, X. Wang, Focused ultrasound-augmented
targeting delivery of nanosonosensitizers from homogenous exosomes for enhanced sonodynamic
cancer therapy, Theranostics. 9 (2019) 5261-5281, http://doi.org/10.7150/thno.33183.
59
[78] T. Yong, X.X. Zhang, N. Bie, H. Zhang, X.X. Zhang, F. Li, A. Hakeem, J. Hu, L. Gan, H.A. Santos,
X. Yang, Tumor exosome-based nanoparticles are efficient drug carriers for chemotherapy, Nat.
Commun. 10 (2019) 3838-3838, http://doi.org/10.1038/s41467-019-11718-4.
[79] Z. Naseri, R.K. Oskuee, M.R. Jaafari, M.F. Moghadam, Exosome-mediated delivery of functionally
active miRNA-142-3p inhibitor reduces tumorigenicity of breast cancer in vitro and in vivo, Int. J.
Nanomedicine. 13 (2018) 7727-7747, http://doi.org/10.2147/IJN.S182384.
[80] F. Pi, D.W. Binzel, T.J. Lee, Z. Li, M. Sun, P. Rychahou, H. Li, F. Haque, S. Wang, C.M. Croce, B.
Guo, B.M. Evers, P. Guo, Nanoparticle orientation to control RNA loading and ligand display on
extracellular vesicles for cancer regression, Nat. Nanotechnol. 13 (2018) 82-89,
http://doi.org/10.1038/s41565-017-0012-z.
[81] Y. Wan, L. Wang, C. Zhu, Q. Zheng, G. Wang, J. Tong, Y. Fang, Y. Xia, G. Cheng, X. He, S.-Y.
Zheng, Aptamer-Conjugated Extracellular Nanovesicles for Targeted Drug Delivery, Cancer Res. 78
(2018) 798-808, http://doi.org/10.1158/0008-5472.CAN-17-2880.
[82] J.H. Wang, A.V. Forterre, J. Zhao, D.O. Frimannsson, A. Delcayre, T.J. Antes, B. Efron, S.S. Jeffrey,
M.D. Pegram, A.C. Matin, Anti-her2 scfv-directed extracellular vesicle-mediated mRNA-based gene
delivery inhibits growth of her2-positive human breast tumor xenografts by prodrug activation, Mol.
Cancer Ther. 17 (2018) 1133-1142, http://doi.org/10.1158/1535-7163.MCT-17-0827.
[83] Y. Hong, G.H. Nam, E. Koh, S. Jeon, G.B. Kim, C. Jeong, D.H. Kim, Y. Yang, I.S. Kim, Exosome as
a Vehicle for Delivery of Membrane Protein Therapeutics, PH20, for Enhanced Tumor Penetration and
Antitumor Efficacy, Adv. Funct. Mater. 28 (2018) 1-9, http://doi.org/10.1002/adfm.201703074.
[84] W.M. Usman, T.C. Pham, Y.Y. Kwok, L.T. Vu, V. Ma, B. Peng, Y.S. Chan, L. Wei, S.M. Chin, A.
Azad, A.B.-L. He, A.Y.H. Leung, M. Yang, N. Shyh-Chang, W.C. Cho, J. Shi, M.T.N. Le, Efficient RNA
drug delivery using red blood cell extracellular vesicles, Nat. Commun. 9 (2018) 2359-2359,
[85] Y. Wang, X. Chen, B. Tian, J. Liu, L. Yang, L. Zeng, T. Chen, A. Hong, X. Wang, Nucleolin-targeted
Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer, Theranostics. 7
(2017) 1360-1372, http://doi.org/10.7150/thno.16532.
[86] M. Hadla, S. Palazzolo, G. Corona, I. Caligiuri, V. Canzonieri, G. Toffoli, F. Rizzolio, Exosomes
increase the therapeutic index of doxorubicin in breast and ovarian cancer mouse models,
Nanomedicine. 11 (2016) 2431-2441, http://doi.org/10.2217/nnm-2016-0154.
[87] G. Toffoli, M. Hadla, G. Corona, I. Caligiuri, S. Palazzolo, S. Semeraro, A. Gamini, V. Canzonieri, F.
Rizzolio, Exosomal doxorubicin reduces the cardiac toxicity of doxorubicin, Nanomedicine. 10 (2015)
2963-2971, http://doi.org/10.2217/nnm.15.118.
[88] Y. Yang, Y. Chen, F. Zhang, Q. Zhao, H. Zhong, Increased anti-tumour activity by exosomes derived
from doxorubicin-treated tumour cells via heat stress, Int. J. Hyperthermia. 31 (2015) 498-506,
[89] Y. Tian, S. Li, J. Song, T. Ji, M. Zhu, G.J. Anderson, J. Wei, G. Nie, A doxorubicin delivery platform
using engineered natural membrane vesicle exosomes for targeted tumor therapy, Biomaterials. 35
(2014) 2383-2390, http://doi.org/10.1016/j.biomaterials.2013.11.083.
[90] S. Ohno, M. Takanashi, K. Sudo, S. Ueda, A. Ishikawa, N. Matsuyama, K. Fujita, T. Mizutani, T.
Ohgi, T. Ochiya, N. Gotoh, M. Kuroda, Systemically injected exosomes targeted to EGFR deliver
antitumor microRNA to breast cancer cells, Mol. Ther. 21 (2013) 185-191,
http://doi.org/10.1038/mt.2012.180.
[91] J. Liu, Z. Ye, M. Xiang, B. Chang, J. Cui, T. Ji, L. Zhao, Q. Li, Y. Deng, L. Xu, G. Wang, L. Wang, Z.
Wang, Functional extracellular vesicles engineered with lipid-grafted hyaluronic acid effectively reverse
cancer drug resistance, Biomaterials. 223 (2019) 119475-119475,
http://doi.org/10.1016/j.biomaterials.2019.119475.
[92] F. Xiong, X. Ling, X. Chen, J. Chen, J. Tan, W. Cao, L. Ge, M. Ma, J. Wu, Pursuing Specific
Chemotherapy of Orthotopic Breast Cancer with Lung Metastasis from Docking Nanoparticles Driven by
Bioinspired Exosomes, Nano Lett. 19 (2019) 3256-3266, http://doi.org/10.1021/acs.nanolett.9b00824.
[93] L. Zhao, C. Gu, Y. Gan, L. Shao, H. Chen, H. Zhu, Exosome-mediated siRNA delivery to suppress
postoperative breast cancer metastasis, J. Control Release. 318 (2020) 1-15,
[94] K.O. Jung, H. Jo, J.H. Yu, S.S. Gambhir, G. Pratx, Development and MPI tracking of novel hypoxia-
targeted theranostic exosomes, Biomaterials. 177 (2018) 139-148,
[95] Y. Hong, Y.K. Kim, G.B. Kim, G.H. Nam, S.A. Kim, Y. Park, Y. Yang, I.S. Kim, Degradation of tumour
stromal hyaluronan by small extracellular vesicle-PH20 stimulates CD103+ dendritic cells and in
combination with PD-L1 blockade boosts anti-tumour immunity, J. Extracell. Vesicles. 8 (2019),
60
[96] C. Gong, J. Tian, Z. Wang, Y. Gao, X. Wu, X. Ding, L. Qiang, G. Li, Z. Han, Y. Yuan, S. Gao,
Functional exosome-mediated co-delivery of doxorubicin and hydrophobically modified microRNA 159
for triple-negative breast cancer therapy, J. Nanobiotechnology. 17 (2019) 1-18,
[97] E. Bagheri, K. Abnous, S.A. Farzad, S.M. Taghdisi, M. Ramezani, M. Alibolandi, Targeted
doxorubicin-loaded mesenchymal stem cells-derived exosomes as a versatile platform for fighting
against colorectal cancer, Life Sci. 261 (2020) 118369-118369, http://doi.org/10.1016/j.lfs.2020.118369.
[98] X. Wang, H. Zhang, H. Yang, M. Bai, T. Ning, T. Deng, R. Liu, Q. Fan, K. Zhu, J. Li, Y. Zhan, G.
Ying, Y. Ba, Exosome‐delivered circRNA promotes glycolysis to induce chemoresistance through the
miR‐122‐PKM2 axis in colorectal cancer, Mol. Oncol. 14 (2020) 539-555, http://doi.org/10.1002/1878-
0261.12629.
[99] Z. Zheng, Z. Li, C. Xu, B. Guo, P. Guo, Folate-displaying exosome mediated cytosolic delivery of
siRNA avoiding endosome trapping, J. Control Release. 311-312 (2019) 43-49,
[100] Y. Li, Y. Gao, C. Gong, Z. Wang, Q. Xia, F. Gu, C. Hu, L. Zhang, H. Guo, S. Gao, A33 antibody-
functionalized exosomes for targeted delivery of doxorubicin against colorectal cancer, Nanomedicine.
14 (2018) 1973-1985, http://doi.org/10.1016/j.nano.2018.05.020.
[101] G. Liang, Y. Zhu, D.J. Ali, T. Tian, H. Xu, K. Si, B. Sun, B. Chen, Z. Xiao, Engineered exosomes
for targeted co-delivery of miR-21 inhibitor and chemotherapeutics to reverse drug resistance in colon
cancer, J. Nanobiotechnology. 18 (2020) 10-10, http://doi.org/10.1186/s12951-019-0563-2.
[102] E. Cho, G.-H. Nam, Y. Hong, Y.K. Kim, D.-H. Kim, Y. Yang, I.-S. Kim, Comparison of exosomes
and ferritin protein nanocages for the delivery of membrane protein therapeutics, J. Control Release. 279
(2018) 326-335, http://doi.org/10.1016/j.jconrel.2018.04.037.
[103] E. Koh, E.J. Lee, G.H. Nam, Y. Hong, E. Cho, Y. Yang, I.S. Kim, Exosome-SIRPα, a CD47 blockade
increases cancer cell phagocytosis, Biomaterials. 121 (2017) 121-129,
[104] W. Fan, X.-D. Tian, E. Huang, J.-J. Zhang, Exosomes from CIITA-Transfected CT26 Cells Enhance
Anti-tumor Effects, Asian Pac. J. Cancer Prev. 14 (2013) 987-991,
http://doi.org/10.7314/APJCP.2013.14.2.987.
[105] M. Garofalo, A. Villa, D. Crescenti, M. Marzagalli, L. Kuryk, P. Limonta, V. Mazzaferro, P. Ciana,
Heterologous and cross-species tropism of cancer-derived extracellular vesicles, Theranostics. 9 (2019)
5681-5693, http://doi.org/10.7150/thno.34824.
[106] J. Peng, J. Zhao, Y. Zhao, P. Wu, L. Gou, S. Fu, P. Chen, Y. Lu, L. Yang, HeLa Cell-Derived
Paclitaxel-Loaded Microparticles Efficiently Inhibit the Growth of Cervical Carcinoma, Int. J.
Nanomedicine Volume. 15 (2020) 6409-6420, http://doi.org/10.2147/IJN.S246659.
[107] J. Wang, Y. Dong, Y. Li, W. Li, K. Cheng, Y. Qian, G. Xu, X. Zhang, L. Hu, P. Chen, W. Du, X.
Feng, Y.-D. Zhao, Z. Zhang, B.-F. Liu, Designer Exosomes for Active Targeted Chemo-Photothermal
Synergistic Tumor Therapy, Adv. Funct. Mater. 28 (2018) 1707360-1707360,
http://doi.org/10.1002/adfm.201707360.
[108] F. Aqil, R. Munagala, J. Jeyabalan, A.K. Agrawal, R. Gupta, Exosomes for the Enhanced Tissue
Bioavailability and Efficacy of Curcumin, AAPS. J. 19 (2017) 1691-1702, http://doi.org/10.1208/s12248-
017-0154-9.
[109] W. Zhang, Z.-L. Yu, M. Wu, J.-G. Ren, H.-F. Xia, G.-L. Sa, J.-Y. Zhu, D.-W. Pang, Y.-F. Zhao, G.
Chen, Magnetic and Folate Functionalization Enables Rapid Isolation and Enhanced Tumor-Targeting
of Cell-Derived Microvesicles, ACS Nano. 11 (2017) 277-290, http://doi.org/10.1021/acsnano.6b05630.
[110] A.K.A. Silva, J. Kolosnjaj-Tabi, S. Bonneau, I. Marangon, N. Boggetto, K. Aubertin, O. Clément,
M.F. Bureau, N. Luciani, F. Gazeau, C. Wilhelm, Magnetic and photoresponsive theranosomes:
Translating cell-released vesicles into smart nanovectors for cancer therapy, ACS Nano. 7 (2013) 4954-
4966, http://doi.org/10.1021/nn400269x.
[111] H. Tao, H. Xu, L. Zuo, C. Li, G. Qiao, M. Guo, L. Zheng, M. Leitgeb, X. Lin, Exosomes-coated bcl-
2 siRNA inhibits the growth of digestive system tumors both in vitro and in vivo, Int. J. Biol. Macromol.
161 (2020) 470-480, http://doi.org/10.1016/j.ijbiomac.2020.06.052.
[112] S. Pan, L. Pei, A. Zhang, Y. Zhang, C. Zhang, M. Huang, Z. Huang, B. Liu, L. Wang, L. Ma, Q.
Zhang, D. Cui, Passion fruit-like exosome-PMA/Au-BSA@Ce6 nanovehicles for real-time fluorescence
