ExRNA

Fu et al. ExRNA (2019) 1:24

https://doi.org/10.1186/s41544-019-0024-y

REVIEW Open Access

Recent progress in microRNA-based

delivery systems for the treatment of
human disease
Yong Fu1, Jiangning Chen1,2 and Zhen Huang1*

Abstract
MicroRNAs (miRNAs) are naturally occurring, small non-coding RNAs that mediate posttranscriptional regulation. Based
on the level of sequence complementarity, miRNAs lead to the degradation of target mRNAs or the suppression of
mRNA translation, thereby inhibiting the synthesis of proteins and achieving the regulation of genes. miRNAs, which
exhibit tissue- and temporal- specific expression, are important negative regulatory RNAs that decrease the levels of
other functional genes. miRNAs play a crucial role in disease progression and prognosis and thus exhibit potential for
developing novel therapeutics. Due to the instability of miRNAs and their complex environment, including degradation
by nucleases in vivo, the safety and effectiveness of miRNA delivery has become the focus of recent attention.
Therefore, we discuss some representative advances related to the application of viral- and nonviral-mediated miRNA
delivery systems and provide a new perspective on the future of miRNA-based therapeutic strategies.
Keywords: microRNA, microRNA delivery, Viral vectors, Nonviral vectors

Background Briefly, RNA polymerase II transcribes miRNA genes,

MicroRNAs (miRNAs) comprise a group of small leading to the formation of long precursor transcripts
non-coding RNAs 18~25 nucleotides (nt) in length that named primary miRNAs (pri-miRNAs), which have
posttranscriptionally regulate gene expression via bind- stem-loop structures consisting of hundreds of nucleo-
ing to the 3′-untranslated regions (3′-UTRs) of target tides. In the nucleus, each pri-miRNA is processed by
gene mRNA [1, 2]. Most miRNAs have highly conserved ribonuclease Drosha into a 70- to 100-nt hairpin struc-
sequences and are tissue- and temporal-specific [3]. ture, denoted a premiRNA. Then, the premiRNA is trans-
Reports have demonstrated that miRNAs participate in ported into the cytoplasm by a shuttle system composed
different physiological responses, including development, of Exportin 5 and Ran. There, each premiRNA is further
organogenesis, viral defense, hematopoietic processes, cleaved into a double-stranded miRNA duplex containing
cell proliferation/apoptosis and fat metabolism [4–8]. In 22 nt by Dicer and each mature miRNA strand binds to
1993, the first miRNA known as lin-4 was discovered in the miRNA-induced silencing complex (miRISC); how-
the nematode Caenorhabditis elegans via genetic screen- ever, the antisense miRNA strand (also known as
ing [9]. This small RNA can suppress the expression miRNA*) is subsequently degraded. The miRISC complex
level of nuclear protein LIN-14 and thus regulates the containing a mature miRNA strand can bind to the
development of nematodes [10]. Since that study, large 3′-UTR of target gene mRNA. This specific binding
numbers of miRNAs have been discovered in humans, between miRNA and target mRNA leads to the repression
mice, zebrafish, fruit flies, Arabidopsis thaliana, rice and of protein synthesis and the subsequent degradation of the
other animals and plants. targeted mRNA [11] (Fig. 1).
Usually, miRISC recognizes mRNA through comple-
mentary base pairing of the miRNA with the target gene
* Correspondence: [email protected]
mRNA. Under some circumstances, the binding between
1
State Key Laboratory of Pharmaceutical Biotechnology, School of Life miRISC and target gene mRNAs does not require per-
Sciences, Nanjing University, Nanjing 210093, Jiangsu, China fect pairing [12]. Moreover, reports have indicated that
Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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Fu et al. ExRNA (2019) 1:24 Page 2 of 14

Fig. 1 Schematic illustration of the biogenesis and function of miRNA

miRNA can also bind to the 5′-UTR of target genes to other plasma components such as high-density lipopro-
[13]. The binding of miRISC to an mRNA can lead to ei- tein (HDL) particles and RNA-binding proteins [19, 20].
ther the repression or promotion of translation, although These circulating miRNAs can enter recipient cells and
the latter is quite rare [14]. decrease the protein levels of target genes [21].

