Journal of Controlled Release 272 (2018) 159–168

Contents lists available at ScienceDirect

Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review article

Nanotherapeutics in oral and parenteral drug delivery: Key learnings and T

future outlooks as we think small
⁎
Puneet Tyagi, J. Anand Subramony
MedImmune, LLC, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Nanotechnology ushered the field of medicine in to a new era. Miniaturization, increased surface area, and the
Nanotechnology unique physicochemical properties in the nano dimension were explored for new applications. Pharmaceutical
Immunotherapy industry picked up the technology and early success came fast for oral drug delivery through improvement in
Sustained release dissolution properties of the active molecules. Many products were launched using the nanocrystal technology
Nanorobots
on the oral side. Further development of polymeric nanoparticles led to wide spread research of nanocarriers for
EPR
parenteral delivery. While considerable efforts have gone in the last two decades for testing nanoparticles for
Immuno-oncology
Nanocrystals tumor targeting, delivery into tumors has remained challenging and suboptimal. Inadequate in vivo models that
Drug targeting didn't accurately reflect the age and vascularity of human tumors, and inability to reproducibly target ther-
API (active pharmaceutical ingredient) apeutic drugs to the tissue of interest due to intrinsic biodistribution of the particles and hence side effects,
limited the number of studies that advanced to the clinic. Our article addresses the questions commonly asked by
scientific researchers in nanomedicine: “Has nanoparticle technology yielded on its initial promise that scientists
predicted towards improving therapeutic index and avoid toxicity by delivering molecules to target tissues or
was it more of wishful thinking that had several roadblocks?” We answer this question by linking the relevance
of nanoparticles to cancer immunotherapy. The advent of immunotherapy has begun to show the potential
applicability of nanoparticles in a different light, to target the immune system. In this approach, nanoparticles
may positively influence the immune system rather than create the targeted “magic bullet”. Utilizing the in-
trinsic properties of nanoparticles for immune targeting as opposed to targeting the tumor can bring about a
positive difference due to the underlying complex cancer mechanisms that can potentially overlap with the
heterogeneous biodistribution of nanoparticles towards improving the acquired and innate immune responses. In
this review, we have followed the progress of nanotechnology in pharmaceutical applications with key insights
from oral and parenteral drug delivery, and how to modify our thinking to better utilize nanoparticles for
immuno-oncology. In contrast to conventional “local” tumor targeting by nanoparticles, we propose a new
mechanism whereby nanoparticles trigger priming of the T cells towards tumor destruction. The heterogenous
biodistribution of nanoparticles lends itself to stimulating immune cells systemically in a “global” manner and
with the right therapeutic combinations will be able to trigger tumor antigens to continually activate, retain
memory effects and destroy tumor cells.

1. Introduction functionality of APIs and excipients. Particularly for oral therapy, na-
noparticle technologies can be used to process APIs by controlling
Nanotechnology offers new possibilities for creating novel materials particle size through bottoms-up crystallization or top-down milling to
and for the development of unique pharmaceutical dosage forms and create unique nano-sized materials. The substantial improvements in
drug delivery systems. Nanoparticle systems are generally classified as the dissolution of oral APIs by nanoparticles has led to enhanced ab-
nano-sized active drug particles or nano sized substrates that can en- sorption and bioavailability [2]. For parenteral dosage forms, such as
capsulate active pharmaceutical ingredients (API) within, complexed or injections containing nanosuspensions, nanoparticles can increase the
conjugated, or synthesized in the nano-dimension. According to the volume of distribution and hence alter the pharmacokinetic (PK)
U.S. Food and Drug Administration (FDA), a nanoparticle's dimension is properties of active molecules [3].
1–100 nm [1]. Nanotechnologies have been used to improve the Here is what we know and believe nanoparticles can do:

⁎
Corresponding author at: One MedImmune Way, Gaithersburg, MD 20878, USA.
E-mail address: [email protected] (J.A. Subramony).

https://doi.org/10.1016/j.jconrel.2018.01.009
Received 6 December 2017; Received in revised form 11 January 2018; Accepted 11 January 2018
Available online 19 January 2018
0168-3659/ © 2018 Elsevier B.V. All rights reserved.
P. Tyagi, J.A. Subramony Journal of Controlled Release 272 (2018) 159–168