imaging and enhanced targeted photodynamic therapy with deep penetration and superior retention
behavior in tumor, Biomaterials. 230 (2020) 119606-119606,
[113] X. Wang, H. Zhang, M. Bai, T. Ning, S. Ge, T. Deng, R. Liu, L. Zhang, G. Ying, Y. Ba, Exosomes
Serve as Nanoparticles to Deliver Anti-miR-214 to Reverse Chemoresistance to Cisplatin in Gastric
Cancer, Mol. Ther. 26 (2018) 774-783, http://doi.org/10.1016/j.ymthe.2018.01.001.
61
[114] H. Zhang, M. Bai, T. Deng, R. Liu, X. Wang, Y. Qu, J. Duan, L. Zhang, T. Ning, S. Ge, H. Li, L.
Zhou, Y. Liu, G. Ying, D. Huang, Y. Ba, Cell-derived microvesicles mediate the delivery of miR-29a/c to
suppress angiogenesis in gastric carcinoma, Cancer Lett. 375 (2016) 331-339,
http://doi.org/10.1016/j.canlet.2016.03.026.
[115] J. Li, N. Li, J. Wang, M1 macrophage-derived exosome-encapsulated cisplatin can enhance its
anti-lung cancer effect, Minerva Med. (2020), http://doi.org/10.23736/S0026-4806.20.06564-7.
[116] M. Garofalo, A. Villa, N. Rizzi, L. Kuryk, B. Rinner, V. Cerullo, M. Yliperttula, V. Mazzaferro, P.
Ciana, Extracellular vesicles enhance the targeted delivery of immunogenic oncolytic adenovirus and
paclitaxel in immunocompetent mice, J. Control Release. 294 (2019) 165-175,
[117] J. Ma, Y. Zhang, K. Tang, H. Zhang, X. Yin, Y. Li, P. Xu, Y. Sun, R. Ma, T. Ji, J. Chen, S. Zhang,
T. Zhang, S. Luo, Y. Jin, X. Luo, C. Li, H. Gong, Z. Long, J. Lu, Z. Hu, X. Cao, N. Wang, X. Yang, B.
Huang, Reversing drug resistance of soft tumor-repopulating cells by tumor cell-derived
chemotherapeutic microparticles, Cell Res. 26 (2016) 713-727, http://doi.org/10.1038/cr.2016.53.
[118] F. Aqil, R. Munagala, J. Jeyabalan, A.K. Agrawal, A.H. Kyakulaga, S.A. Wilcher, R.C. Gupta, Milk
exosomes - Natural nanoparticles for siRNA delivery, Cancer Lett. 449 (2019) 186-195,
[119] M. Guo, F. Wu, G. Hu, L. Chen, J. Xu, P. Xu, X. Wang, Y. Li, S. Liu, S. Zhang, Q. Huang, J. Fan,
Z. Lv, M. Zhou, L. Duan, T. Liao, G. Yang, K. Tang, B. Liu, X. Liao, X. Tao, Y. Jin, Autologous tumor cell–
derived microparticle-based targeted chemotherapy in lung cancer patients with malignant pleural
effusion, Sci. Transl. Med. 11 (2019) 1-16, http://doi.org/10.1126/scitranslmed.aat5690.
[120] M. Garofalo, A. Villa, N. Rizzi, L. Kuryk, V. Mazzaferro, P. Ciana, Systemic administration and
targeted delivery of immunogenic oncolytic adenovirus encapsulated in extracellular vesicles for cancer
therapies, Viruses. 10 (2018), http://doi.org/10.3390/v10100558.
[121] M. Garofalo, H. Saari, P. Somersalo, D. Crescenti, L. Kuryk, L. Aksela, C. Capasso, M. Madetoja,
K. Koskinen, T. Oksanen, A. Mäkitie, M. Jalasvuori, V. Cerullo, P. Ciana, M. Yliperttula, Antitumor effect
of oncolytic virus and paclitaxel encapsulated in extracellular vesicles for lung cancer treatment, J.
Control Release. 283 (2018) 223-234, http://doi.org/10.1016/j.jconrel.2018.05.015.
[122] A.K. Agrawal, F. Aqil, J. Jeyabalan, W.A. Spencer, J. Beck, B.W. Gachuki, S.S. Alhakeem, K.
Oben, R. Munagala, S. Bondada, R.C. Gupta, Milk-derived exosomes for oral delivery of paclitaxel,
Nanomedicine. 13 (2017) 1627-1636, http://doi.org/10.1016/j.nano.2017.03.001.
[123] R. Munagala, F. Aqil, J. Jeyabalan, A.K. Agrawal, A.M. Mudd, A.H. Kyakulaga, I.P. Singh, M.V.
Vadhanam, R.C. Gupta, Exosomal formulation of anthocyanidins against multiple cancer types, Cancer
Lett. 393 (2017) 94-102, http://doi.org/10.1016/j.canlet.2017.02.004.
[124] R. Munagala, F. Aqil, J. Jeyabalan, R.C. Gupta, Bovine milk-derived exosomes for drug delivery,
Cancer Lett. 371 (2016) 48-61, http://doi.org/10.1016/j.canlet.2015.10.020.
[125] F. Aqil, H. Kausar, A.K. Agrawal, J. Jeyabalan, A.-H. Kyakulaga, R. Munagala, R. Gupta, Exosomal
formulation enhances therapeutic response of celastrol against lung cancer, Exp. Mol. Med. 101 (2016)
12-21, http://doi.org/10.1016/j.yexmp.2016.05.013.
[126] M.S. Kim, M.J. Haney, Y. Zhao, V. Mahajan, I. Deygen, N.L. Klyachko, E. Inskoe, A. Piroyan, M.
Sokolsky, O. Okolie, S.D. Hingtgen, A.V. Kabanov, E.V. Batrakova, Development of exosome-
encapsulated paclitaxel to overcome MDR in cancer cells, Nanomedicine. 12 (2016) 655-664,
http://doi.org/10.1016/j.nano.2015.10.012.
[127] H. Nie, X. Xie, D. Zhang, Y. Zhou, B. Li, F.F. Li, F.F. Li, Y. Cheng, H. Mei, H. Meng, L. Jia, Use of
lung-specific exosomes for miRNA-126 delivery in non-small cell lung cancer, Nanoscale. 12 (2020) 877-
887, http://doi.org/10.1039/c9nr09011h.
[128] Q. Lin, M. Qu, B. Zhou, H.K. Patra, Z. Sun, Q. Luo, W. Yang, Y. Wu, Y. Zhang, L. Li, L. Deng, L.
Wang, T. Gong, Q. He, L. Zhang, X. Sun, Z. Zhang, Exosome-like nanoplatform modified with targeting
ligand improves anti-cancer and anti-inflammation effects of imperialine, J. Control Release. 311-312
[129] M.S. Kim, M.J. Haney, Y. Zhao, D. Yuan, I. Deygen, N.L. Klyachko, A.V. Kabanov, E.V. Batrakova,
Engineering macrophage-derived exosomes for targeted paclitaxel delivery to pulmonary metastases: in
vitro and in vivo evaluations, Nanomedicine. 14 (2018) 195-204,
[130] D. Hao, Y. Li, G. Zhao, M. Zhang, Soluble fms-like tyrosine kinase-1-enriched exosomes suppress
the growth of small cell lung cancer by inhibiting endothelial cell migration, Thorac. Cancer. 10 (2019)
1962-1972, http://doi.org/10.1111/1759-7714.13175.
[131] G. Lou, L. Chen, C. Xia, W. Wang, J. Qi, A. Li, L. Zhao, Z. Chen, M. Zheng, Y. Liu, MiR-199a-
modified exosomes from adipose tissue-derived mesenchymal stem cells improve hepatocellular
62
carcinoma chemosensitivity through mTOR pathway, J. Exp. Clin. Cancer. Res. 39 (2020) 1-9,
[132] D. Wang, Y. Yao, J. He, X. Zhong, B. Li, S. Rao, H. Yu, S. He, X. Feng, T. Xu, B. Yang, T. Yong,
L. Gan, J. Hu, X. Yang, Engineered Cell‐Derived Microparticles Bi 2 Se 3 /DOX@MPs for Imaging Guided
Synergistic Photothermal/Low‐Dose Chemotherapy of Cancer, Adv. Sci. 7 (2020) 1901293-1901293,
http://doi.org/10.1002/advs.201901293.
[133] H. Qi, C. Liu, L. Long, Y. Ren, S. Zhang, X. Chang, X. Qian, H. Jia, J. Zhao, J. Sun, X. Hou, X.
Yuan, C. Kang, Blood Exosomes Endowed with Magnetic and Targeting Properties for Cancer Therapy,
ACS Nano. 10 (2016) 3323-3333, http://doi.org/10.1021/acsnano.5b06939.
[134] K. Tang, Y. Zhang, H. Zhang, P. Xu, J. Liu, J. Ma, M. Lv, D. Li, F. Katirai, G.-X. Shen, G. Zhang,
Z.-H. Feng, D. Ye, B. Huang, Delivery of chemotherapeutic drugs in tumour cell-derived microparticles,
Nat. Commun. 3 (2012) 1282-1282, http://doi.org/10.1038/ncomms2282.
[135] T. Wu, Y. Qi, D. Zhang, Q. Song, C. Yang, X. Hu, Y. Bao, Y. Zhao, Z. Zhang, Bone Marrow Dendritic
Cells Derived Microvesicles for Combinational Immunochemotherapy against Tumor, Adv. Funct. Mater.
27 (2017) 1703191-1703191, http://doi.org/10.1002/adfm.201703191.
[136] L. Rivoltini, C. Chiodoni, P. Squarcina, M. Tortoreto, A. Villa, B. Vergani, M. Bürdek, L. Botti, I.
Arioli, A. Cova, G. Mauri, E. Vergani, B. Bianchi, P. Della Mina, L. Cantone, V. Bollati, N. Zaffaroni, A.M.
Gianni, M.P. Colombo, V. Huber, TNF-Related Apoptosis-Inducing Ligand (TRAIL)–Armed Exosomes
Deliver Proapoptotic Signals to Tumor Site, Clin. Cancer Res. 22 (2016) 3499-3512,
http://doi.org/10.1158/1078-0432.CCR-15-2170.
[137] D. Ingato, J.A. Edson, M. Zakharian, Y.J. Kwon, Cancer Cell-Derived, Drug-Loaded Nanovesicles
Induced by Sulfhydryl-Blocking for Effective and Safe Cancer Therapy, ACS Nano. 12 (2018) 9568-9577,
http://doi.org/10.1021/acsnano.8b05377.
[138] M. Zhuang, X. Chen, D. Du, J. Shi, M. Deng, Q. Long, X. Yin, Y. Wang, L. Rao, SPION decorated
exosome delivery of TNF-α to cancer cell membranes through magnetism, Nanoscale. 12 (2020) 173-
188, http://doi.org/10.1039/c9nr05865f.
[139] F.H. Shamili, H.R. Bayegi, Z. Salmasi, K. Sadri, M. Mahmoudi, M. Kalantari, M. Ramezani, K.
Abnous, Exosomes derived from TRAIL-engineered mesenchymal stem cells with effective anti-tumor
activity in a mouse melanoma model, Int. J. Pharm. 549 (2018) 218-229,
http://doi.org/10.1016/j.ijpharm.2018.07.067.
[140] M. Morishita, Y. Takahashi, A. Matsumoto, M. Nishikawa, Y. Takakura, Exosome-based tumor
antigens–adjuvant co-delivery utilizing genetically engineered tumor cell-derived exosomes with
immunostimulatory CpG DNA, Biomaterials. 111 (2016) 55-65,
[141] G. Chen, J.-Y. Zhu, Z.-L. Zhang, W. Zhang, J.-G. Ren, M. Wu, Z.-Y. Hong, C. Lv, D.-W. Pang, Y.-
F. Zhao, Transformation of Cell-Derived Microparticles into Quantum-Dot-Labeled Nanovectors for
Antitumor siRNA Delivery, Angew. Chem. Int. Ed. Engl. 54 (2015) 1036-1040,
http://doi.org/10.1002/anie.201410223.
[142] H. Liu, M. Shen, D. Zhao, D. Ru, Y. Duan, C. Ding, H. Li, The Effect of Triptolide-Loaded Exosomes
on the Proliferation and Apoptosis of Human Ovarian Cancer SKOV3 Cells, Biomed. Res. Int. 2019
(2019) 1-14, http://doi.org/10.1155/2019/2595801.
[143] S.M. Kim, Y. Yang, S.J. Oh, Y. Hong, M. Seo, M. Jang, Cancer-derived exosomes as a delivery
platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting, J. Control Release. 266 (2017)
8-16, http://doi.org/10.1016/j.jconrel.2017.09.013.
[144] F. Aqil, J. Jeyabalan, A.K. Agrawal, A.-H. Kyakulaga, R. Munagala, L. Parker, R.C. Gupta,
Exosomal delivery of berry anthocyanidins for the management of ovarian cancer, Food Funct. 8 (2017)
4100-4107, http://doi.org/10.1039/C7FO00882A.
[145] L. Xu, F.N. Faruqu, Y.M. Lim, K.Y. Lim, R. Liam-Or, A.A. Walters, P. Lavender, D. Fear, C.M. Wells,
J. Tzu-Wen Wang, K.T. Al-Jamal, Exosome-mediated RNAi of PAK4 prolongs survival of pancreatic
cancer mouse model after loco-regional treatment, Biomaterials. 264 (2021) 120369-120369,