Tissue and temporal specificity Cross-kingdom regulation

The expression of most miRNAs occurs in a tissue- and Emerging evidence has uncovered the ability of small
temporal- specific manner [3]. Recently, miRNA expres- non-coding RNAs to transform from one species to other
sion profiling studies of multiple tumor types have species. Professor Zhang and his team revealed an import-
revealed that aberrantly expressed miRNAs are beneficial ant function of miRNA: cross-kingdom regulation [22].
for the classification, diagnosis, staging, and prognosis of Their results revealed that exogenous plant miRNAs could
disease [15]. The analysis of sequencing data from 27 be detected in both tissues and sera from different animals
different organs/tissues of Arabidopsis also proved that after the oral intake of plants. Subsequently, miRNA-2911,
most miRNAs are widely expressed, whereas a fraction an atypical honeysuckle-encoded miRNA, was found to
of miRNAs exhibit tissue-specific expression patterns directly target various influenza A viruses, inhibiting virus
[16]. replication and ultimately rescuing weight loss in viral
infected mice [23]. In 2017, Professor Zhang and co-
Circulating miRNA workers again reported that plant miRNAs enriched in
Recent research has reported that a large number of larval beebread regulated the development of honeybee
stable miRNAs derived from various organs/tissue exist caste [24]. Interestingly, a recent report by Saima et al.
in body fluids, and these miRNAs are promising as suggested the potential cross-kingdom regulation of
novel biomarkers for the diagnosis of cancer and plant-derived miRNAs and indicated that miRNAs from
other immune-related diseases via expression profil- the parasitic plant Cuscuta campestris could target the
ing. miRNA-21 was the first miRNA discovered in mRNAs of host Arabidopsis thaliana, leading to mRNA
serum [17]. In addition to working inside cells, miR- cleavage, which ultimately inhibited mRNA accumulation
NAs also communicate remotely in the form of circulat- [25]. These new modes of cross-species regulation may be
ing miRNAs [18]. Emerging evidence has indicated that involved in symbiotic and pathogenic relationships among
circulating miRNAs are localized in microvesicles or bind different kinds of organisms [26, 27].
Fu et al. ExRNA (2019) 1:24 Page 3 of 14

As mounting reports document that miRNAs work as oligonucleotides, methylphosphonate-containing oligonu-

extensive regulators of various types of physiological activ- cleotides, boranophosphate-containing oligonucleotides,
ity (e.g., hematopoiesis, tumorigenesis, tumor metastasis, 2′-O-methyl-(2′-O-Me) or 2′-O-methoxyethyl oligonucleo-
fat metabolism and intestinal mucosal homeostasis), inter- tides (2′-O-MOE), 2′-fluoro oligonucleotides (2′-F), locked
est in developing miRNA-based medicine has dramatically nucleic acid (LNA) oligonucleotides, peptide nucleic acids
increased [28–31]. However, the half-life of miRNAs is (PNAs), phosphorodiamidate morpholino oligomers
short because of the presence of nucleases [32]. Moreover, (PMOs) and other chemical modifications, such as Cy3-,
due to their polarity, miRNAs have difficulty passing cholesterol-, biotin- and amino-modified oligonucleotides
through the phospholipid bilayer cell membrane; thus, (Fig. 2).
miRNA cannot rapidly penetrate the vascular endothe- Phosphorothioate-, methylphosphonate-, or borano
lium and is retained in blood storage organs, including the phosphate-containing oligonucleotides substitute a sul-
liver and spleen, and it is ultimately excreted by the fur, methyl, or borano group, respectively, for the
kidneys. To solve this problem, a large number of vectors α-oxygen of the phosphate, in an attempt to overcome
have been developed to deliver miRNAs. Here, we provide the stability issue [34].
novel insight into the promise of miRNA-based The introduction of a 2′-O-methyl or 2′-O-methoxyethyl
therapeutic approaches and the development of viral and group to the ribose moiety of a phosphorothioate oligoribo-
nonviral vectors, including therapeutic applications for nucleotide dramatically enhances binding stability and
modified miRNAs and the challenges of vector protects oligonucleotides from nuclease degradation.
construction. 2′-fluoro-oligoribonucleotides contain a fluorine molecule
bound to the 2′-oxygen of the ribose [35].
Therapeutic approaches involving miRNA LNAs are RNA analogs that introduce a 2′,4′ methy-
Usually, naked RNA is highly susceptible to degradation by lene bridge in the ribose to form a bicyclic nucleotide
abundant ribonucleases in the blood and to phagocytosis [36]. PNA is an artificially synthesized polymer similar
by the reticuloendothelial system (RES). Chemical modifi- to DNA or RNA that is composed of repeating
cations can increase the stability of oligonucleotides for in N-(2-aminoethyl)-glycine units linked by peptide bonds
vivo delivery. Antisense oligonucleotide (ASO) technology [37]. PMOs contain morpholine rings that are linked
was introduced for functionally studying miRNA, and the through phosphorodiamidate groups [38].
ASOs that are used to silence miRNA are called Terminal chemical modifications, including Cy3-,
anti-miRNA oligonucleotides (AMOs) [33]. Chemical cholesterol-, biotin- and amino-modified oligonucleo-
modifications include phosphorothioate-containing tides, can increase the stability and tracer function of

Fig. 2 Chemical modifications improve stability, biodistribution, cellular uptake and delivery efficiency and increase the tracer function of
oligonucleotides. (0) Unmodified RNA; (1) phosphorothioate-, (2) methylphosphonate-, or (3) boranophosphate-containing oligonucleotides
containing a sulfur, methyl, or borano group, respectively; (4) 2′-O-methyl, (5) 2′-O-methoxyethyl, (6) or 2′-fluoro introduced into the 2′ oxygen of
the ribose; (7) LNAs; (8) PNAs; (9) PMOs; and terminal chemical modifications, including (10) Cy3-, (11) cholesterol-, (12) biotin- and (13) amino-
modified oligonucleotides could increase the stability and tracer function of oligonucleotides for in vivo delivery
Fu et al. ExRNA (2019) 1:24 Page 4 of 14