Nanoparticles offer improved dissolution capabilities for the APIs on System (BCS) [7] dictates the suitability of active pharmaceutical in-
the oral side and enable better partition of the APIs in the blood stream gredients as candidates for oral dosage form development. Poor drug
in the case of parenteral due to albumin and other binding mechanisms. solubility of APIs is a major concern that limits the pharmaceutical
Because many small-molecule drug candidates have potential toxicity development of oral dosage forms; about 40–60% of the early-stage
due to high serum concentration (Cmax) upon injection and during discovery molecules are poorly soluble [8,9]. Poor solubility can also
systemic circulation, encapsulating them in a nanoparticle delivery lead to erratic dissolution velocity and, eventually, unpredictable ab-
system allows their release be modulated via controlled release. sorption and poor bioavailability.
Similarly, the rapid clearance of an API on injection can be overcome by Reductions in particle size to micron and submicron levels have
suitable encapsulation and surface decoration of the nanoparticle with been adopted in the last couple of decades to enhance rate of dissolu-
appropriate functional groups for slow release and increased circula- tion [10]. In comparison with micronization, reduction to the nano-size
tion. Nanoparticle-based therapeutics can potentially decrease systemic has proven to be a more powerful approach because it increases sa-
toxicity and could improve efficacy by targeting a drug to a tumor site turation solubility and dissolution velocity. The relationship between
or by offsetting the drug's release [4]. Active drug targeting using na- the drug's saturation solubility and the particle size is explained by the
noparticles, another approach for preventing systemic toxicity in APIs, Ostwald–Freundlich eq. [11]:
involves anchoring the nanotherapeutics using a targeting ligand that Cs 2σV
can be an antibody fragment, peptide or a small molecule through log =
Cα 2.303RTρr
surface functionalization. Active targeting relies on the interaction of
targeting ligands with the overexpressed receptors (such as folates and where Cs is the saturation solubility, Cα is the solubility of the solid, σ is
transferrin) on the tumor cell surface. Active targeting helps with in- the interfacial tension of the substance, V is the molar volume of the
ternalization of the API and its uptake into the tumor. Several pub- particle material, R is the gas constant, T is the absolute temperature, ρ
lications have described the use of active targeting and the mechanisms is the density of the solid, and r is the radius.
of uptake [5]. The effect of particle size on solubility is not likely to be substantial
However, data from the descriptive literature presents a different for larger micron-sized particles, but it is pronounced for nano-sized
picture. Even though several years of research have been devoted to the materials [12,13]. Furthermore, increasing saturation solubility im-
use of nanoparticles for parenteral applications, the ability to create proves the concentration gradient between the gastrointestinal tract
reproducible tumor targeting nanoparticle has remained elusive due to and blood, boosting the drug's passive absorption.
various challenges. A recent review has looked into many of these Reducing particles to nano-size also increases the drug's dissolution
challenges in a systematic way [6]. While looking at these difficulties, velocity, as explained by the Noyes-Whitney equation [14]:
we discuss alternate ways of utilizing nanoparticles in immunotherapy dx DA
due to their intrinsic features during biodistribution rather than where = × (Cs − Ct )
dt hd
we would like them to go. We anticipate that the next wave of na-
notherapeutics in immuno-oncology would require particles that can where dx/dt is the dissolution velocity, D is the diffusion coefficient, A
tackle and participate in competing events that go on during tumor- is the surface area, hd is the diffusional distance, Cs is the saturation
igenesis, such as parallel processing of mechanistic pathways through solubility, and Ct is the concentration around the particles.
different cell types (macrophages, dendritic cells), and heterogeneous
signal transduction occurring away from the tumor in bone marrow and 2.1. Approaches to prepare nanocrystals for oral drug delivery
other places via killer cells for triggering positive immune responses.
In this review, we track the progress of nanotechnology in the last Nanocrystal preparations can be broadly categorized into those that
few decades (Fig. 1) which can be broadly classified into 4 different are top down, in which large particles are broken down by attrition or
waves and describe the proven utility of nanoparticle technologies for cavitation, and those that are bottom up, in which nanocrystals are
oral products, nanocarriers for parenteral delivery and active targeting, created by precipitation. Of the two approaches, the top-down ap-
and the next wave of utility in cancer immunotherapy. proach using milling is by far the most commonly used.

2.1.1. Top-down approach

2. Nanocrystals for oral delivery In this approach, large drug crystals are reduced to nano-size by a
milling process or high-pressure homogenization. The classical wet-
Oral dosage forms such as tablets and capsules have been the choice milling method is NanoCrystal Technology, wherein a ball mill, con-
of drug products over the years. Biopharmaceutical Classification taining beads made of ceramic, stainless steel, or glass, generate shear
force and impact to reduce the particle size of the drug suspension
[3,15]. To stabilize nano-sized particles, stabilizers such as particle
surface modifiers (surfactants) or hydro-colloids (hydroxyl propyl me-
thyl cellulose) are added, which coat the surface of the nanoparticles to
prevent crystal aggregation. NanoCrystal Technology was first used to
develop sirolimus tablets, which was FDA approved in 1999 [16]. Many
other drug products, such as fenofibrate (Tricor) and aprepitant
(Emend), were subsequently developed using the nano-milling tech-
nology. Table 1 shows the list of products developed with nano-sizing
technologies.
High-pressure homogenization is a high-energy disintegration pro-
cess that involves passing the drug suspension through an orifice under
Fig. 1. Evolution of nanotherapeutics. Nanocrystals (Table 1) were utilized for bioa-
vailability enhancement of oral drugs. Liposomes and other nanoparticle systems were
high pressure. Particle size can be reduced through particle collision
developed as nanocarriers to improve solubility, distribution, and reduce toxicity of drugs and/or cavitation [17]. Particle size reduction can be achieved by jet
(Table 2). While research continues on active targeting, current focus is more on deli- stream homogenizers, such as microfluidization (Microfluidizer, Mi-
vering macromolecules such as RNA and DNA. The future of nanotherapeutics is expected crofluidics Inc., USA), or by piston gap homogenization, where nano-
to be immune targeting by nanoparticle robots, influenced by new learnings in immune crystals are produced using pressures of up to 1500 bar. In the micro-
biology.
fluidization process, frontal collision of fluid streams under high

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Table 1
List of FDA approved oral drug products that utilize nanosizing technologies.