[146] Y.J. Li, J.Y. Wu, J.M. Wang, X.B. Hu, J.X. Cai, D.X. Xiang, Gemcitabine loaded autologous
exosomes for effective and safe chemotherapy of pancreatic cancer, Acta. Biomater. 101 (2020) 519-
530, http://doi.org/10.1016/j.actbio.2019.10.022.
[147] M. Mendt, S. Kamerkar, H. Sugimoto, K.M. McAndrews, C.-C. Wu, M. Gagea, S. Yang, E.V.R.
Blanko, Q. Peng, X. Ma, J.R. Marszalek, A. Maitra, C. Yee, K. Rezvani, E. Shpall, V.S. LeBleu, R. Kalluri,
Generation and testing of clinical-grade exosomes for pancreatic cancer, JCI Insight. 3 (2018) 1-22,
http://doi.org/10.1172/jci.insight.99263.
63
[148] S. Kamerkar, V.S. LeBleu, H. Sugimoto, S. Yang, C.F. Ruivo, S.A. Melo, J.J. Lee, R. Kalluri,
Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer, Nature. 546 (2017)
498-503, http://doi.org/10.1038/nature22341.
[149] Y. Ding, F. Cao, H. Sun, Y. Wang, S. Liu, Y. Wu, Q. Cui, W.T. Mei, F. Li, Exosomes derived from
human umbilical cord mesenchymal stromal cells deliver exogenous miR-145-5p to inhibit pancreatic
ductal adenocarcinoma progression, Cancer Lett. 442 (2019) 351-361,
[150] Y. Binenbaum, E. Fridman, Z. Yaari, N. Milman, A. Schroeder, G. Ben David, T. Shlomi, Z. Gil,
Transfer of miRNA in Macrophage-Derived Exosomes Induces Drug Resistance in Pancreatic
Adenocarcinoma, Cancer Res. 78 (2018) 5287-5299, http://doi.org/10.1158/0008-5472.CAN-18-0124.
[151] L. Qiao, S. Hu, K. Huang, T. Su, Z. Li, A. Vandergriff, J. Cores, P.U. Dinh, T. Allen, D. Shen, H.
Liang, Y. Li, K. Cheng, Tumor cell-derived exosomes home to their cells of origin and can be used as
Trojan horses to deliver cancer drugs, Theranostics. 10 (2020) 3474-3487,
[152] Y.Y. Zhang, L. Li, J. Yu, D. Zhu, Y.Y. Zhang, X. Li, H. Gu, C.Y. Zhang, K. Zen, Microvesicle-
mediated delivery of transforming growth factor β1 siRNA for the suppression of tumor growth in mice,
Biomaterials. 35 (2014) 4390-4400, http://doi.org/10.1016/j.biomaterials.2014.02.003.
[153] J. Li, Y. Zhang, Y. Liu, X. Dai, W. Li, X. Cai, Y. Yin, Q. Wang, Y. Xue, C. Wang, D. Li, D. Hou, X.
Jiang, J. Zhang, K. Zen, X. Chen, C.-Y. Zhang, Microvesicle-mediated Transfer of MicroRNA-150 from
Monocytes to Endothelial Cells Promotes Angiogenesis, J. Biol. Chem. 288 (2013) 23586-23596,
http://doi.org/10.1074/jbc.M113.489302.
[154] Z. Li, H. Wang, H. Yin, C. Bennett, H.g. Zhang, P. Guo, Arrowtail RNA for Ligand Display on Ginger
Exosome-like Nanovesicles to Systemic Deliver siRNA for Cancer Suppression, Sci. Rep. 8 (2018) 1-11,
[155] D. Bellavia, S. Raimondo, G. Calabrese, S. Forte, M. Cristaldi, A. Patinella, L. Memeo, M. Manno,
S. Raccosta, P. Diana, G. Cirrincione, G. Giavaresi, F. Monteleone, S. Fontana, G.D. Leo, R. Alessandro,
Interleukin 3- receptor targeted exosomes inhibit in vitro and in vivo chronic myelogenous Leukemia cell
growth, Theranostics. 7 (2017) 1333-1345, http://doi.org/10.7150/thno.17092.
[156] J. Wang, Q. Jiang, O.D. Faleti, C.M. Tsang, M. Zhao, G. Wu, S.W. Tsao, M. Fu, Y. Chen, T. Ding,
T. Chong, Y. Long, X. Yang, Y. Zhang, Y. Cai, H. Li, M. Peng, X. Lyu, X. Li, Exosomal Delivery of
AntagomiRs Targeting Viral and Cellular MicroRNAs Synergistically Inhibits Cancer Angiogenesis, Mol.
Ther. Nucleic Acids. 22 (2020) 153-165, http://doi.org/10.1016/j.omtn.2020.08.017.
[157] Y. Si, S. Kim, E. Zhang, Y. Tang, R. Jaskula‐Sztul, J.M. Markert, H. Chen, L. Zhou, X. Liu, Targeted
Exosomes for Drug Delivery: Biomanufacturing, Surface Tagging, and Validation, Biotechnol. J. 15
(2020) 1900163-1900163, http://doi.org/10.1002/biot.201900163.
[158] J.J. He, J.J. He, L. Min, Y. He, H. Guan, J. Wang, X. Peng, Extracellular vesicles transmitted
miR‐31‐5p promotes sorafenib resistance by targeting MLH1 in renal cell carcinoma, Int. J. Cancer. 146
(2020) 1052-1063, http://doi.org/10.1002/ijc.32543.
[159] A. Mizrak, M.F. Bolukbasi, G.B. Ozdener, G.J. Brenner, S. Madlener, E.P. Erkan, T. Ströbel, X.O.
Breakefield, O. Saydam, Genetically engineered microvesicles carrying suicide mRNA/protein inhibit
schwannoma tumor growth, Mol. Ther. 21 (2013) 101-108, http://doi.org/10.1038/mt.2012.161.
[160] P. Huang, L. Wang, Q. Li, X. Tian, J. Xu, J. Xu, Y. Xiong, G. Chen, H. Qian, C. Jin, Y. Yu, K. Cheng,
L. Qian, Y. Yang, Atorvastatin enhances the therapeutic efficacy of mesenchymal stem cells-derived
exosomes in acute myocardial infarction via up-regulating long non-coding RNA H19, Cardiovasc. Res.
116 (2019) 353-367, http://doi.org/10.1093/cvr/cvz139.
[161] Q. Luo, D. Guo, G. Liu, G. Chen, M. Hang, M. Jin, Exosomes from MiR-126-Overexpressing Adscs
Are Therapeutic in Relieving Acute Myocardial Ischaemic Injury, Cell Physiol. Biochem. 44 (2018) 2105-
2116, http://doi.org/10.1159/000485949.
[162] J. Ma, Y. Zhao, L. Sun, X.X. Sun, X. Zhao, X.X. Sun, H. Qian, W. Xu, W. Zhu, Exosomes Derived
from Akt -Modified Human Umbilical Cord Mesenchymal Stem Cells Improve Cardiac Regeneration and
Promote Angiogenesis via Activating Platelet-Derived Growth Factor D, Stem Cells Transl. Med. 6 (2017)
51-59, http://doi.org/10.5966/sctm.2016-0038.
[163] J. Ni, X. Liu, Y. Yin, P. Zhang, Y.W. Xu, Z. Liu, Exosomes derived from TIMP2-modified human
umbilical cord mesenchymal stem cells enhance the repair effect in rat model with myocardial infarction
possibly by the Akt/ SFRP2 pathway, Oxid. Med. Cell. Longev. 2019 (2019),
http://doi.org/10.1155/2019/1958941.
[164] Y. Song, C. Zhang, J. Zhang, Z. Jiao, N. Dong, G. Wang, Z. Wang, L. Wang, Localized injection of
miRNA-21-enriched extracellular vesicles effectively restores cardiac function after myocardial infarction,
Theranostics. 9 (2019) 2346-2360, http://doi.org/10.7150/thno.29945.
64
[165] J.Y. Kang, H.H. Park, H. Kim, D. Mun, H.H. Park, N. Yun, B. Joung, Human peripheral
blood‑derived exosomes for microRNA delivery, Int. J. Mol. Med. 43 (2019) 2319-2328,
http://doi.org/10.3892/ijmm.2019.4150.
[166] X.H. Gong, H. Liu, S.J. Wang, S.W. Liang, G.G. Wang, Exosomes derived from
SDF1‐overexpressing mesenchymal stem cells inhibit ischemic myocardial cell apoptosis and promote
cardiac endothelial microvascular regeneration in mice with myocardial infarction, J. Cell Physiol. 234
(2019) 13878-13893, http://doi.org/10.1002/jcp.28070.
[167] Q. Chen, Y. Liu, X. Ding, Q. Li, F. Qiu, M. Wang, Z. Shen, H. Zheng, G. Fu, Bone marrow
mesenchymal stem cell-secreted exosomes carrying microRNA-125b protect against myocardial
ischemia reperfusion injury via targeting SIRT7, Mol. Cell. Biochem. 465 (2020) 103-114,
http://doi.org/10.1007/s11010-019-03671-z.
[168] Y. Zhao, Y. Gan, G. Xu, K. Hua, D. Liu, Exosomes from MSCs overexpressing microRNA-223-3p
attenuate cerebral ischemia through inhibiting microglial M1 polarization mediated inflammation, Life Sci.
260 (2020) 118403-118403, http://doi.org/10.1016/j.lfs.2020.118403.
[169] T. Tian, H.-x. Zhang, C.-p. He, S. Fan, Y.-l. Zhu, C. Qi, N.-p. Huang, Z.-d. Xiao, Z.-h. Lu, B.A.
Tannous, J. Gao, Surface functionalized exosomes as targeted drug delivery vehicles for cerebral
ischemia therapy, Biomaterials. 150 (2018) 137-149, http://doi.org/10.1016/j.biomaterials.2017.10.012.
[170] J. Yang, S. Wu, L. Hou, D. Zhu, S. Yin, G. Yang, Y. Wang, Therapeutic Effects of Simultaneous
Delivery of Nerve Growth Factor mRNA and Protein via Exosomes on Cerebral Ischemia, Mol. Ther.
Nucleic Acids. 21 (2020) 512-522, http://doi.org/10.1016/j.omtn.2020.06.013.
[171] J. Yang, X. Zhang, X. Chen, L. Wang, G. Yang, Exosome Mediated Delivery of miR-124 Promotes
Neurogenesis after Ischemia, Mol. Ther. Nucleic Acids. 7 (2017) 278-287,
[172] R. He, Y. Jiang, Y. Shi, J. Liang, L. Zhao, Curcumin-laden exosomes target ischemic brain tissue
and alleviate cerebral ischemia-reperfusion injury by inhibiting ROS-mediated mitochondrial apoptosis,
Mater. Sci. Eng. C. 117 (2020), http://doi.org/10.1016/j.msec.2020.111314.
[173] X. Huang, J. Ding, Y. Li, W. Liu, J. Ji, H. Wang, X. Wang, Exosomes derived from PEDF modified
adipose-derived mesenchymal stem cells ameliorate cerebral ischemia-reperfusion injury by regulation
of autophagy and apoptosis, Exp. Cell Res. 371 (2018) 269-277,
http://doi.org/10.1016/j.yexcr.2018.08.021.
[174] A. Kalani, P. Chaturvedi, P.K. Kamat, C. Maldonado, P. Bauer, I.G. Joshua, S.C. Tyagi, N. Tyagi,
Curcumin-loaded embryonic stem cell exosomes restored neurovascular unit following ischemia-
reperfusion injury, Int. J. Biochem. Cell Biol. 79 (2016) 360-369,
http://doi.org/10.1016/j.biocel.2016.09.002.
[175] C. Han, J. Zhou, B. Liu, C. Liang, X. Pan, Y. Zhang, Y. Zhang, Y. Wang, L. Shao, B. Zhu, J. Wang,
Q. Yin, X.Y. Yu, Y. Li, Delivery of miR-675 by stem cell-derived exosomes encapsulated in silk fibroin
hydrogel prevents aging-induced vascular dysfunction in mouse hindlimb, Mater. Sci. Eng. C. 99 (2019)
322-332, http://doi.org/10.1016/j.msec.2019.01.122.
[176] W. Sun, P. Zhao, Y. Zhou, C. Xing, L. Zhao, Z. Li, L. Yuan, Ultrasound targeted microbubble
destruction assisted exosomal delivery of miR-21 protects the heart from chemotherapy associated
cardiotoxicity, Biochem. Biophys. Res. Commun. 532 (2020) 60-67,
http://doi.org/10.1016/j.bbrc.2020.05.044.
[177] X. Wang, H. Gu, W. Huang, J. Peng, Y. Li, L. Yang, D. Qin, K. Essandoh, Y. Wang, T. Peng, G.-C.
Fan, Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function
and Angiogenesis in Diabetic Mice, Diabetes. 65 (2016) 3111-3128, http://doi.org/10.2337/db15-1563.
[178] R. Reshke, J.A. Taylor, A. Savard, H. Guo, L.H. Rhym, P.S. Kowalski, M.T. Trung, C. Campbell,
W. Little, D.G. Anderson, D. Gibbings, Reduction of the therapeutic dose of silencing RNA by packaging