oligonucleotides for in vivo delivery [39, 40]. In practical RNAs that bind to the seed region, which allows them
applications, multiple modifications are used together to to block a family of miRNAs containing the same seed
increase the stability, delivery and cellular uptake effi- sequence. As competitive inhibitors, miRNA sponges
ciency of oligonucleotides in vivo. exhibit similar inhibition efficiency with short nucleotide
To change the expression levels of target genes, fragments.
miRNA-based therapies include the following two types:
(a) miRNA suppression therapy when the target gene is miRNA replacement therapy
downregulated and (b) miRNA replacement therapy miRNA mimics are synthetic double-stranded miRNA-like
when the target gene is downregulated (Fig. 3). RNA molecules that can simulate endogenous miRNAs
and bind to target gene mRNA, which ultimately leads to
miRNA suppression therapy posttranscriptional repression.
miRNA suppression therapy can remove miRNA sup- miRNA agomirs are artificial double-stranded miRNA
pression of a target mRNA, thus increasing the mRNA mimics with more chemical modifications. The antisense
expression level. AMOs bind to the miRNA sense strand of an agomir has the same modification as that of
strand, block interactions between miRISC and its target the antagomir. Compared with miRNA mimics, these
mRNA, prevent the degradation of the mRNA, and thus chemical modifications enhance the stability and activity
allow the mRNA to be translated. To improve the inhib- of miRNA agomirs. Therefore, agomirs can also be used
ition efficiency, multiple chemical modifications are ap- to upregulate their corresponding miRNAs in special
plied to enhance the affinity and stability of AMOs, tissues and to investigate the biological function of
including miRNA inhibitors and miRNA antagomirs. miRNA in vivo.
miRNA inhibitors (also denoted as anti-miRNAs) are miRNA precursors (also known as pre-miRNA) are
single-stranded RNA molecules. These anti-miRNAs can chemically modified single-stranded RNA fragments that
specifically bind to endogenous miRNA and abolish its are synthesized to simulate mature miRNAs. These
activity. miRNA inhibitors are mainly used in vitro in miRNA precursors are transfected into cells via a com-
combination with Lipofectamine transfection reagent to mercial reagent or electroporation similar to siRNAs.
investigate the biological function of miRNA via “los- After entering cells, miRNA precursors are cleaved by
s-of-function” experiments. the Dicer enzyme and transformed into mature miRNAs.
Antagomirs are single-stranded RNA molecules with Therefore, pre-miRNAs can be used to investigate the
specific chemical modifications. 2-Phosphorothioates are biological function of miRNA via “gain-of-function”
introduced at the 5′ end and the cholesterol group, and experiments.
4-phosphorothioates are introduced at the 3′ end. More- miRNA-expressing plasmids can also induce the
over, 2′-methoxy groups are introduced into the full upregulation of miRNA as they carry a fluorescent
length oligonucleotides [41]. These chemical modifica- reporter that can help investigators verify the expression
tions enhance the stability and cellular uptake efficiency and localization of miRNA. For example, Takara Bio
of antagomirs [42]. Therefore, these miRNA antagomirs constructed the pmR-ZsGreen1 and pmR-mCherry
can be used in vivo via either local or systemic adminis- vectors, which link a selective miRNA expression cas-
tration to downregulate the corresponding endogenous sette with a bright green or red fluorescent reporter
miRNA levels. gene, respectively.
miRNA masks are 22-nt single-stranded oligoribonu- Although many chemical modifications increase the sta-
cleotides with 2′-O-methyl-modifications [43]. Unlike bility of miRNAs, this effect may not be sufficient for in
AMOs, a miRNA mask does not directly bind to target vivo applications. An efficient delivery system is generally
miRNA. Instead, the miRNA mask can interact with accepted to be essential for developing miRNA-based ther-
miRNA binding sites localized in the 3′-UTR of target apeutics. In this review, we divide vectors into two types:
gene mRNA through a fully complementary mechanism. viral vectors (1) and nonviral carriers. Nonviral carriers are
The miRNA mask approach is an important supplement divided into six categories: (2) inorganic material-based de-
to AMOs, which are useful for investigating the total livery systems, (3) lipid-based nanocarriers, (4) polymeric
biological function of a specific miRNA; however, vectors/dendrimer-based vectors, (5) cell-derived mem-
miRNA masks are more suitable for studying the influ- brane vesicles and (6) 3D scaffold-based delivery systems
ence of miRNA on specific pathways containing a target (Fig. 4).
gene.
miRNA sponges are usually plasmid-encoding copies Viral vectors for miRNA and anti-miRNA oligonucleotide
that contain binding sites complementary to the seed re- delivery
gion of the target miRNA [44]. After transfection into Viral vectors can efficiently transfer genes into target
cells, these plasmids can transcribe high levels of sponge cells. Various viral vectors have been constructed to
Fu et al. ExRNA (2019) 1:24 Page 5 of 14