Drug Trade name/year of approval Indication Technology used

Sirolimus Rapamune/1999 prophylaxis of organ rejection in patients aged ≥ 13 years receiving renal Top-down milling (NanoCrystal
transplants Technology)
Aprepitant Emend/2003 Nausea and vomiting associated with cancer chemotherapy
Fenofibrate Tricor/2001 Hypercholesterolemia or mixed dyslipidemia
Megestrol acetate Megace ES/2005 Anorexia, cachexia or significant weight loss in AIDS
Fenofibrate Triglide/2005 Hypercholesterolemia or mixed dyslipidemia Top-down high-pressure homogenization
Griseofulvin Gris-PEG/2003 Ring worm infections Bottom-up precipitation
Nabilone Cesamet/2006 Nausea and vomiting associated with cancer chemotherapy Bottom-up precipitation

pressure inside the interaction chamber reduces particle size by gen- 2.1.3. Combination approach
erating shear forces, particle collision, and cavitation forces. Nekkanti A combination of a pretreatment (bottoms-up) approach followed
et al. evaluated the Microfluidics M110-P Microfluidizer for a BCS class by a high-energy (top-down) process is used in NanoEdge technology
II drug and observed that particle size reduction depends on various [27]. In the first step, crystals are precipitated, and the resulting sus-
process parameters, such as the number of homogenization cycles, pension is subjected to high-pressure homogenization. SmartCrystal
homogenization pressure, and stabilizer concentration [18]. At a con- technology combines several different processes based on drug prop-
stant homogenization pressure of 30,000 psi, increasing the number of erties. The advantage of this technology is that it can create particles
cycles reduced particle size significantly. Furthermore, particle size smaller than 100 nm. Such small nanocrystals are difficult to produce
reduction was significantly greater at 30,000 psi homogenization with the top-down approach alone. SmartCrystal technology uses spray-
pressure than 10,000 psi after 60 homogenization cycles. Increasing the drying, lyophilization, precipitation, or bead milling for the pretreat-
surfactant concentration from 10 to 12 mg/ml also played an important ment step, followed by the main treatment with high-pressure homo-
role in particle size reduction through particle stabilization. genization [28].
In piston-gap homogenizers, the aqueous drug surfactant dispersion
is forced by a piston through a tiny homogenization gap (5–20 μm) 2.2. Key learnings and future considerations from oral nanocrystal dosage
under high pressure. Gas bubbles (known as cavitation) are formed and forms
imploded when the suspension passes from a cylinder with a larger
diameter than the width of the resulting homogenization gap. The high Nanoparticle approach on the oral side has relied on the ability to
shear forces, the turbulent flow, and the powerful shockwaves gener- improve the dissolution properties of the API towards enhanced
ated by cavitation reduce the drug particle's size. PharmaSol's drug bioabsorption. Scalability has been easier on the top down nanosizing
nanocrystal technology is another piston gap homogenizer that is useful approach as evidenced by the number of products prepared by top-
for reducing the particle size of thermolabile drugs because it uses low down methods that are commercially available [23]. On the other hand,
vapor-pressure dispersion media (such as mixtures of water and ethanol the number of products prepared by bottom-up methods is very limited
or water and glycerol) for homogenization, which helps with processing as evidenced in. Table 1. Part of the reason for the challenges in the
at low temperatures because the amount of cavitation in the homo- bottom up approach might be due to the fact that controlled crystal-
genization gap is very small [19–21]. lization of the actives in the nanodimension due to precipitation
ContraSol™ uses nanotechnology to overcome the limitations of method is very sensitive to a number of variables that are fundamental
poorly soluble drugs. ContraSol™ blends a poorly soluble drug within a to crystal growth.
water-soluble excipient. On exposure to water, the blend generates The number of drug products using the nanocrystal technologies is
nanoparticles of the drug, which are stabilized by the soluble excipient continually on the rise over the years. Nanocrystals have accounted
[22]. for > 20% of FDA submissions since their invention in the 1970s. This
steady percentage indicates that the nanocrystal platform is versatile,
easy to scale up, safe, and effective. This proportion is expected to re-
2.1.2. Bottom-up approach main steady because nanocrystals are a promising tool for increasing
In this approach, the drug is dissolved in a solvent and then pre- the number of poorly soluble drugs in the drug development process In
cipitated by the addition of an antisolvent. Drawbacks to the bottom-up the five year period from 2010 to 2015, 29% of nanomaterial in-
approach include the use of solvents and lack of control of the solid vestigational new drug applications, and abbreviated new drug appli-
state of the crystals [23]. For example, the hydrosols technology has the cations received by the FDA were nanocrystals [29]. The only nano-
advantage of producing crystalline drug nanoparticles, but the drug material drug products for which the FDA received more applications
must be soluble in at least one solvent and the process requires organic were liposomes (more about this later), which accounted for 35% of
solvents that need to be removed. Auweter et al. developed the pre- nanomaterial submissions to the FDA. In the future, development of
cipitation technology, Nanomorph®, in which amorphous drug nano- targeted nanocrystals with enhanced therapeutic effects and “designer”
particles are obtained that have higher saturation solubility and a faster nanocrystals that have controlled crystal morphology will be vital to the
dissolution rate than crystalline nanoparticles [24]. However, conver- advancement of new dosage forms [29]. We would go one step further
sion to the crystalline state decreases bioavailability. Another category with the idea that designer nanocrystals may also help preserve specific
of bottom-up techniques is based on supercritical fluid technologies, in polymorphic form needed for improved bio pharmaceutics in humans.
which gas antisolvent recrystallization and rapid expansion of super-
critical solution are employed to generate nanocrystals. In gas anti-
3. Nanocarriers in parenteral drug delivery
solvent recrystallization, drug solution in an organic solvent is satu-
rated with supercritical fluid (such as supercritical carbon dioxide),
It is important to note that several commercial products have been
causing the drug to precipitate [25]. The other supercritical fluid
developed on the oral side based on nanoparticles technologies by
technique, rapid expansion of supercritical solution [26], uses the su-
improving absorption aspects of the API. For example, dissolution of
percritical fluid as a solvent. The drug precipitates when the pressure
APIs is integral to enhanced absorption and pharmacokinetics and that
decreases.
is the precise attribute that is being achieved using nanomilling

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Table 2
List of nanotherapeutics approved by FDA that utilize nanoparticle technologies for parenteral drug delivery. Those in clinical trials can be found in several recent references (see
references [6,39,40]).