it in extracellular vesicles via a pre-microRNA backbone, Nat. Biomed. Eng. 4 (2020) 52-68,
[179] H. Wang, H. Sui, Y. Zheng, Y. Jiang, Y. Shi, J. Liang, L. Zhao, Curcumin-primed exosomes potently
ameliorate cognitive function in AD mice by inhibiting hyperphosphorylation of the Tau protein through
the AKT/GSK-3β pathway, Nanoscale. 11 (2019) 7481-7496, http://doi.org/10.1039/C9NR01255A.
[180] L. Alvarez-Erviti, Y. Seow, H. Yin, C. Betts, S. Lakhal, M.J. Wood, Delivery of siRNA to the mouse
brain by systemic injection of targeted exosomes, Nat. Biotechnol. 29 (2011) 341-345,
http://doi.org/10.1038/nbt.1807.
[181] M. Izco, J. Blesa, M. Schleef, M. Schmeer, R. Porcari, R. Al-shawi, S. Ellmerich, M.D. Toro, C.
Gardiner, Y. Seow, A. Reinares-sebastian, R. Forcen, J.P. Simons, V. Bellotti, J.M. Cooper, L. Alvarez-
erviti, Systemic Exosomal Delivery of shRNA Minicircles Prevents Parkinsonian Pathology, Mol. Ther.
27 (2019) 1-12, http://doi.org/10.1016/j.ymthe.2019.08.010.
65
[182] X. Ren, Y. Zhao, F. Xue, Y. Zheng, H. Huang, W. Wang, Y. Chang, H. Yang, J. Zhang, Exosomal
DNA Aptamer Targeting α-Synuclein Aggregates Reduced Neuropathological Deficits in a Mouse
Parkinson’s Disease Model, Mol. Ther. Nucleic Acids. 17 (2019) 726-740,
[183] R. Kojima, D. Bojar, G. Rizzi, G.C.E. Hamri, M.D. El-Baba, P. Saxena, S. Ausländer, K.R. Tan, M.
Fussenegger, Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo
for Parkinson's disease treatment, Nat. Commun. 9 (2018), http://doi.org/10.1038/s41467-018-03733-8.
[184] M. Qu, Q. Lin, L. Huang, Y.Y. Fu, L. Wang, S. He, Y.Y. Fu, S. Yang, Z. Zhang, L. Zhang, X. Sun,
Dopamine-loaded blood exosomes targeted to brain for better treatment of Parkinson's disease, J.
Control Release. 287 (2018) 156-166, http://doi.org/10.1016/j.jconrel.2018.08.035.
[185] M.J. Haney, N.L. Klyachko, Y. Zhao, R. Gupta, E.G. Plotnikova, Z. He, T. Patel, A. Piroyan, M.
Sokolsky, A.V. Kabanov, E.V. Batrakova, Exosomes as drug delivery vehicles for Parkinson's disease
therapy, J Control Release. 207 (2015) 18-30, http://doi.org/10.1016/j.jconrel.2015.03.033.
[186] J.M. Cooper, P.B.O. Wiklander, J.Z. Nordin, R. Al-Shawi, M.J. Wood, M. Vithlani, A.H.V. Schapira,
J.P. Simons, S. El-Andaloussi, L. Alvarez-Erviti, Systemic exosomal siRNA delivery reduced alpha-
synuclein aggregates in brains of transgenic mice, Mov. Disord. 29 (2014) 1476-1485,
http://doi.org/10.1002/mds.25978.
[187] M.C. Didiot, L.M. Hall, A.H. Coles, R.A. Haraszti, B.M. Godinho, K. Chase, E. Sapp, S. Ly, J.F.
Alterman, M.R. Hassler, D. Echeverria, L. Raj, D.V. Morrissey, M. DiFiglia, N. Aronin, A. Khvorova,
Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing, Mol.
Ther. 24 (2016) 1836-1847, http://doi.org/10.1038/mt.2016.126.
[188] F. Hosseini Shamili, M. Alibolandi, H. Rafatpanah, K. Abnous, M. Mahmoudi, M. Kalantari, S.M.
Taghdisi, M. Ramezani, Immunomodulatory properties of MSC-derived exosomes armed with high
affinity aptamer toward mylein as a platform for reducing multiple sclerosis clinical score, J. Control
Release. 299 (2019) 149-164, http://doi.org/10.1016/j.jconrel.2019.02.032.
[189] S.C. Tao, T. Yuan, Y.L. Zhang, W.J. Yin, S.C. Guo, C.Q. Zhang, Exosomes derived from miR-140-
5p-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and
prevent osteoarthritis of the knee in a rat model, Theranostics. 7 (2017) 180-195,
[190] Y. Liang, X. Xu, X. Li, J. Xiong, B. Li, L. Duan, D. Wang, J. Xia, Chondrocyte-Targeted MicroRNA
Delivery by Engineered Exosomes toward a Cell-Free Osteoarthritis Therapy, ACS. Appl. Mater.
Interfaces. 12 (2020) 36938-36947, http://doi.org/10.1021/acsami.0c10458.
[191] Z. Chen, H. Wang, Y. Xia, F. Yan, Y. Lu, Therapeutic Potential of Mesenchymal Cell–Derived
miRNA-150-5p–Expressing Exosomes in Rheumatoid Arthritis Mediated by the Modulation of MMP14
and VEGF, J. Immunol. 201 (2018) 2472-2482, http://doi.org/10.4049/jimmunol.1800304.
[192] K. Bryniarski, W. Ptak, A. Jayakumar, K. Pullmann, M.J. Caplan, A. Chairoungdua, J. Lu, B.D.
Adams, E. Sikora, K. Nazimek, S. Marquez, S.H. Kleinstein, P. Sangwung, Y. Iwakiri, E. Delgato, F.
Redegeld, B.R. Blokhuis, J. Wojcikowski, A.W. Daniel, T. Groot Kormelink, P.W. Askenase, Antigen-
specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector
T cells to inhibit contact sensitivity, J. Allergy Clin. Immunol. 132 (2013) 170-181,
http://doi.org/10.1016/j.jaci.2013.04.048.
[193] X. Zhuang, X. Xiang, W. Grizzle, D. Sun, S. Zhang, R.C. Axtell, S. Ju, J. Mu, L. Zhang, L. Steinman,
D. Miller, H.-G. Zhang, Treatment of Brain Inflammatory Diseases by Delivering Exosome Encapsulated
Anti-inflammatory Drugs From the Nasal Region to the Brain, Mol. Ther. 19 (2011) 1769-1779,
http://doi.org/10.1038/mt.2011.164.
[194] J.P. de Rivero Vaccari, F. Brand, S. Adamczak, S.W. Lee, J. Perez-Barcena, M.Y. Wang, M.R.
Bullock, W.D. Dietrich, R.W. Keane, Exosome-mediated inflammasome signaling after central nervous
system injury, J. Neurochem. 136 (2016) 39-48, http://doi.org/10.1111/jnc.13036.
[195] J. Yang, C.-Z. Zhou, R. Zhu, H. Fan, X.-X. Liu, X.-Y. Duan, Q. Tang, Z.-X. Shou, D.-M. Zuo, miR-
200b-containing microvesicles attenuate experimental colitis associated intestinal fibrosis by inhibiting
epithelial-mesenchymal transition, J. Gastroenterol. Hepatol. 32 (2017) 1966-1974,
http://doi.org/10.1111/jgh.13797.
[196] D. Sun, X. Zhuang, X. Xiang, Y. Liu, S. Zhang, C. Liu, S. Barnes, W. Grizzle, D. Miller, H.-G. Zhang,
A Novel Nanoparticle Drug Delivery System: The Anti-inflammatory Activity of Curcumin Is Enhanced
When Encapsulated in Exosomes, Mol. Ther. 18 (2010) 1606-1614, http://doi.org/10.1038/mt.2010.105.
[197] X. Yang, S. Meng, H. Jiang, T. Chen, W. Wu, Exosomes derived from interleukin-10-treated
dendritic cells can inhibit trinitrobenzene sulfonic acid-induced rat colitis, Scand. J. Gastroenterol. 45
(2010) 1168-1177, http://doi.org/10.3109/00365521.2010.490596.
66
[198] D. Wen, Y. Peng, D. Liu, Y. Weizmann, R.I. Mahato, Mesenchymal stem cell and derived exosome
as small RNA carrier and Immunomodulator to improve islet transplantation, J. Control Release. 238
[199] W. Sun, C. Xing, L. Zhao, P. Zhao, G. Yang, L. Yuan, Ultrasound Assisted Exosomal Delivery of
Tissue Responsive mRNA for Enhanced Efficacy and Minimized Off-Target Effects, Mol. Ther. Nucleic
Acids. 20 (2020) 558-567, http://doi.org/10.1016/j.omtn.2020.03.016.
[200] R.H. Nanjundappa, R. Wang, Y. Xie, C.S. Umeshappa, J. Xiang, Novel CD8 + T cell-based vaccine
stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic
HLA-A2 mice, Vaccine. 30 (2012) 3519-3525, http://doi.org/10.1016/j.vaccine.2012.03.075.
[201] Y. Cheng, J.S. Schorey, Extracellular vesicles deliver Mycobacterium RNA to promote host
immunity and bacterial killing, EMBO. Rep. 20 (2019) 1-16, http://doi.org/10.15252/embr.201846613.
[202] M. Tapparo, S. Bruno, F. Collino, G. Togliatto, M.C. Deregibus, P. Provero, S. Wen, P.J.
Quesenberry, G. Camussi, Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-
Engineered Mesenchymal Stromal Cells, Int. J. Mol. Sci. 20 (2019) 2381-2381,
http://doi.org/10.3390/ijms20102381.
[203] H. Wang, B. Wang, A. Zhang, F. Hassounah, Y. Seow, M. Wood, F. Ma, J.D. Klein, S.R. Price,
X.H. Wang, Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice,
Mol. Ther. 27 (2019) 571-583, http://doi.org/10.1016/j.ymthe.2019.01.008.
[204] Y. Wang, X. Lu, J. He, W. Zhao, Influence of erythropoietin on microvesicles derived from
mesenchymal stem cells protecting renal function of chronic kidney disease, Stem Cell Res. Ther. 6
(2015) 1-14, http://doi.org/10.1186/s13287-015-0095-0.
[205] S. Zhang, L. Jiang, H. Hu, H. Wang, X. Wang, J. Jiang, Y. Ma, J. Yang, Y. Hou, D. Xie, Q. Zhang,
Pretreatment of exosomes derived from hUCMSCs with TNF-α ameliorates acute liver failure by inhibiting
the activation of NLRP3 in macrophage, Life Sci. 246 (2020) 117401-117401,
http://doi.org/10.1016/j.lfs.2020.117401.
[206] D. Lainšček, L. Kadunc, M.M. Keber, I.H. Bratkovič, R. Romih, R. Jerala, Delivery of an Artificial
Transcription Regulator dCas9-VPR by Extracellular Vesicles for Therapeutic Gene Activation, ACS.
Synth. Biol. 7 (2018) 2715-2725, http://doi.org/10.1021/acssynbio.8b00192.
[207] Z. Li, X. Zhou, M. Wei, X. Gao, L. Zhao, R. Shi, W. Sun, Y. Duan, G. Yang, L. Yuan, In Vitro and in
Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or
CRISPR/dCas9, Nano Lett. 19 (2019) 19-28, http://doi.org/10.1021/acs.nanolett.8b02689.
[208] Q. Pan, V. Ramakrishnaiah, S. Henry, S. Fouraschen, P.E. de Ruiter, J. Kwekkeboom, H.W.
Tilanus, H.L.A. Janssen, L.J.W. van der Laan, Hepatic cell-to-cell transmission of small silencing RNA
can extend the therapeutic reach of RNA interference (RNAi), Gut. 61 (2012) 1330-1339,
http://doi.org/10.1136/gutjnl-2011-300449.
[209] N. Ran, X. Gao, X. Dong, J. Li, C. Lin, M. Geng, H. Yin, Effects of exosome-mediated delivery of
myostatin propeptide on functional recovery of mdx mice, Biomaterials. 236 (2020) 119826-119826,
[210] X. Gao, N. Ran, X. Dong, B. Zuo, R. Yang, Q. Zhou, H.M. Moulton, Y. Seow, H. Yin, Anchor peptide
captures, targets, and loads exosomes of diverse origins for diagnostics and therapy, Sci. Transl. Med.
10 (2018) eaat0195-eaat0195, http://doi.org/10.1126/scitranslmed.aat0195.
[211] S.-C. Tao, S.-C. Guo, M. Li, Q.-F. Ke, Y.-P. Guo, C.-Q. Zhang, Chitosan Wound Dressings
Incorporating Exosomes Derived from MicroRNA-126-Overexpressing Synovium Mesenchymal Stem
Cells Provide Sustained Release of Exosomes and Heal Full-Thickness Skin Defects in a Diabetic Rat