Fig. 3 miRNA. (a) Endogenous miRNA with normal function; (b) miRNA inhibition therapy using miRNA inhibitors, miRNA antagomirs, miRNA
masks and miRNA sponges; (c) miRNA replacement therapy using miRNA mimics, miRNA agomirs, miRNA precursors and miRNA-expressing
plasmids. The dotted lines represent the modified structure of miRNA antagomirs and miRNA agomirs

mediate RNA interference (RNAi) because they can Adenoviral vectors

transfer genes into different tissues/organs and cause Adenoviruses (Ad), which are derived from the Adeno-
long-term gene expression. As viral vectors possess dis- viridae family, are nonenveloped viruses that contain lin-
tinct characteristics, some vectors are more suitable for ear double-stranded DNA genomes of ~ 36 kb in length
certain purposes than others. Here, we introduce four with two inverted terminal repeats (ITRs) at its termini
widely used viral vectors for miRNA delivery including [45].
adenovirus vectors, adeno-associated virus vectors, To upregulate transgene efficiency and reduce im-
retroviral vectors, and lentivirus vectors. munogenicity in vivo, all of the viral protein-coding
Fu et al. ExRNA (2019) 1:24 Page 6 of 14

Fig. 4 Different types of vectors used for miRNA delivery. Vectors are divided into two types: viral vectors (1) and nonviral vectors. Nonviral
vectors are divided into six categories: (2) inorganic material-based delivery systems, (3) lipid-based nanocarriers, (4) polymeric vectors/dendrimer-
based vectors, (5) cell-derived membrane vesicles and (6) 3D scaffold-based delivery systems

Fig. 5 Chemical structures of different polymers used for miRNA delivery. (a) Structure diagram of different charged lipids (DSDAP, DOTAP, DSPC
and DSPE). (b) Structure diagram of polymeric vectors (PLL, PEI, PLGA, chitosan, β-cyclodextrin and PAMAM)
Fu et al. ExRNA (2019) 1:24 Page 7 of 14

sequences were deleted to construct helper-dependent vector encoding miRNA-133b improved functional
Ad vectors (HD AdVs) [46]. Moreover, the natural hepa- recovery in spinal cord-injured mice [59]. In another
totropism of Ad makes it potentially advantageous for study, a lentiviral vector-mediated miRNA-101 sponge
liver-targeted gene delivery [47]. HD AdVs are therefore was prepared, and the intrahippocampal injection of LVs
applied to efficiently deliver cassettes encoding primary mitigated the overproduction of soluble β-amyloid pre-
miRNAs into liver tissue. Recently, Mohube et al. found cursor protein (sAPPβ) in hippocampal neurons [60].
the short-term blockade of Hepatitis B Virus (HBV) rep- Despite their high delivery efficiency, viral vectors also
lication in vivo via the expression of anti-HBV have disadvantages, including low loading capacity, high
pri-miRNA mimics (pri-miRNA-122/5, pri-miRNA-31/5, toxicity and strong immunogenicity [61]. Therefore,
or pri-miRNA-31/5–8-9) by HD AdVs [48]. various nonviral vectors have been designed and con-
Oncolytic adenoviruses are considered suitable vectors structed based on actual needs. Their low toxicity and
for transferring therapeutic genes for tumor immuno- high biocompatibility make nonviral vectors a useful
therapy due to their commendably tumor-restricted complement to viral vectors.
replication abilities [49]. Cheng et al. generated an
oncolytic adenoviral vector named AdCN205 to coex- Nonviral vectors for miRNA and anti-miRNA
press interleukin-24 (IL-24) and miRNA-34a, and they oligonucleotide delivery
achieved better antitumor effects in experimental hepa- Inorganic material-based delivery systems
tocellular carcinoma (HCC) models [50]. However, the Inorganic materials, including gold nanoparticles
major disadvantage of HD AdVs is their powerful stimu- (AuNPs), mesoporous silicon, graphene oxide and Fe3O4--
lation of host innate and adaptive immune responses, mediated NPs, are widely used in nanotechnologies and
which may limit the widespread use of this vector [50]. have been developed as vectors to deliver miRNA. Func-
tional groups such as thiol and amino groups can be easily
Adeno-associated viral vectors attached to the surface of AuNPs, and these chemically
Adeno-associated viruses (AAV) from the Parvoviridae modified AuNPs have been employed as miRNA vehicles
family are nonenveloped viruses with single-stranded [62]. Jia et al. reported the covalent conjugation of
DNA genomes [51]. Sustained gene expression has been thiol-modified antagomir-miRNA-155 to AuNPs, and the
observed in different organs of mice after AAV treatment administration of miRNA-155-AuNPs via tail vein injec-
[52]. Recently, Yu Miyazaki and colleagues reported that a tion promoted M2 macrophage polarization, reduced
novel therapeutic approach based on an AAV vector plas- inflammatory mediators and ultimately recovered cardiac
mid encoding miRNA-196a ameliorated spinal and bulbar function in an ovariectomized (OVX) diabetic murine
muscular atrophy (SBMA) symptoms by downregulating model [63].
Elav-like family member 2 (CELF2) [53]. Mesoporous silica nanoparticles (MSNs) have several
advantages, such as large surface area and pore volume,
Retroviral vectors easy surface modification, thermal stability and favorable
Retroviruses (RVs) are enveloped viruses that can carry biocompatibility. Therefore, MSNs are considered to
two copies of a single-stranded RNA [54]. Most retro- be promising miRNA carriers [64]. Recently, Li et al.
viral vectors are constructed based on moloney murine demonstrated that anti-miRNA-155-loaded MSNs
leukemia virus (MMLV), which has a simple genome could be conjugated to polymerized dopamine (PDA)
that encodes env, pol and gag and is flanked by long ter- and the aptamer AS1411 to fabricate a nanocomplex
minal repeats (LTRs) [55]. When a virus infects host (MSNs-anti-miRNA-155@PDA-Apt) [65]. Treatment
cells, double-stranded DNA is formed by the reverse with MSNs-anti-miRNA-155@PDA-Apt effectively
transcriptase enzyme and then integrated into the host inhibited tumor growth in a colorectal cancer (CRC)
genome, which ultimately leads to the persistent expres- mouse model [65].
sion of the inserted gene fragment [56]. In a recent Graphene oxide (GO) is widely applied to deliver nu-
study, the administration of MMLV encoding miRNA-21 cleic acids in vivo. The unique honeycomb network of
(MMLV-miR-21) significantly improved miRNA-21 GO enables it to adsorb nucleobases [66]. In a recent
expression levels in adult mouse cardiac fibroblasts com- study, researchers developed a Cy3-labeled antisense
pared with those of the MMLV-ctrl group [57]. miRNA-21 PNA probe loaded onto hyaluronic acid
(HA)-conjugated GO, and this novel delivery system
Lentiviral vectors specifically targeted CD44-positive MBA-MB231 cells
Lentiviruses (LVs), which are similar to RVs, can stably and excited fluorescence via interactions with endogen-
insert themselves into the genomes of recipient cells, ous miRNA-21 [67].
which leads to sustained gene expression [58]. Recently, A Fe3O4-based delivery nanovector was developed for
evidence emerged that the administration of a lentivirus miRNA-100-mediated fibroblast growth factor receptor
Fu et al. ExRNA (2019) 1:24 Page 8 of 14