Product name Formulation/drug encapsulated Indication Year of approval

Doxil Liposomes/doxorubicin HIV-associated Kaposi sarcoma, ovarian cancer, multiple myeloma 1995
DaunoXome Liposomes/Daunorubicin HIV-associated Kaposi sarcoma 1996
AmBisome Liposomes/Amphotericin B Systemic fungal infection 1997
DepoCyt Liposomes/Cytarabine Lymphomatous meningitis 1997
Visudyne Liposomes/Verteporfin Classic subfoveal choroidal neovascularization due to age-related macular degeneration, pathologic 2000
myopia, or presumed ocular histoplasmosis
Abraxane Albumin nanoparticles/ Breast cancer 2007
Paclitaxel
Exparel Liposomes/bupivacaine Post-surgical anesthesia 2011
Marqibo Liposomes/vincristine sulfate Philadelphia chromosome–negative acute lymphoblastic leukemia 2012
Onivyde Liposomes/Irinotecan Advanced (metastatic) pancreatic cancer 2015

technologies. The widespread interest of nanoparticle formulations on have a structure and lipid composition similar to cell membranes, and
the parenteral side came about due to the potential of improved drug they are nontoxic and nonimmunogenic because of their natural lipid
targeting to the site of tumors as well as improved solubility/partition composition [37]. Intravenously administered liposomes are cleared
in the blood stream without having to use several excipients. rapidly from the bloodstream because of opsonization. Coating the li-
Nanocarrier systems have been evaluated for parenteral drug delivery posomal surface with inert polymers, such as polyethylene glycol, slows
also due to the fact that they offer enhanced drug protection, controlled down clearance by allowing liposomes to remain in blood circulation
release, extended circulation, compared with their free-drug counter- for several days. Doxil was the first FDA-approved liposomal product
parts. (Table 2), and it features PEGylation (attaching poly ethylene glycol
Nanotechnology has been extensively tested for parenteral delivery chains) of liposome containing encapsulated doxorubicin [38].
of several molecules (Table 2). Some of these molecules have toxicity Micelles are 5–100 nm colloidal particles, often consisting of am-
because of uncontrolled systemic distribution or non-target effects, or phiphilic surface-active agents, which have two distinct regions, a hy-
they require localized delivery through nanoparticle approaches. For drophilic head group and a hydrophobic tail. At concentrations above
example, doxorubicin, an effective anticancer drug, has serious side the critical micelle concentration of the surface-active agents, ag-
effects, such as cardiotoxicity, and its use is therefore limited [30]. gregation and self-assembly occur, and micelles are formed [41]. Above
Encapsulation of doxorubicin in nanocarriers, such as liposomal and the critical micelle concentration, hydrophobic ends tend to arrange
polymer-mediated delivery systems, can potentially improve drug tar- themselves in an entropically favorable state and establish hydrogen
geting and increase serum residence time, and reduce their side effects. bond networks with the surrounding water [41]. Many micellar for-
FDA approved the first “nano” particle based delivery system, a 130 nm mulations are being tested in clinical trials.
albumin-bound paclitaxel nanoparticle, in January 2005 for the treat- Polymeric nanocarriers consist of a spherical polymeric matrix that
ment of breast cancer. Albumin has several unique features that makes encapsulates the drug. A typical example is poly (lactic acid-co-glycolic
it particularly appropriate as a vehicle for targeted drug delivery in acid) polymer based particles. However, other forms of nanoparticles
oncology. It is a natural carrier of systemic hydrophobic molecules, have also been considered for drug targeting [42]. such as dendrimers
such as hormones, and its noncovalent binding characteristics allow it are also under development [43–45].
to transport hydrophobic molecules to the cell surface [31]. Once al- The release of drug particles from nanocarrier upon parenteral in-
bumin is in close proximity to the cell surface, it can assist with jection can be explained as follows and is depicted pictorially in Fig. 2.
transcytosis of albumin-bound molecules. A preclinical study by Desai In most cases, the drug is evenly distributed throughout the nanocarrier
et al. showed that endothelial cell surface binding and transcytosis of matrix and slowly released by diffusion and/or erosion of the nano-
paclitaxel were markedly higher for the albumin-bound, nanoparticle carrier [46]. Diffusion-controlled release through porous structure can
form of paclitaxel compared with cremophor-based paclitaxel [32]. be explained by extending the well-known Higuchi equation
This study also demonstrated that tumor accumulation of paclitaxel 1/2
Dε
from the albumin-bound, nanoparticle form of paclitaxel was sig- Q = ⎡ (2CT − εCS )CS t⎤
nificantly higher than with cremophor-based paclitaxel. Albumin ⎣ τ ⎦
binding can also improve drug substance solubility. Since this study was Where Q is the amount of drug released at time t, D is the diffusion
published, several nanotherapeutic systems have been under in- coefficient of the drug in the polymer matrix, CT is the total con-
vestigation to explore albumin binding, but none has progressed as a centration and CS the solubility of the drug in the polymer matrix, ε is
drug product till now [33]. We will now look at the various nanocarrier the porosity of the matrix and τ-the tortuosity of the system. However,
types in the following sections. most of the current polymeric nanocarriers are degradable in nature
and have a decreasing surface area and thus some modifications to the
3.1. Types of nanocarriers above mentioned Higuchi equation is required [47]. Furthermore, sol-
vation of the drug therapeutic (once on the surface or out of the na-
Nanocarriers have a distinct advantage for drug delivery because of noparticle) has been suggested to be more important than diffusion
their unique composition, structure, and surface properties [34,35]. [48]. Single dose of a sustained release particle formulation can release
This diversity permits the carriers to be modified for different targets therapeutics for longer duration and hence reduce the frequency of
and achieve different circulation. The most common designs for nano- injections via the process of surface and bulk erosion eventually leading
carrier drug delivery systems include liposomes, micelles, polymeric to complete degradation of the particles.
nanoparticles, and dendrimers and Table 2 list examples of FDA-ap-
proved nanocarrier drug products. 3.2. Passive tumor targeting with nanocarriers
Liposomes are spherical vesicles made of lipid layers that can be
unilamellar or multilamellar based on the number of lipid bilayers [36]. Passive targeting of nanocarriers containing drug molecules to
Liposomes are an attractive platform for drug delivery because they tumor tissues, by exploitation of abnormal tumor physiology and