Model, Stem Cells Transl. Med. 6 (2017) 736-747, http://doi.org/10.5966/sctm.2016-0275.
[212] S.S. Yerneni, S. Lathwal, P. Shrestha, H. Shirwan, K. Matyjaszewski, L. Weiss, E.S. Yolcu, P.G.
Campbell, S.R. Das, Rapid On-Demand Extracellular Vesicle Augmentation with Versatile
Oligonucleotide Tethers, ACS Nano. 13 (2019) 10555-10565, http://doi.org/10.1021/acsnano.9b04651.
[213] M. Bader, Rat Models of Cardiovascular Diseases, 2010, pp. 403-414, http://doi.org/10.1007/978-
1-60327-389-3_27.
[214] M.E. Castañeda-Lopez, I. Garza-Veloz, J.M. Ortiz-Rodriguez, R. Castañeda-Miranda, L.O. Solis-
Sanchez, H.R. Vega-Carrillo, M.d.R. Martinez-Blanco, F. Trejo-Vazquez, G. Ornelas-Vargas, I.P.
Rodriguez-Sanchez, H.A. Guerrero-Osuna, I. Delgado-Enciso, O.G. Meza-Zavala, M.d.l.L. Martinez-
Fierro, Animal Models of Rheumatoid Arthritis, InTech2018, http://doi.org/ 10.5772/intechopen.72554.
[215] Hason, Bartůněk, Zebrafish Models of Cancer—New Insights on Modeling Human Cancer in a
Non-Mammalian Vertebrate, Genes. 10 (2019) 935-935, http://doi.org/10.3390/genes10110935.
[216] S.E. Emam, A.S. Abu Lila, N.E. Elsadek, H. Ando, T. Shimizu, K. Okuhira, Y. Ishima, M.A. Mahdy,
F.-e.S. Ghazy, T. Ishida, Cancer cell-type tropism is one of crucial determinants for the efficient systemic
delivery of cancer cell-derived exosomes to tumor tissues, Eur. J. Pharm. Biopharm. 145 (2019) 27-34,
http://doi.org/10.1016/j.ejpb.2019.10.005.
67
[217] J.M. Gudbergsson, K. Jønsson, J.B. Simonsen, K.B. Johnsen, Systematic review of targeted
extracellular vesicles for drug delivery – Considerations on methodological and biological heterogeneity,
J. Control Release. 306 (2019) 108-120, http://doi.org/10.1016/j.jconrel.2019.06.006.
[218] R. Huey, S. Hawthorne, P. McCarron, The potential use of rabies virus glycoprotein-derived
peptides to facilitate drug delivery into the central nervous system: a mini review, J. Drug Target. 25
(2017) 379-385, http://doi.org/10.1080/1061186X.2016.1223676.
[219] E. van der Pol, A.N. Boing, P. Harrison, A. Sturk, R. Nieuwland, Classification, functions, and clinical
relevance of extracellular vesicles, Pharmacol. Rev. 64 (2012) 676-705,
http://doi.org/10.1124/pr.112.005983.
[220] E. Van Der Pol, A.G. Hoekstra, A. Sturk, C. Otto, T.G. Van Leeuwen, R. Nieuwland, Optical and
non-optical methods for detection and characterization of microparticles and exosomes, J. Thromb.
Haemost. 8 (2010) 2596-2607, http://doi.org/10.1111/j.1538-7836.2010.04074.x.
[221] R.A. Dragovic, C. Gardiner, A.S. Brooks, D.S. Tannetta, D.J. Ferguson, P. Hole, B. Carr, C.W.
Redman, A.L. Harris, P.J. Dobson, P. Harrison, I.L. Sargent, Sizing and phenotyping of cellular vesicles
using Nanoparticle Tracking Analysis, Nanomedicine. 7 (2011) 780-788,
[222] V. Filipe, A. Hawe, W. Jiskoot, Critical evaluation of Nanoparticle Tracking Analysis (NTA) by
NanoSight for the measurement of nanoparticles and protein aggregates, Pharm. Res. 27 (2010) 796-
[223] A.S. Lawrie, A. Albanyan, R.A. Cardigan, I.J. Mackie, P. Harrison, Microparticle sizing by dynamic
light scattering in fresh-frozen plasma, Vox Sang. 96 (2009) 206-212, http://doi.org/10.1111/j.1423-
0410.2008.01151.x.
[224] E. van der Pol, F.A.W. Coumans, A.E. Grootemaat, C. Gardiner, I.L. Sargent, P. Harrison, A. Sturk,
T.G. van Leeuwen, R. Nieuwland, Particle size distribution of exosomes and microvesicles determined
by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse
sensing, J. Thromb. Haemost. 12 (2014) 1182-1192, http://doi.org/10.1111/jth.12602.
[225] F.A.W. Coumans, A.R. Brisson, E.I. Buzas, F. Dignat-George, E.E.E. Drees, S. El-Andaloussi, C.
Emanueli, A. Gasecka, A. Hendrix, A.F. Hill, R. Lacroix, Y. Lee, T.G. van Leeuwen, N. Mackman, I.
Mäger, J.P. Nolan, E. van der Pol, D.M. Pegtel, S. Sahoo, P.R.M. Siljander, G. Sturk, O. de Wever, R.
Nieuwland, Methodological Guidelines to Study Extracellular Vesicles, Circ. Res. 120 (2017) 1632-1648,
http://doi.org/10.1161/CIRCRESAHA.117.309417.
[226] J. Webber, A. Clayton, How pure are your vesicles?, J. Extracell. Vesicles. 2 (2013) 19861-19861,
http://doi.org/10.3402/jev.v2i0.19861.
[227] O.P. Wiklander, J.Z. Nordin, A. O'Loughlin, Y. Gustafsson, G. Corso, I. Mager, P. Vader, Y. Lee,
H. Sork, Y. Seow, N. Heldring, L. Alvarez-Erviti, C.I. Smith, K. Le Blanc, P. Macchiarini, P. Jungebluth,
M.J. Wood, S.E. Andaloussi, Extracellular vesicle in vivo biodistribution is determined by cell source,
route of administration and targeting, J. Extracell. Vesicles. 4 (2015) 26316,
[228] J.W. Wang, Y.N. Zhang, S.K. Sze, S.M. van de Weg, F. Vernooij, A.H. Schoneveld, S.H. Tan, H.H.
Versteeg, L. Timmers, C.S.P. Lam, D.P.V. de Kleijn, Lowering Low-Density Lipoprotein Particles in
Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles, Int. J. Mol. Sci. 19
(2017), http://doi.org/10.3390/ijms19010094.
[229] S. Senapati, A.K. Mahanta, S. Kumar, P. Maiti, Controlled drug delivery vehicles for cancer
treatment and their performance, Signal Transduct. Target. Ther. 3 (2018) 1-19,
[230] S. Svenson, Clinical translation of nanomedicines, Curr. Opin. Solid State Mater. Sci. 16 (2012)
287-294, http://doi.org/10.1016/j.cossms.2012.10.001.
[231] M. Çağdaş, A.D. Sezer, S. Bucak, Liposomes as Potential Drug Carrier Systems for Drug Delivery,
InTech 2014, http://doi.org/ 10.5772/58459.
[232] T.M. Allen, C.B. Hansen, D.E.L. de Menezes, Pharmacokinetics of long-circulating liposomes, Adv.
Drug. Deliv. Rev. 16 (1995) 267-284, http://doi.org/10.1016/0169-409X(95)00029-7.
[233] E. Gentile, F. Cilurzo, L. Di Marzio, M. Carafa, C. Anna Ventura, J. Wolfram, D. Paolino, C. Celia,
Liposomal chemotherapeutics, Future Oncol. 9 (2013) 1849-1859, http://doi.org/10.2217/fon.13.146.
[234] S. Walker, S. Busatto, A. Pham, M. Tian, A. Suh, K. Carson, A. Quintero, M. Lafrence, H. Malik,
M.X. Santana, J. Wolfram, Extracellular vesicle-based drug delivery systems for cancer treatment,
Theranostics. 9 (2019) 8001-8017, http://doi.org/10.7150/thno.37097.
[235] M. Merino, S. Zalba, M.J. Garrido, Immunoliposomes in clinical oncology: State of the art and future
perspectives, J. Control Release. 275 (2018) 162-176, http://doi.org/10.1016/j.jconrel.2018.02.015.
68
[236] J.C. Kraft, J.P. Freeling, Z. Wang, R.J.Y. Ho, Emerging Research and Clinical Development Trends
of Liposome and Lipid Nanoparticle Drug Delivery Systems, J. Pharm. Sci. 103 (2014) 29-52,
http://doi.org/10.1002/jps.23773.
[237] E. Beltrán-Gracia, A. López-Camacho, I. Higuera-Ciapara, J.B. Velázquez-Fernández, A.A.
Vallejo-Cardona, Nanomedicine review: clinical developments in liposomal applications, Cancer
Nanotechnol. 10 (2019) 11-11, http://doi.org/10.1186/s12645-019-0055-y.
[238] T. Olusanya, R. Haj Ahmad, D. Ibegbu, J. Smith, A. Elkordy, Liposomal Drug Delivery Systems and
Anticancer Drugs, Molecules. 23 (2018) 907-907, http://doi.org/10.3390/molecules23040907.
[239] Y.H. Choi, H.-K. Han, Nanomedicines: current status and future perspectives in aspect of drug
delivery and pharmacokinetics, J. Pharm. Investig. 48 (2018) 43-60, http://doi.org/10.1007/s40005-017-
0370-4.
[240] S. Wilhelm, A.J. Tavares, Q. Dai, S. Ohta, J. Audet, H.F. Dvorak, W.C.W. Chan, Analysis of
nanoparticle delivery to tumours, Nat. Rev. Mater. 1 (2016) 16014-16014,
http://doi.org/10.1038/natrevmats.2016.14.
[241] M. Longmire, P.L. Choyke, H. Kobayashi, Clearance properties of nano-sized particles and
molecules as imaging agents: considerations and caveats, Nanomedicine. 3 (2008) 703-717,
http://doi.org/10.2217/17435889.3.5.703.
[242] A. Nagayasu, K. Uchiyama, H. Kiwada, The size of liposomes: a factor which affects their targeting
efficiency to tumors and therapeutic activity of liposomal antitumor drugs, Adv. Drug. Deliv. Rev. 40
(1999) 75-87, http://doi.org/10.1016/S0169-409X(99)00041-1.
[243] G.J.R. Charrois, T.M. Allen, Rate of biodistribution of STEALTH® liposomes to tumor and skin:
influence of liposome diameter and implications for toxicity and therapeutic activity, Biochim. Biophys.
Acta. Biomembr. 1609 (2003) 102-108, http://doi.org/10.1016/S0005-2736(02)00661-2.
[244] J. Patel, Liposomal doxorubicin: Doxil®, J. Oncol. Pharm. Pract. 2 (1996) 201-210,
http://doi.org/10.1177/107815529600200402.
[245] J. Wang, D. Chen, E.A. Ho, Challenges in the development and establishment of exosome-based
drug delivery systems, J. Control Release. (2020) 1-13, http://doi.org/10.1016/j.jconrel.2020.10.020.
[246] M.F. Attia, N. Anton, J. Wallyn, Z. Omran, T.F. Vandamme, An overview of active and passive
targeting strategies to improve the nanocarriers efficiency to tumour sites, J. Pharm. Pharmacol. 71
(2019) 1185-1198, http://doi.org/10.1111/jphp.13098.
[247] S. Hua, S.Y. Wu, The use of lipid-based nanocarriers for targeted pain therapies, Front. Pharmacol.
4 NOV (2013) 1-7, http://doi.org/10.3389/fphar.2013.00143.
[248] C. Rothkopf, A. Fahr, G. Fricker, G.L. Scherphof, J.A. Kamps, Uptake of phosphatidylserine-
containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver, Biochim
Biophys Acta. 1668 (2005) 10-16, http://doi.org/10.1016/j.bbamem.2004.10.013.
[249] J.A. Kamps, H.W. Morselt, P.J. Swart, D.K. Meijer, G.L. Scherphof, Massive targeting of liposomes,
surface-modified with anionized albumins, to hepatic endothelial cells, Proc. Natl. Acad. Sci. U S A. 94
(1997) 11681-11685, http://doi.org/10.1073/pnas.94.21.11681.
[250] T. Imai, Y. Takahashi, M. Nishikawa, K. Kato, M. Morishita, T. Yamashita, A. Matsumoto, C.
Charoenviriyakul, Y. Takakura, Macrophage-dependent clearance of systemically administered B16BL6-