3 (FGFR3) regulation. The nanocomplex, named murine breast cancer model [72]. Using the thin film
PMMNCs-miR-100, contained mesoporous magnetic hydration approach, these novel liposomes were assem-
clusters linked by ternary polymers (poly(γ-glutamic bled with the 1,2-dioleoyl-3-trimethylammonium-pro-
acid) (γ-PGA), polyethylenimine (PEI), or polyacrylic pane (DOTAP), soybean phosphatidylcholine (SPC),
acid (PAA)) for delivering miRNA in vivo [68]. Due to DSPE-PEG2000-[D]-H6L9 and DSPE-PEG2000 (Fig. 5a).
its polycation polymer-functionalized mesoporous
structure, the miRNA-loading ability and tumor cell up- Polymeric vectors
take efficiency of the nanocomplex were greatly in- Polyethylenimines (PEIs) are rich in amine groups and
creased [68]. Additionally, systemic administration of are positively charged. Thus, they can bind to small
PMMNCs-miRNA-100 combined with conventional do- RNAs to form nanosized complexes, which prevent
cetaxel chemotherapy significantly enhanced the antitu- RNA degradation and promote cellular uptake and intra-
mor therapeutic effects compared with that of docetaxel cellular release [73] (Fig. 5b). At present, branched or
alone in FGFR3-mediated patient-derived xenografts linear PEIs with different molecular weights ranging
(PDXs) [68]. from 100 Da to approximately 1000 kDa can be pur-
chased [74]. Previous studies have shown that branched
Lipid-based nanocarriers 25 kDa PEIs were more effective at transferring
Lipids can be easily chemically modified to conjugate mmu-miRNA-494-3p into mouse embryonic fibroblast
with targeting moieties and fluorescent probes. There- (MEF) cells than Lipofectamine 2000 [75]. Huang et al.
fore, lipid-based nanocarriers are widely used for deliver- and Shi et al. proved that complexes of branched PEIs
ing nucleic acids in vivo. Cationic lipids are amphiphilic (25 kDa) with the miRNA-141 precursor or the
molecules composed of a hydrophilic head and a hydro- miRNA-31 precursor could significantly enhance colon
phobic tail [69], and they can currently be selected from tissue miRNA-141 or miRNA-31 expression levels, re-
commercially available products, such as Lipofectamine®. spectively, through intracolonic administration [76, 77].
Many studies have validated the use of cationic lipo- Due to its toxicity, the application of PEI is limited in
somes as carriers for transporting miRNA in vivo. At current clinical research. PEG, a non-ionic and hydro-
present, a large number of cationic lipids have been syn- philic polymer, can impair the toxicity of PEI when cova-
thesized for nucleic acid drug delivery; however, low lently linked to it. A large number of studies have
delivery efficiency is the main obstacle that limits their confirmed that PEGylation enhances the biocompatibil-
clinical application. To overcome this obstacle, novel ity of delivery systems based on PEI. Recently, research
lipids have been synthesized and new methods for was reported in which PEG/PEI nanoparticles were
constructing lipid nanocomplexes have been developed. employed as a nonviral vector for miRNA-150 transfec-
Subsequently, polyethylene glycol (PEG), a frequently tion, and these nanoscale complexes addressed the issue
used functional group, was conjugated to cationic lipids of poor transfection efficiency and instability in human
to escape phagocytosis of the RES when administered leukemia cells [78]. In addition to PEG, other polymers
systemically [70]. such as poly(L-lysine) (PLL) can also be used for PEI
Recently, a report by Tokyo University researchers modification (Fig. 5b). Gao et al. indicated that a
demonstrated that miRNA-126-loaded PEG-modified PEI-PLL/miRNA-21 sponge or PEI-PLL/anti-miRNA-21
liposomes combined with entrap ultrasound (named treatment could effectively reduce miRNA-21 levels in
“bubble liposomes”) promote angiogenesis and improved MCF-7 cells [79].
blood flow in an experimental hindlimb ischemia model As an FDA approved biomaterial, poly(lactide-co-gly-
[71]. Using the reversed-phase evaporation method, bub- colide) (PLGA) is a copolymer of poly lactic acid (PLA)
ble liposomes were synthesized using 1,2-distearoyl-sn-- and poly glycolic acid [80] (Fig. 5b). Due to its favorable
glycero-phosphatidylcholine (DSPC), 1,2-distearoyl-3- biocompatibility and well-documented utility for sus-
dimethylammonium-propane (DSDAP) and 1,2-distear- tained drug release, PLGA has been frequently used in
oylphosphatidylethanolamine-methoxy-polyethylene gly- the clinic. PLGA NPs are taken up by cells via endo-
col (PEG2000) (Fig. 5a). Then, the liposome suspension cytosis, and the loading drug is released inside cells.
was pressurized with perfluoropropane gas (an Nucleic acid drug delivery systems based on PLGA
echo-contrast gas) and placed in a bath sonicator, even- NPs have improved therapeutic effects due to their
tually forming bubble liposomes [71]. excellent drug release properties. Previous studies
In another study, Zhang et al. constructed a novel sys- have shown that treatment with nanoparticles com-
tem containing miRNA-10b antagomirs and paclitaxel posed of monomethoxy PEG, PLGA, PLL, lactobionic
via a pH-responsive liposome modified with the anti- acid, vascular endothelial growth factor antibodies
microbial peptide [D]-H6L9 (D-Lip) that could delay 4 and has-miRNA-99a mimics could suppress tumor
T1 tumor growth and reduce lung metastases in a growth in an experimental HCC model [81].
Fu et al. ExRNA (2019) 1:24 Page 9 of 14