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Fig. 2. Comparison of pharmacokinetic profiles of con-

ventional immediate release (red) and sustained release
formulation (blue). Arrows in red and blue indicate the
time of dosing for conventional and sustained release for-
mulations, respectively. The enlarged section depicts the
interfacial diffusion limitations during release of active
from nanoparticle formulations. Cs, Ci, and Cb represent the
drug concentration at surface, interface, and boundary
layer. ks and kd are solvation and diffusion constants, re-
spectively. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version
of this article.)

vasculature, has been explored in preclinical models wherein pre- on the age of the tumor and variability between preclinical species
ferential accumulation of nanoparticles has been observed in tumors during the drug development process. Recently there was an editorial
[49]. In an early study, Matsumura and Maeda showed that proteins in [56] article that further questioned our understanding of “enhanced
the molecular weight range of 15,000–70,000 g/mol can effectively permeability” wherein they highlight other extravasation mechanism as
accumulate in a solid tumor [50]. Size is a significant factor in tumor a key contributor to nanoparticle accumulation in tumors. Wong et al.
accumulation and preventing diffusion back into the systemic circula- [57] and Ulmschneider and Searson [53] addressed the varying vas-
tion. In general, particles of around 400 nm are removed more slowly culator issue by quantifying the vascularity. However, the vascularity
from the circulation than larger ones [51]. However, along with particle index can vary between patients during a trial, and, hence, it is very
size, other characteristics of nanocarriers must be taken in to account to difficult to develop accurate correlations between experimental in-
achieve targeting such as surface charge/zeta potential, circulation time vestigative systems towards developing a controlled drug product/for-
etc. For example, instability of polymeric micelles in blood results in mulation that can deliver therapeutic quantities to the tumor site re-
their complete release within 15 min [52]. producibly to achieve a label claim for the product.
Preferential accumulation of nanocarriers in tumors tissues (also
known as passive targeting or enhanced permeation and retention, EPR 4. Actively targeted nanoparticles and gene delivery
effect) is attributed to the leaky vasculature of the tumor tissues [53],
which in turn results from imbalance of angiogenic factors at the tumor Active targeting of nanocarriers [58] has been attempted with the
site [54]. Discrepancy in angiogenic factors results in porous, dis- use of targeting ligands (peptides, small molecules or antibodies) that
organized vessels that lack tight junctions. In addition to a leaky vas- interacts with receptors on the surfaces of tumor cells based on the idea
culature, tumors do not have a well-developed lymphatic system, which that targeting ligands will serve as “zip codes” to send the drugs to the
leads to nanocarrier accumulation within the tumor tissue [55]. This appropriate tissue site. Once the nanocarrier containing the active
leaky vasculature and impaired lymphatic clearance mechanism is the molecule reaches the tumor site by systemic circulation, the receptor
supposed primary cause of passive targeting of nanocarriers in human and ligand interact with one another to enable uptake via endocytosis.
tumors [54]. However, this mechanism alone cannot be used to develop In oncology, particularly for the treatment of solid tumors, active tar-
robust drug products because the vascularity index can vary, depending geting is advantageous because it can lead to reductions in side effects