derived exosomes from the blood circulation in mice, J. Extracell. Vesicles. 4 (2015) 26238-26238,
[251] T. Smyth, M. Kullberg, N. Malik, P. Smith-Jones, M.W. Graner, T.J. Anchordoquy, Biodistribution
and delivery efficiency of unmodified tumor-derived exosomes, J. Control Release. 199 (2015) 145-155,
[252] R. van der Meel, M.H. Fens, P. Vader, W.W. van Solinge, O. Eniola-Adefeso, R.M. Schiffelers,
Extracellular vesicles as drug delivery systems: lessons from the liposome field, J. Control Release. 195
[253] P. Milla, F. Dosio, L. Cattel, PEGylation of Proteins and Liposomes: a Powerful and Flexible
Strategy to Improve the Drug Delivery, Curr. Drug Metab. 13 (2012) 105-119,
http://doi.org/10.2174/138920012798356934.
[254] S.A.A. Kooijmans, L.A.L. Fliervoet, R. van der Meel, M. Fens, H.F.G. Heijnen, P.M.P. van Bergen
En Henegouwen, P. Vader, R.M. Schiffelers, PEGylated and targeted extracellular vesicles display
enhanced cell specificity and circulation time, J. Control Release. 224 (2016) 77-85,
[255] D.E. Murphy, O.G. de Jong, M. Brouwer, M.J. Wood, G. Lavieu, R.M. Schiffelers, P. Vader,
Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking, Exp. Mol.
Med. 51 (2019) 32-32, http://doi.org/10.1038/s12276-019-0223-5.
69
[256] M. Germain, M.E. Meyre, L. Poul, M. Paolini, C. Berjaud, F. Mpambani, M. Bergere, L. Levy, A.
Pottier, Priming the body to receive the therapeutic agent to redefine treatment benefit/risk profile, Sci.
Rep. 8 (2018) 1-11, http://doi.org/10.1038/s41598-018-23140-9.
[257] N.R.M. Saunders, M.S. Paolini, O.S. Fenton, L. Poul, J. Devalliere, F. Mpambani, A. Darmon, M.
Bergère, O. Jibault, M. Germain, R. Langer, A Nanoprimer To Improve the Systemic Delivery of siRNA
and mRNA, Nano Lett. 20 (2020) 4264-4269, http://doi.org/10.1021/acs.nanolett.0c00752.
[258] D.C. Watson, D. Bayik, A. Srivatsan, C. Bergamaschi, A. Valentin, G. Niu, J. Bear, M. Monninger,
M. Sun, A. Morales-Kastresana, J.C. Jones, B.K. Felber, X. Chen, I. Gursel, G.N. Pavlakis, Efficient
production and enhanced tumor delivery of engineered extracellular vesicles, Biomaterials. 105 (2016)
195-205, http://doi.org/10.1016/j.biomaterials.2016.07.003.
[259] E.T.M. Dams, P. Laverman, W.J.G. Oyen, G. Storm, G.L. Scherphof, J.W.M. Van Der Meer, F.H.M.
Corstens, O.C. Boerman, Accelerated blood clearance and altered biodistribution of repeated injections
of sterically stabilized liposomes, J. Pharmacol. Exp. Ther. 292 (2000) 1071-1079.
[260] A.S. Abu Lila, H. Kiwada, T. Ishida, The accelerated blood clearance (ABC) phenomenon: Clinical
challenge and approaches to manage, J. Control Release. 172 (2013) 38-47,
[261] Y. Mima, Y. Hashimoto, T. Shimizu, H. Kiwada, T. Ishida, Anti-PEG IgM Is a Major Contributor to
the Accelerated Blood Clearance of Polyethylene Glycol-Conjugated Protein, Mol. Pharm. 12 (2015)
2429-2435, http://doi.org/10.1021/acs.molpharmaceut.5b00144.
[262] A.E. Hansen, A.L. Petersen, J.R. Henriksen, B. Boerresen, P. Rasmussen, D.R. Elema, P.M.a.
Rosenschöld, A.T. Kristensen, A. Kjær, T.L. Andresen, Positron Emission Tomography Based
Elucidation of the Enhanced Permeability and Retention Effect in Dogs with Cancer Using Copper-64
Liposomes, ACS Nano. 9 (2015) 6985-6995, http://doi.org/10.1021/acsnano.5b01324.
[263] H. Koide, T. Asai, K. Hatanaka, T. Urakami, T. Ishii, E. Kenjo, M. Nishihara, M. Yokoyama, T.
Ishida, H. Kiwada, N. Oku, Particle size-dependent triggering of accelerated blood clearance
phenomenon, Int. J. Pharm. 362 (2008) 197-200, http://doi.org/10.1016/j.ijpharm.2008.06.004.
[264] T. Ishihara, M. Takeda, H. Sakamoto, A. Kimoto, C. Kobayashi, N. Takasaki, K. Yuki, K.I. Tanaka,
M. Takenaga, R. Igarashi, T. Maeda, N. Yamakawa, Y. Okamoto, M. Otsuka, T. Ishida, H. Kiwada, Y.
Mizushima, T. Mizushima, Accelerated blood clearance phenomenon upon repeated injection of peg-
modified pla-nanoparticles, Pharm. Res. 26 (2009) 2270-2279, http://doi.org/10.1007/s11095-009-9943-
x.
[265] T. Ishihara, T. Maeda, H. Sakamoto, N. Takasaki, M. Shigyo, T. Ishida, H. Kiwada, Y. Mizushima,
T. Mizushima, Evasion of the accelerated blood clearance phenomenon by coating of nanoparticles with
various hydrophilic polymers, Biomacromolecules. 11 (2010) 2700-2706,
http://doi.org/10.1021/bm100754e.
[266] Z. Zhang, Y. Chu, C. Li, W. Tang, J. Qian, X. Wei, W. Lu, T. Ying, C. Zhan, Anti-PEG scFv corona
ameliorates accelerated blood clearance phenomenon of PEGylated nanomedicines, J. Control Release.
330 (2021) 493-501, http://doi.org/10.1016/j.jconrel.2020.12.047.
[267] E. Őrfi, T. Mészáros, M. Hennies, T. Fülöp, L. Dézsi, A. Nardocci, L. Rosivall, P. Hamar, B.W.
Neun, M.A. Dobrovolskaia, J. Szebeni, G. Szénási, Acute physiological changes caused by complement
activators and amphotericin b-containing liposomes in mice, Int. J. Nanomedicine. 14 (2019) 1563-1573,
http://doi.org/10.2147/IJN.S187139.
[268] J. Szebeni, Complement activation-related pseudoallergy: A new class of drug-induced acute
immune toxicity, Toxicology. 216 (2005) 106-121, http://doi.org/10.1016/j.tox.2005.07.023.
[269] L.R. Sharma, A. Subedi, B.K. Shah, Anaphylaxis to pegylated liposomal doxorubicin: A case report,
West Indian Med. J. 63 (2014) 376-377, http://doi.org/10.7727/wimj.2013.270.
[270] G. Milosevits, J. Szebeni, S. Krol, Exosomes: potential model for complement-stealth delivery
systems, Eur. J. Nanomed. 7 (2015), http://doi.org/10.1515/ejnm-2015-0005.
[271] G. Ferrandina, M. Ludovisi, D. Lorusso, S. Pignata, E. Breda, A. Savarese, P. Del Medico, L.
Scaltriti, D. Katsaros, D. Priolo, G. Scambia, Phase III trial of gemcitabine compared with pegylated
liposomal doxorubicin in progressive or recurrent ovarian cancer, J. Clin. Oncol. 26 (2008) 890-896,
http://doi.org/10.1200/JCO.2007.13.6606.
[272] M.A. Morse, J. Garst, T. Osada, S. Khan, A. Hobeika, T.M. Clay, N. Valente, R. Shreeniwas, M.A.
Sutton, A. Delcayre, D.-H.H. Hsu, J.-B.B. Le Pecq, H.K. Lyerly, A phase I study of dexosome
immunotherapy in patients with advanced non-small cell lung cancer, J. Transl. Med. 3 (2005) 9-9,
http://doi.org/10.1186/1479-5876-3-9.
[273] B. Escudier, T. Dorval, N. Chaput, F. André, M.P. Caby, S. Novault, C. Flament, C. Leboulaire, C.
Borg, S. Amigorena, C. Boccaccio, C. Bonnerot, O. Dhellin, M. Movassagh, S. Piperno, C. Robert, V.
Serra, N. Valente, J.B. Le Pecq, A. Spatz, O. Lantz, T. Tursz, E. Angevin, L. Zitvogel, Vaccination of
70
metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: Results of the first
phase 1 clinical trial, J. Transl. Med. 3 (2005) 1-13, http://doi.org/10.1186/1479-5876-3-10.
[274] K.B. Johnsen, J.M. Gudbergsson, M. Duroux, T. Moos, T.L. Andresen, J.B. Simonsen, On the use
of liposome controls in studies investigating the clinical potential of extracellular vesicle-based drug
delivery systems – A commentary, J. Control Release. 269 (2018) 10-14,
[275] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat. Rev. Drug Discov.
4 (2005) 145-160, http://doi.org/10.1038/nrd1632.
[276] I.E. Allijn, B.M.S. Czarny, X. Wang, S.Y. Chong, M. Weiler, A.E. da Silva, J.M. Metselaar, C.S.P.
Lam, G. Pastorin, D.P.V. de Kleijn, G. Storm, J.W. Wang, R.M. Schiffelers, Liposome encapsulated
berberine treatment attenuates cardiac dysfunction after myocardial infarction, J. Control Release. 247
[277] S. Sindhwani, A.M. Syed, J. Ngai, B.R. Kingston, L. Maiorino, J. Rothschild, P. MacMillan, Y. Zhang,
N.U. Rajesh, T. Hoang, J.L.Y. Wu, S. Wilhelm, A. Zilman, S. Gadde, A. Sulaiman, B. Ouyang, Z. Lin, L.
Wang, M. Egeblad, W.C.W. Chan, The entry of nanoparticles into solid tumours, Nat. Mater. 19 (2020)
566-575, http://doi.org/10.1038/s41563-019-0566-2.
[278] B.M. Bell, I.D. Kirk, S. Hiltbrunner, S. Gabrielsson, J.J. Bultema, Designer exosomes as next-
generation cancer immunotherapy, Nanomedicine. 12 (2016) 163-169,
[279] Y. Yang, X. Tai, K. Shi, S. Ruan, Y. Qiu, Z. Zhang, B. Xiang, Q. He, A New Concept of Enhancing
Immuno-Chemotherapeutic Effects Against B16F10 Tumor via Systemic Administration by Taking
Advantages of the Limitation of EPR Effect, Theranostics. 6 (2016) 2141-2160,
[280] J. Wolfram, M. Ferrari, Clinical cancer nanomedicine, Nano Today. 25 (2019) 85-98,
http://doi.org/10.1016/j.nantod.2019.02.005.
[281] D.R. Beckford Vera, S.D. Fontaine, H.F. VanBrocklin, B.R. Hearn, R. Reid, G.W. Ashley, D.V.
Santi, PET Imaging of the EPR Effect in Tumor Xenografts Using Small 15 nm Diameter Polyethylene
Glycols Labeled with Zirconium-89, Mol. Cancer Ther. 19 (2020) 673-679, http://doi.org/10.1158/1535-
7163.MCT-19-0709.
[282] A. Hoshino, B. Costa-Silva, T.-L. Shen, G. Rodrigues, A. Hashimoto, M. Tesic Mark, H. Molina, S.
Kohsaka, A. Di Giannatale, S. Ceder, S. Singh, C. Williams, N. Soplop, K. Uryu, L. Pharmer, T. King, L.
Bojmar, A.E. Davies, Y. Ararso, T. Zhang, H. Zhang, J. Hernandez, J.M. Weiss, V.D. Dumont-Cole, K.
Kramer, L.H. Wexler, A. Narendran, G.K. Schwartz, J.H. Healey, P. Sandstrom, K. Jørgen Labori, E.H.
Kure, P.M. Grandgenett, M.A. Hollingsworth, M. de Sousa, S. Kaur, M. Jain, K. Mallya, S.K. Batra, W.R.
Jarnagin, M.S. Brady, O. Fodstad, V. Muller, K. Pantel, A.J. Minn, M.J. Bissell, B.A. Garcia, Y. Kang,
V.K. Rajasekhar, C.M. Ghajar, I. Matei, H. Peinado, J. Bromberg, D. Lyden, Tumour exosome integrins
determine organotropic metastasis, Nature. 527 (2015) 329-335, http://doi.org/10.1038/nature15756.
[283] M. Tsuchihashi, H. Harashima, H. Kiwada, Development of a pharmacokinetic/pharmacodynamic
(PK/PD)-simulation system for doxorubicin in long circulating liposomes in mice using peritoneal P388,