Chitosan is a linear molecule with randomly distributed PAMAM dendrimers were the first synthetic polymers
β-(1 → 4)-linked D-glucosamine and N-acetyl-D-glucosa- with dendritic structures. Through a divergent method,
mine [82] (Fig. 5b). As a natural biocompatible and PAMAM dendrimers were developed from ethylenedi-
mucoadhesive polysaccharide, chitosan has little cytotox- amine or ammonia initiator core reagents [94]. Due to
icity and can prevent nucleic acid degradation. Macro- the positive charge on their surface, PAMAM dendri-
phages express high levels of galactose/N-acetyl- mers can condense nucleic acid molecules. Unlike unde-
galactosamine-specific lectin (MGL), which can mediate gradable PEI, PAMAM dendrimers are biodegradable
endocytosis [83]. Therefore, galactosylated low molecular polymers that exhibit relatively low genotoxicity and
weight chitosan (G-LMWC) is synthesized using chitosan cytotoxicity.
and lactic acid for targeting macrophages [84–86]. Zou et Recently, Wang et al. demonstrated a novel carrier de-
al. developed a colonic macrophage-targeted nucleic acid noted as NGO-PEG-dendrimers for miRNA delivery.
delivery system based on the G-LMWC/ASO nanocomplex NGO-PEG-dendrimers/anti-miRNA-21 were fabricated by
[87]. Huang et al. reported that G-LMWC combined with conjugating PAMAM dendrimers and PEG-functionalized
miRNA-16 precursors increased colonic macrophage nanographene oxide (NGO) to 2′-O-methyl-modified
miRNA-16 levels and alleviated the colitis symptoms of anti-miRNA-21 [95]. The intravenous injection of
2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated mice NGO-PEG-dendrimers/anti-miRNA-21 caused a remark-
through intracolonical injection [88]. able increase in bioluminescence signals within tumor areas
β-cyclodextrin is composed of homogeneous cyclic through a luciferase reporter [95].
α1,4-linked D-glucopyranose units in a seven-member
ring [89] (Fig. 5b). β-carbohydrate-based polymers have Cell-derived membrane vesicles
been used to enhance the efficiency of miRNA delivery Distant intercellular communication is crucial for the
systems. Recently, Zeng et al. constructed a novel deliv- maintenance of cellular environmental homeostasis in
ery vector composed of miRNA-34a mimics and matrix multicellular organisms. Recent reports have shown that
metalloproteinase-2 (MMP2)-cleavable substrate peptides distant cell-to-cell communication also occurs via extra-
[90]. In this system, enzyme-cleavable PEG derivatives are cellular vesicles (EVs) [96]. EVs are considered important
linked to cationic β-cyclodextrin/PEI, decreasing the factors involved in intercellular communication and are
cytotoxicity of PEI and condensing the therapeutic also used as biomarkers and drug carriers [97].
cargos, which resulted in excellent tumor targeting Wang et al. reported that AS1411, DNA aptamer-
capability and antitumor activity in 4 T1 xenograft modified EVs-loaded with Cy5-labeled let-7, could effect-
tumor models [90]. ively accumulate in tumor tissues and suppress tumor
Polymeric micelles are obtained by linking a hydro- growth when injected intravenously [98]. In another
philic polymer with a hydrophobic polymer. The most study, researchers demonstrated that the systemic ad-
widely used hydrophilic polymers contain PEG, poly ministration of brain metastatic cancer cell-derived EVs
(L-amino acids), poly (propylene glycol), biodegradable containing miRNA-181c promoted brain metastasis and
polyesters and phospholipids, polyorthoesters, and destruction of the blood-brain barrier (BBB) [99].
long-chain fatty acids [91]. Recently, researchers de- Based on their molecular profiles and intracellular ori-
signed a novel dual stimulus-sensitive mixed polymeric gins, three main types of EVs are typically found: exo-
micelles codelivery system for the delivery of the doxo- somes, microvesicles and apoptotic bodies. Exosomes
rubicin and endogenous tumor suppressor miRNA-34a are nanoscale vesicles that contribute to intercellular
into cancer cells [92]. Two stimulus-sensitive compo- communication, antigen presentation and RNA shuttling
nents, an MMP2-repsonsive doxorubicin conjugate and (mainly mRNA and miRNA). These membrane vesicles
a glutathione-responsive miRNA-34a conjugate, were as- (40–120 nm in diameter) are derived from late endo-
sembled to form single NPs and then linked with PEG somes [100]. Emerging data suggests that exosomes
for long-term blood circulation and with cell-penetrating could mediate intercellular communication through the
peptide (CPP)-TATp to improve intracellular uptake in a transfer of bioactive molecules such as miRNAs and the
3D spheroid model of tumor mass [92]. protection of encapsulated small RNAs from ribonucle-
ases (RNases) in bodily fluids [101]. Recently, Lee et al.
Dendrimer-based vectors developed a novel single-step in situ detection method
Dendrimers are three-dimensional, hyperbranched for exosome miRNAs using a nanosized fluorescent
globular nanopolymeric materials. Due to their narrow oligonucleotide probe they called a–“molecular beacon”
polydispersity index and modification with multiple [102].
functional groups, dendrimers have unique advantages Additionally, exosomes have low cytotoxicity and neg-
compared with other polymers and are widely used in ligible antigenicity. Therefore, they are ideal vehicles for
different fields [93]. nucleic acid drugs. As they can circumvent endocytosis
Fu et al. ExRNA (2019) 1:24 Page 10 of 14