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involving nontumor cells [59]. Several studies have evaluated active amount of materials and hence with lesser side effect potential. The
targeting in preclinical models containing overexpressed tumor cell subsequent sections would highlight some of the key mechanistic dif-
receptors for folate and transferrin [34]. These targeted nanocarriers ferences between the two approaches.
have shown significant improvements in tumor cell cytotoxicity com- Immunotherapy treats diseases by inducing, enhancing, or sup-
pared with nontargeted nanocarriers [60]. Beletskii et al. [42] delivered pressing an immune response. Cancer utilizes complex regulatory net-
folate-conjugated doxorubicin loaded liposomes and observed high works to suppress immunity. Hence a multipronged approach is re-
accumulation in murine lung carcinoma (M109R-HiFR) cell line [61]. quired to overcome immune suppression. In contrast to conventional
In contrast, free doxorubicin diffused out of the cells quickly. This ac- tumor targeted nanoparticles, which require targeting to tumors by
cumulation is especially important because many tumor cells exhibit using receptors (such as αVβ3 integrin) overexpressed on tumors, na-
multidrug resistance by rapid efflux of the drugs. nomedicine for immuno-oncology will be used to effectively target the
However, the extent of targeting depends on various factors, in- immune cascade [67] in bringing about antitumor response either by
cluding the specificity of the targeted receptor to tumor cells versus amplifying immunoresponse or by improving costimulatory signals.
non-tumor cells and availability of the receptor on the tumor cells. The Nanoparticles have the potential to improve immune response
general concentration distribution of the receptor on the tumor site vs [68,69] and modulate receptor clustering [55]. In recent years, im-
non tumor location is also important to promote active targeting. munotherapy has become great interest due to its promise to treat
Furthermore, cancer cells can also alter receptor expression over time, various forms of cancer [70]. Results from anti CTLA-4 and anti-PD-1/
resulting in lack of targeting [62]. Even if the nanocarriers accumulate PDL-1 antibody therapies have brought forward the power of im-
preferentially at the tumor site, delivering the drug might be challen- munotherapy through immune targeting and T Cell activation [71]. Key
ging because endosomes (vesicles pinched off from the cell membrane differences between immune targeting and tumor targeting is that in
into the cytoplasm) play a role in breaking down entrapped materials the former immune systems is primed through a number of mechanistic
once the tumor internalizes the nanocarrier [60]. For nanotherapeutics, pathways. Mechanisms by which one can prime and fine tune activation
understanding the translational aspects during drug development (from of T cells and antigen presenting cells (APC) are therefore very im-
in vitro, ex vivo, to preclinical studies to studies in humans) as well as portant. Approaches to augment immune response will involve ampli-
biology of the membranes/cells such as receptor density, volume of fying costimulatory signals, block immune suppression as well as op-
distribution of the particle, stability of the active molecule, are im- timize output from primed T cells. Furthermore, sustained activation
portant for successful development as drug products. The configuration from T cells can also be obtained by using co-stimulatory targets that
of the ligand with respect to the active molecule in terms of binding and can prolong the activation. In the literature, researchers have used
interaction of the ligand with the receptor due to conformational ro- OX40 [72] and GITR [73] agonists for costimulatory effects.
tations and docking can also play a significant role in internalization.
Nanoparticles have shown great potential for delivery of genetic 5.1. Methodology for immune targeting by nanotherapeutics
material including DNA and mRNA. Nanoparticles can protect genetic
material from degradation in vivo and can also help in endocytosis and While creating nanomedicines for immuno-oncology, the nano-
endosomal escape of the material. Many nanoparticle based systems particles containing immune activation agents will influence and
have been assessed for gene delivery [63]. In 2008, CALAA-01 was the trigger various components of the immune cascade to bring about
first nanoparticle to be approved for cancer clinical trials to treat pa- collective antitumor activity from various locations. This mechanism of
tients with solid tumors that were refractory to standard-of-care priming immune cells that are far from the disease site/tumor tissue to
therapies [63]. Since then different platforms including lipid nano- bring about antitumor response is a new approach that can fit well with
carriers, polyethyleneimine nanoparticles, and cyclodextrin nano- the increased volume of distribution of nanoparticles and their het-
particles have been tested in humans to evaluate the safety of gene erogenous bio-distribution upon injection which is an intrinsic property
delivery. of nanoparticulate systems. Choosing the right immuno- oncology agent
With regard to some new entrants in the nanoparticle field, various is key to bring positive aspects of this approach. In the immune cascade
nanostructured materials, such as carbon nanotubes [64], silica nano- routine, nanoparticles would trigger various cell types such as T-cells,
particles [65], and 3DNA nanostructures [66] of different shapes, are memory cells or dendritic cells to provide collective antitumor re-
currently under evaluation for drug delivery and other nanomedicinal sponse. Nanoparticles are also known to have a propensity to be taken
uses, and they can serve as future flag bearers for nanomaterials. These up by macrophages, get deposited in bone marrow and spleen to elicit
promising nanostructured materials are worthy of future scientific at- immune production and antitumor immunity. This phenomenon of
tention because of their scalability. bringing specific immune-activation by nanotherapeutics to bring about
cancer immunity is akin to “cooperative action from different locations”
5. Nanomedicines for immunotherapy or nanoparticles working as catalysts from various organs/locations to
trigger immune response. Nanoparticles have the potential to bring
The recent trends in the treatment of cancer is moving from a tra- about these effects when used with the right molecules and hence ef-
ditional chemotherapy towards cancer immunotherapy, whereby we fectively address the complex cancer regulatory network. Both innate
are maximizing the power of the immune system to fight cancer. and acquired immune responses can be improved in this mechanism.
Therefore, we need to look at the nanoparticle opportunity in a dif- The tunability aspects of drug release from nanoparticles, ability to load
ferent light for cancer immunotherapy and possibility to utilize and different type of agonists/antagonists, and co-stimulatory molecules
leverage what we know about their intrinsic properties particularly, makes nanoparticles as ideal tools for immune targeting.
their ADME. In order to improve the success rate of nanomedicine in Another aspect of the nanoparticle approach for immuno-oncology
oncology, we must first adapt the utility of nanoparticles to the un- is that specific combinations and “fine tuning” of the composition can
derlying requirements of tumor biology and address what are those enable a rapid high throughput screening using appropriate ex vivo cell
aspects that we can positively influence matching the intrinsic physi- lines to measure and quantify various immune cascade signals (im-
cochemical aspects of nanoparticles. munomonitoring). Since the focus of such studies would be to better
For instance, in traditional chemotherapy, high concentrations of understand cellular uptake and understanding immune stimulatory
the active is required to inhibit tumorigenesis with potential side effects signals, the methodology is very different than the previous studies
(both targeted and non-targeted). In the new immuno-oncology ap- required for passive targeting and tumor pharmacokinetics that relied
proach, the role of nanoparticles would be to effectively modulate the on in vivo models most of them without a common baseline and hence
immune system to elicit anti-tumor response while using smaller leading to clinical translational challenges. Thus, immuno-