J. Control Release. 61 (1999) 9-19, http://doi.org/10.1016/S0168-3659(99)00103-0.
[284] A. Gabizon, R. Catane, B. Uziely, B. Kaufman, T. Safra, R. Cohen, F. Martin, A. Huang, Y.
Barenholz, Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin
encapsulated in polyethylene-glycol coated liposomes, Cancer Res. 54 (1994) 987-992.
[285] F. Martin, A. Huang, B. Uziely, B. Kaufman, T. Safra, Prolonged Circulation Time and Enhanced
Accumulation in Malignant Exudates of Doxorubicin Encapsulated in Polyethylene-glycol Coated
Liposomes, Cancer Res. 54 (1994) 987-992.
[286] J. Zhang, F. Leifer, S. Rose, D.Y. Chun, J. Thaisz, T. Herr, M. Nashed, J. Joseph, W.R. Perkins,
K. DiPetrillo, Amikacin liposome inhalation suspension (ALIS) penetrates non-tuberculous mycobacterial
biofilms and enhances amikacin uptake into macrophages, Front. Microbiol. 9 (2018) 1-12,
http://doi.org/10.3389/fmicb.2018.00915.
[287] C. Charoenviriyakul, Y. Takahashi, M. Morishita, A. Matsumoto, M. Nishikawa, Y. Takakura, Cell
type-specific and common characteristics of exosomes derived from mouse cell lines: Yield,
physicochemical properties, and pharmacokinetics, Eur. J. Pharm. Sci. 96 (2017) 316-322,
http://doi.org/10.1016/j.ejps.2016.10.009.
[288] K.W. Witwer, Extracellular vesicles versus synthetic nanoparticles for drug delivery, Nat. Rev.
Mater., http://doi.org/10.1038/s41578-020-00277-6.
[289] R. Munter, K. Kristensen, D. Pedersbaek, J.B. Larsen, J.B. Simonsen, T.L. Andresen, Dissociation
of fluorescently labeled lipids from liposomes in biological environments challenges the interpretation of
uptake studies, Nanoscale. 10 (2018) 22720-22724, http://doi.org/10.1039/c8nr07755j.
71
[290] P. Gangadaran, C.M. Hong, B.-C. Ahn, Current Perspectives on In Vivo Noninvasive Tracking of
Extracellular Vesicles with Molecular Imaging, Biomed. Res. Int. 2017 (2017) 1-11,
http://doi.org/10.1155/2017/9158319.
[291] H. Choi, D.S. Lee, Illuminating the physiology of extracellular vesicles, Stem Cell Res. Ther. 7
(2016) 55, http://doi.org/10.1186/s13287-016-0316-1.
[292] W. Luo, Y. Dai, Z. Chen, X. Yue, K.C. Andrade-Powell, J. Chang, Spatial and temporal tracking of
cardiac exosomes in mouse using a nano-luciferase-CD63 fusion protein, Commun. Biol. 3 (2020) 114-
[293] A.B. Keener, How extracellular vesicles can enhance drug delivery, Nature. 582 (2020) S14-S15,
http://doi.org/10.1038/d41586-020-01769-9.
[294] S.A.A. Kooijmans, S. Stremersch, K. Braeckmans, S.C. de Smedt, A. Hendrix, M.J.A. Wood, R.M.
Schiffelers, K. Raemdonck, P. Vader, Electroporation-induced siRNA precipitation obscures the
efficiency of siRNA loading into extracellular vesicles, J. Control Release. 172 (2013) 229-238,
[295] D.S. Sutaria, J. Jiang, O.A. Elgamal, S.M. Pomeroy, M. Badawi, X. Zhu, R. Pavlovicz, A.C.P.
Azevedo-Pouly, J. Chalmers, C. Li, M.A. Phelps, T.D. Schmittgen, Low active loading of cargo into
engineered extracellular vesicles results in inefficient miRNA mimic delivery, J. Extracell. Vesicles. 6
(2017) 1333882-1333882, http://doi.org/10.1080/20013078.2017.1333882.
[296] S. Bosch, L. de Beaurepaire, M. Allard, M. Mosser, C. Heichette, D. Chretien, D. Jegou, J.M. Bach,
Trehalose prevents aggregation of exosomes and cryodamage, Sci. Rep. 6 (2016) 36162,
http://doi.org/10.1038/srep36162.
[297] D.E. Murphy, O.G. de Jong, M.J.W. Evers, M. Nurazizah, R.M. Schiffelers, P. Vader, Natural or
Synthetic RNA Delivery: A Stoichiometric Comparison of Extracellular Vesicles and Synthetic
Nanoparticles, Nano Lett. (2021) acs.nanolett.1c00094-acs.nanolett.00091c00094,
http://doi.org/10.1021/acs.nanolett.1c00094.
[298] A. Vandergriff, K. Huang, D. Shen, S. Hu, M.T. Hensley, T.G. Caranasos, L. Qian, K. Cheng,
Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide, Theranostics.
8 (2018) 1869-1878, http://doi.org/10.7150/thno.20524.
[299] X.T.T. Dang, J.M. Kavishka, D.X. Zhang, M. Pirisinu, M.T.N. Le, Extracellular Vesicles as an
Efficient and Versatile System for Drug Delivery, Cells. 9 (2020) 2191-2191,
http://doi.org/10.3390/cells9102191.
[300] O.G. de Jong, D.E. Murphy, I. Mäger, E. Willms, A. Garcia-Guerra, J.J. Gitz-Francois, J. Lefferts,
D. Gupta, S.C. Steenbeek, J. van Rheenen, S. El Andaloussi, R.M. Schiffelers, M.J.A. Wood, P. Vader,
A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated
functional transfer of RNA, Nat. Commun. 11 (2020) 1113-1113, http://doi.org/10.1038/s41467-020-
14977-8.
[301] W.J. Goh, S. Zou, C.K. Lee, Y.-H. Ou, J.-W. Wang, B. Czarny, G. Pastorin, EXOPLEXs: Chimeric
Drug Delivery Platform from the Fusion of Cell-Derived Nanovesicles and Liposomes,
Biomacromolecules. 19 (2018) 22-30, http://doi.org/10.1021/acs.biomac.7b01176.
[302] Y.-H. Ou, S. Zou, W.J. Goh, S.Y. Chong, G. Venkatesan, M.G. Wacker, G. Storm, J.-W. Wang, B.
Czarny, G. Pastorin, E.C.Y. Woon, Micro cell vesicle technology (mCVT): a novel hybrid system of gene
delivery for hard-to-transfect (HTT) cells, Nanoscale. 12 (2020) 18022-18030,
http://doi.org/10.1039/D0NR03784B.
[303] J. Gilleron, W. Querbes, A. Zeigerer, A. Borodovsky, G. Marsico, U. Schubert, K. Manygoats, S.
Seifert, C. Andree, M. Stöter, H. Epstein-Barash, L. Zhang, V. Koteliansky, K. Fitzgerald, E. Fava, M.
Bickle, Y. Kalaidzidis, A. Akinc, M. Maier, M. Zerial, Image-based analysis of lipid nanoparticle–mediated
siRNA delivery, intracellular trafficking and endosomal escape, Nat. Biotechnol. 31 (2013) 638-646,
http://doi.org/10.1038/nbt.2612.
[304] B.S. Joshi, M.A. de Beer, B.N.G. Giepmans, I.S. Zuhorn, Endocytosis of Extracellular Vesicles and
Release of Their Cargo from Endosomes, ACS Nano. 14 (2020) 4444-4455,
http://doi.org/10.1021/acsnano.9b10033.
[305] E. Bonsergent, G. Lavieu, Content release of extracellular vesicles in a cell‐free extract, FEBS.
Lett. 593 (2019) 1983-1992, http://doi.org/10.1002/1873-3468.13472.
[306] R.L. Webb, E.E. Kaiser, S.L. Scoville, T.A. Thompson, S. Fatima, C. Pandya, K. Sriram, R.L.
Swetenburg, K. Vaibhav, A.S. Arbab, B. Baban, K.M. Dhandapani, D.C. Hess, M.N. Hoda, S.L. Stice,
Human Neural Stem Cell Extracellular Vesicles Improve Tissue and Functional Recovery in the Murine
Thromboembolic Stroke Model, Transl. Stroke Res. 9 (2018) 530-539, http://doi.org/10.1007/s12975-
017-0599-2.
[307] Z. Han, S. Liu, Y. Pei, Z. Ding, Y. Li, X. Wang, D. Zhan, S. Xia, T. Driedonks, K.W. Witwer, R.G.
Weiss, P.C.M. Zijl, J.W.M. Bulte, L. Cheng, G. Liu, Highly efficient magnetic labelling allows MRI tracking
72
of the homing of stem cell‐derived extracellular vesicles following systemic delivery, J. Extracell. Vesicles.
10 (2021), http://doi.org/10.1002/jev2.12054.
[308] L. Peruzzotti-Jametti, J.D. Bernstock, G. Manferrari, R. Rogall, E. Fernandez-Vizarra, J.C.
Williamson, A. Braga, A. van den Bosch, T. Leonardi, Á. Kittel, C. Benincá, N. Vicario, S. Tan, C. Bastos,
I. Bicci, N. Iraci, J.A. Smith, P.J. Lehner, E.I. Buzas, N. Faria, M. Zeviani, C. Frezza, A. Brisson, N.J.
Matheson, C. Viscomi, S. Pluchino, Neural stem cells traffic functional mitochondria via extracellular
vesicles to correct mitochondrial dysfunction in target cells, bioRxiv. (2020),
http://doi.org/10.1101/2020.01.29.923441.
[309] J. Silva, L.M.A. Murray, Y. Su, D.F. Mc Auley, C.M. O'Kane, A. Krasnodembskaya, Transfer of
mitochondria from mesenchymal stromal cells through extracellular vesicles improves alveolar epithelial-
capillary barrier in ARDS, European Respiratory Society, pp. 98-98,
http://doi.org/10.1183/23120541.LSC-2020.98.
[310] G. Ikeda, M.R. Santoso, Y. Tada, A.M. Li, E. Vaskova, J.-H. Jung, C. O’Brien, E. Egan, J. Ye, P.C.
Yang, Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell–Derived Cardiomyocytes
Restore Energetics of Ischemic Myocardium, J. Am. Coll. Cardiol. 77 (2021) 1073-1088,
http://doi.org/10.1016/j.jacc.2020.12.060.
[311] P. Wolf, The nature and significance of platelet products in human plasma, Br. J. Haematol. 13
(1967) 269-288, http://doi.org/10.1111/j.1365-2141.1967.tb08741.x.
[312] S. Aaronson, U. Behrens, R. Orner, T.H. Haines, Ultrastructure of intracellular and extracellular
vesicles, membranes, and myelin figures produced by Ochromonas danica, J. Ultrastruct Res. 35 (1971)
418-430, http://doi.org/10.1016/s0022-5320(71)80003-5.
[313] E.G. Trams, C.J. Lauter, N. Salem, Jr., U. Heine, Exfoliation of membrane ecto-enzymes in the
form of micro-vesicles, Biochim Biophys Acta. 645 (1981) 63-70, http://doi.org/10.1016/0005-
2736(81)90512-5.
[314] B.T. Pan, R.M. Johnstone, Fate of the transferrin receptor during maturation of sheep reticulocytes
in vitro: selective externalization of the receptor, Cell. 33 (1983) 967-978, http://doi.org/10.1016/0092-
8674(83)90040-5.
[315] C. Harding, J. Heuser, P. Stahl, Receptor-mediated endocytosis of transferrin and recycling of the
transferrin receptor in rat reticulocytes, J. Cell Biol. 97 (1983) 329-339,
http://doi.org/10.1083/jcb.97.2.329.
[316] L. Zitvogel, A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. Ricciardi-Castagnoli, G.
Raposo, S. Amigorena, Eradication of established murine tumors using a novel cell-free vaccine:
dendritic cell-derived exosomes, Nat. Med. 4 (1998) 594-600, http://doi.org/10.1038/nm0598-594.
[317] T. Pisitkun, R.F. Shen, M.A. Knepper, Identification and proteomic profiling of exosomes in human
urine, Proc. Natl. Acad. Sci. U S A. 101 (2004) 13368-13373, http://doi.org/10.1073/pnas.0403453101.
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Extracellular Vesicles As A Drug Delivery System A Systematic Review of Preclinical Studies