and escape from phagocytosis by the RES, exosomes 3D scaffold-based delivery systems
have high delivery efficacy. In a recent study, endothelial With favorable spatiotemporal control and mechanical
progenitor cell-derived exosomes containing abundant barriers circumventions, 3D biomaterial scaffolds can ef-
miRNA-126-3p and 5p could attenuate organ injury and ficiently maintain the therapeutic effects of miRNA. At
vascular permeability in cecal ligation and puncture present, various 3D-scaffold types have been developed
(CLP)-induced sepsis [103]. In another study, Wen et al. for miRNA delivery, including hydrogels, electrospun fi-
utilized human bone marrow mesenchymal stem cells bers, and other more abundantly porous or spongy
(hBMSCs) and their exosomes (which are rich in siFas 3D-scaffolds.
and anti-miRNA-375) to restrain islet apoptosis and pri- Hydrogels are polymer networks with hydrophilic prop-
mary nonfunction (PNF) during islet transplantation in erties. Researchers have demonstrated that PEGylation
humanized NOD scid gamma (NSG) mice [104]. However, hydrogels constantly release siRNA against noggin and
the large-scale production of exosomes is not easily avail- miRNA-20a mimics and promote encapsulated human
able due to cost. An interesting study showed that bovine bone marrow-derived mesenchymal stem cells (hMSCs)
milk could be used as a scalable source of exosomes that to differentiate into osteoblasts [116]. In another study,
might act as carriers for miRNA delivery [105]. scientists from the Massachusetts Institute of Technology
Microvesicles (MVs, or shedding vesicles) (100–1000 (MIT) showed that a novel self-assembled dual-color
nm in size) are vesicles that are shed from multiple cell RNA-triple-helix hydrogel composed of miRNA-225
types during certain pathological and physiological states mimics and miRNA-221 antagomirs facilitated nearly 90%
[106]. In a recent study, BMSCs were infected with a tumor shrinkage in a triple-negative breast cancer mouse
miRNA-200b-expressing lentivirus, and MVs were model [117].
isolated through a differential centrifugation method. Due to their versatility, electrospun fibers are being ex-
The collected MVs were subsequently used to treat plored for use in many different applications. To target the
TNBS-induced rat intestinal fibrosis [107]. Zhang et al. delivery of miRNA-126 mimics into vascular endothelial
demonstrated the suppressive effect of MVs containing cells (VECs), researchers recently developed a bilayer vas-
miRNA-29a/c on tumor growth in gastric cancer (GC) cular scaffold fabricated by target carriers and electrospun
[108]. Recently, a report by Cui et al. proved that fibrous membranes [118]. The outer layer of poly(ε-capro-
leukemia cell-derived MVs could induce T cell exhaus- lactone) (PCL) and gelatin contributed to mechanical sta-
tion by delivering multiple functional miRNAs [109]. bility, and the inner layer of poly(ethylene glycol)-b-
Platelets, which are derived from bone marrow mega- poly(L-lactide-co-ε-caprolactone) (PELCL), which con-
karyocytes, are fragments with a diameter of 1 to 4 μm tained complexes of miRNA-126 mimics in REDV
that are responsible for the maintenance of vascular peptide-modified trimethyl chitosan-g-poly(ethylene glycol),
integrity and physiology hemostasis [110]. In some cases, regulated the response mediated by VECs [118].
activated platelets can release microparticles (MPs) (a Recently, Zhang et al. demonstrated that a novel hyper-
type of MV), which are small EVs that range from 0.1 to branched polymer (HP) with high miRNA-26-binding abil-
1 μm in size and are from the cytoplasmic membrane ity could self-assemble into nanoscale complexes [119].
[111]. John et al. illustrated that Ago2/miRNA-223 Such an engineered 3D-scaffold was able to induce the
complex-laden platelet-derived cargos could easily enter regeneration of calvarial bone defects in an osteoporotic
human umbilical vein endothelial cells (HUVECs) [111]. mouse model [119]. Irene et al. reported an interesting
Subsequently, Liang et al. further elaborated that platelet- collagen-nanohydroxyapatite miRNA-activated scaffold for
derived MVs containing high levels of miRNA-223 could tissue engineering that could efficiently deliver both
promote lung cancer cell invasion by reducing the level of miRNA antagomirs and miRNA mimics to human mesen-
tumor suppressor EPB41L3 [112]. chymal stem cells [120].
Apoptotic bodies are characteristic membrane blebs
that are released from apoptotic cells. Apoptotic bodies Progress in clinical research on miRNAs as nucleic acid
have the widest spread diameter, which can range from drugs
approximately 50–5000 nm [113]. As they express To date, many miRNA-based therapeutics have been
“eat-me” molecules (e.g., phosphatidylserine), apoptotic used in clinical trials (https://clinicaltrials.gov/ct2/home).
bodies can recruit phagocytes to nearby apoptotic cells, Miravirsen (SPC3649) (Clinical Trials.gov Identifier:
leading to their clearance [114]. Research has demon- NCT02452814), the world’s first miRNA drug candidate,
strated that endothelial cell-derived apoptotic bodies which is currently in clinical testing, was applied to treat
containing high levels of miRNA-126 can trigger the se- hepatitis C in Phase II clinical trials in 2017. Miravirsen is
cretion of chemokine (C-X-C motif ) ligand-12 composed of LNA ribonucleotides whose sequences are
(CXCL12), recruit multiple progenitor cells and protect complementary to miRNA-122 [121]. RG-101, which is in a
mice from atherosclerosis [115]. Phase 1b clinical trial, is a chemically modified
Fu et al. ExRNA (2019) 1:24 Page 11 of 14