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nanomedicines can be designed to target components of immune signaling pathway. Magnetoplasmonic nanoparticles combine the use
system and help the immune system to effectively destroy cancer cells. of magnetism to deliver a small amount of force and plasmonic gold
Also, once activated, a continuous production of T cells can be achieved shell that can be used for imaging. Seo et al. have also shown that by
by memory of the immune system [74]. The approach bears a different force loading, Notch receptors can be unfolded, resulting in the ex-
outlook to treating cancer and can be advantageous in the future as our posure of the buried proteolytic site. Unfolding of the Notch receptor
understanding of immune system and its responses to antitumor treat- and exposure of the proteolytic site is an important step in activation of
ments are progressing rapidly. Notch. These studies depict the potential of using nanoscale force to
Many strategies to target cancers via immune system are under in- alter and evaluate cell signaling pathways. Furthermore, studies such as
vestigation currently. Li et al. [75] have developed a vaccine that by Seo et al. [82] point to multiple innovative applications of magnetic
combines Toll-like receptor (TLR) agonists and antigen-carrying lipid nanoparticles. Although these technologies are in their infancy, they
nanocapsules. The combination vaccine elicited a long-lived memory T hold immense potential for the future of nanotechnology in cancer
cell response in the lung mediated by the APC. Cho et al. [76] de- immunotherapy.
monstrated that dendritic cells can take up superparamagnetic iron
oxide nanoparticles covered with ZnO shell and elicit an anti-tumor
immunity. Li et al.’s [77] work with alpha-alumina (α-Al2O3) nano- 5.3. Nanorobots and molecular machines
particles indicated that α-Al2O3 based cancer therapeutic vaccine can be
used for the delivery of antigens to the APCs. Their findings also sug- Advances in microprocessing to submicron scale have enabled re-
gested that α-Al2O3 can boost the antitumour efficacy of tumor-derived searchers to consider designing molecular machines at a nanoscale.
autophagosomes to deliver antigens to T cells. Min et al. delivered Although molecular machines are conceptual at this stage, they have
tumor-specific proteins to APCs and demonstrated a significant im- potential medical applications in early detection and treatment of dis-
provement in the cytotoxic T cell response [78]. The utility of nano- eases via imaging, sensing, and drug delivery. These molecular ma-
particles for immune targeting has come from the ability of nano- chines in the nano dimensions have the potential to bring innovative
particles for signal transduction which is described in the next section. solutions to healthcare because of their intrinsic biodistribution, disease
recognition and signal transduction properties.
Douglas et al. have developed a nanorobot using DNA strands that
5.2. Nanoparticles for signal transduction activation rearrange their conformation and release the drug load when they reach
the target cells [83]. Freitas et al. use a synthetic material to prepare
Nanotechnology is being explored for its ability to activate signal nanorobots (which they call “pharmacytes”) that can target selected
transduction in many receptors. Receptor clustering is required for the cells and travel through cell membranes [84]. Wu et al. developed a
activation of many receptors that play important roles in the develop- tubular nanorocket made of chitosan and sodium alginate polymers
mental process by binding to proteins such as cytokines and growth with highly efficient driving force [85]. The motion and direction of the
factors [79]. Such receptors can be activated by applying mechanical nanorocket can be adjusted with an external magnetic field. Ma et al.
forces to physically promote clustering and lead to signal transduction have developed a Janus nanomotor using mesoporous silica nano-
[80]. particles smaller than 100 nm [86]. The nanoparticles' high porosity is
Using nanotechnology to manipulate individual receptors, Mannix used for drug loading.
et al. [80] demonstrated that nanoparticles can activate signal trans- Nanorobots can help trigger the immune system to recognize cancer
duction in cells. Mannix et al. triggered the receptor clustering of FcεRI cells and destroy them. Immune targeting nanocarriers can be more
– IgE receptor-antibody complexes present on mast cells to induce effective because the intrinsic immune cell cytotoxicity tend to be tar-
calcium secretion by using superparamagnetic nanoparticles (30 nm) geted to signals from the tumor specific antigens. In this way nanor-
covalently conjugated to dinitrophenyl-lysine ligands. Activation of obots could both sense and eliminate cancer cells. Immune targeting
such nanoparticle mediated receptor clustering can open up new ave- based therapy is not expected to be repetitive as traditional che-
nues to control cellular responses and control cell metabolism [80]. motherapy since the immune system has memory effects to sustain the
Development of such nano-technologies also have potential to be effect via continued generation of T cells that can prevent tumor re-
combined with other segments such as tumor diagnosis [81]. Seo et al. occurrence [74]. The toxicity to healthy cells from chemotherapy can
[82] used magnetoplasmonic nanoparticles to activate Notch receptor be overcome by development of a nanorobot that can distinguish cancer

Fig. 3. Schematic showing possible mechanism by which nanorobots trigger priming of T cell towards tumor destruction. Upon systemic circulation, nanoparticles can interact with
macrophages and dendritic cells to activate T cells and also can unleash natural killer cells based on their biodistribution in the bone marrow. Some of nanoparticles previously localized
in tumors during biodistribution may pick up tumor antigens and can further accumulate in lymph nodes to continually improve immune response via dendritic cell uptake. Nanorobots
can also trigger immune response by identifying tumor antigens and stimulating immune cells systemically.