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Extracellular vesicles as a drug delivery system:A systematic review of pre-

Pol Escudé Martinez de Castilla, Lingjun Tong, Chenyuan Huang,

To appear in: Advanced Drug Delivery Reviews

Received Date: 18 February 2021

© 2021 Elsevier B.V. All rights reserved.

A systematic review of preclinical studies

e Department of Pharmacy, Faculty of Science, National University of Singapore, 117543 Singapore,

g Departments of Chemical and Biomolecular Engineering, and Biomedical Engineering, Faculty of

j Department of Pharmaceutics, Faculty of Science, Utrecht University, 3584 CG Utrecht, the

1These authors contributed equally

*Correspondence to: [email protected] (R.M.S.) and [email protected] (J.W.W.)

Number of words: 108

1.1 Types of EVs: exosomes, microvesicles and apoptotic bodies . . . . . . . . . . . . . . . . …….. 5

1.2 Functions of EVs ...................................................... 5

2. Systematic review of EVs as drug delivery systems in preclinical studies . .............. 7

2.1 Introduction of the systematic review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …. 7

2.2 Approaches for Systematic Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …. 8

2.3 Inclusion and exclusion criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ….. 9

3. Data analysis of systematic review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ……. 10

3.1. Categorization of selected publications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . … 10

3.2. Diving deeper into the tables: analytical interpretation. . . . . . . . . . . . . . . . . . . . . . . . . . ... 30

3.2.1. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …….. 30

3.2.2. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

3.2.3. Donor cell types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.2.4. Active pharmaceutical ingredients (API) associated with EVs . . . . . . . . . . . . . . . . . . . . . 32

3.2.5. API loading procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 33

3.2.6. Surface modification status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.2.7. Size and zeta potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.2.8. Estimation purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.2.9. Biodistribution studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3.2.10. Administration routes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.3. Clinical trials of EVs as DDS: on the way to reach patients . . . . . . . . . . . . . . . . . . . . . . . . 38

4. Liposomes versus EVs for drug delivery: heads up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4.2. Physical features, production and quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4.3. In vivo administration of EVs and liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.3.3. Complement activation-related pseudoallergy (CARPA) upon nanoparticle injection . . . . .42

4.3.4. Biodistribution profiles: passive vs. active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . … 43

4.3.5. Pharmacokinetics and pharmacodynamics (PK/PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

5.1.1. Challenges in production, isolation, purity and characterization . . . . . . . . . . . . . . . . . . . . 45

5.1.2. Misleading information on EV biodistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

5.1.3. Complications in upscaling, loading strategies and storage . . . . . . . . . . . . . . . . . . . . . . . 46

5.1.4. Lack of comparison with liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47

5.2.1. Active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

5.2.2. Improved circulation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

5.2.3. Upgraded loading efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

5.2.4. EVs in intracellular trafficking: Escaping the endosomal system . . . . . . . . . . . . . . . . . . . 48

5.2.5. Crossing the Blood-Brain Barrier (BBB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

5.2.6. Emerging EV platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49

5.3. Defining the niches of EVs in comparison to liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

6. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

1.1. Types of EVs: exosomes, microvesicles and apoptotic bodies

1.2. Functions of EVs

2. Systematic review of EVs as drug delivery systems in preclinical studies

2.2. Approaches for Systematic Review

2.3. Inclusion and exclusion criteria

3. Data analysis of systematic review

Digestive system cancer

Other cardiovascular disease