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Similar to fixed dose combinations in small molecule oral therapy,

particularly for cardiovascular and metabolic disorders, cancer im-
munotherapy in the near future would include mixture of various
combinations including antibodies, small molecules, and cancer vac-
cines that would bring about synergistic effects and improved immune
response towards tumor eradication. Nanorobots with unique surface
functionalization capabilities will have the ability to support multiple
drug combinations. The use of nanoparticles in obtaining antigen pre-
sentation as well as priming of a T-cell towards producing adaptive
immune response will be the key. Ability to mix and match various
receptor targeted or antitumor response inducing nanoparticles will
further provide flexibility to the utility of nanomedicine for immuno-
oncology (Fig. 4). Genetic test including biomarker and tumor imaging
will be used towards carrying out designing of the right proportion of
nanoparticles for optimum response. Precision medicine and digital
data analytic tools will help in designing the right combination and
dose for patients. Nanorobots, as tiny devices, can couple important
aspects of sensing/diagnosis to trigger the release of the right immune
function entity and bring about the best mechanism needed for anti-
tumor response. Versatile nanoparticles systems with these features that
Fig. 4. Schematic showing flexibility for developing immunotherapy combinations based are scalable will certainly have an advantage towards commercializa-
on patient parameters such as tumor phenotype and tumor biomarker information. tion.
Developability and scale up of the nanotherapeutic system while
cells from healthy cells or trigger improved immune response in col- paying attention to critical quality attributes, content uniformity in
lective ways as described in the next paragraph. loading/attaching of the therapeutics in the nanosystem, and stability
Immune targeted nanotherapeutics (nanorobts) (Fig. 3) would of the nanotherapeutics are other important aspects to consider in
comprise nanoparticles targeting the antigen presenting cells (such as bringing nanomedicines to patient from a commercial perspective.
dendritic cells), which are predominantly distributed in the body in
unleashing attack against cancer cells through antigen presentations. 7. Conclusions
Nanorobots containing antibodies intended for tumor cell receptors can
be targeted through dendritic cells. In a parallel event, sensing moieties With the advent of nanotechnology in the last few decades, the
on nanorobots can identify tumor specific antigens and trigger the na- projected future of new applications towards healthcare is expected to
norobot to interact immune cells to trigger a response. In this way offer many improvements for human life and drug delivery in parti-
nanorobot containing sensors can help to act as GPS (global positioning cular. However, if nanomedicines are to realize their full potential, a
system) enabled particle for optimum immune modulation and also act few impediments must be addressed right now. For drug delivery, for
in a closed loop fashion with sensing and delivering a therapeutic (see example, a deeper understanding of the biology and development of
graphical abstract). appropriate disease models and the interaction of cells with nano-
Natural killer (NK) cells originate in the bone marrow and they are carriers is needed, PK characteristics and the fate of nanocarrier in vivo
body's defense mechanisms against cancer cells. Immune targeting na- is also critical to develop PK/PD correlations for immunotherapy while
noparticles may also end up in bone marrow which can then trigger the using nanoparticles.
release of NK cells for destroying the cancer cells in a collective me- The winner of the 2016 Nobel Prize in chemistry, Dr. Bernard L.
chanism. Jang et al. [87] reported the use of iron oxide (Fe3O4) mag- Feringa, envisioned many possible biomedical applications for mole-
netic nanoparticles to trigger natural killer cells to target tumor sites in cular machines, saying “It's a bit early days, of course, but once you are
an early demonstration of a nanorobot. able to control movement, all sort of things are possible. Think about
tiny little robots that the doctor would inject into your bloodstream,
and that would go search for a cancer cell or deliver a drug” [91]. We
6. Future outlook hope that nanotechnology will disrupt cancer immunotherapy with new
treatment possibilities very soon in the near future….
The key to designing future nano delivery systems from a precision
medicine perspective involves understanding specific individual tumor Author contribution
pathophysiology and how nanoparticle based drug delivery systems
would interact with the biology and create immune stimulatory re- Anand Subramony conceived the manuscript idea. Puneet Tyagi and
sponses. Using a disease driven evaluation approach helps to better Anand Subramony wrote the manuscript.
bring about the clinical translational needs of the delivery system.
Understanding the tumor phenotype and variability is critical for better Competing financial interest statement
translational results. Biomarker development and use of companion
diagnostics are important for patient stratification and to assess the The authors declare no competing financial interests.
right outcome parameters. Circulating tumor DNA [88] and tumor cells
[89] are two such tools that can help with patient stratification. Digi- Acknowledgment
tally enhanced immuno monitoring of data at the RNA, protein, and
cellular level together with data collection and mining will help re- Editorial support was provided by Deborah Berlyne.
searches to pick and choose which combinations to adopt, test, and
develop predicitive algorithm to support future clinical and transla- References
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