phosphorothioate oligonucleotide inhibitor that targets by the kidney. The targeting ability and long-term blood
miRNA-122; it is conjugated to a multivalent circulation of miRNA-based delivery systems should be
N-acetylgalactosamine carbohydrate structure that was de- improved to increase delivery efficiency. Therefore, new
signed to enhance uptake via binding to the asialoglycopro- biomaterials should be synthesized and new methods
tein receptor on hepatocytes. However, RG-101 has been should be developed for delivery systems. Emerging evi-
placed on clinical hold as a result of two serious adverse dence has demonstrated that cell-derived membrane
events (SAE) of jaundice. MRX34 (Clinical Trials.gov Iden- vesicles (e.g., exosomes, microvesicles and apoptotic
tifier: NCT01829971), a liposome-encapsulated miRNA- bodies) might act as ideal delivery vectors due to their
34a mimic, was used for patients with advanced solid low cytotoxicity and negligible antigenicity. More im-
tumors in a multicenter Phase I trial. Despite its therapeutic portantly, a deeper and clearer understanding of the bio-
effects, the clinical program was terminated due to logical functions of such systems is imperative.
immune-related adverse events. MesomiR-1 (Clinical Tri-
als.gov Identifier: NCT02369198), a miRNA-16-based Abbreviations
2′-F: 2′-fluoro oligonucleotide; 2′-O-Me: 2′-O-methyl-oligonucleotide; 2′-O-
miRNA mimic encapsulated in nonliving bacterial minicells MOE: 2′-O-methoxyethyl oligonucleotide; 3′-UTR: 3′-untranslated region;
with an anti-EGFR bispecific antibody, was applied for AAV: Adeno-associated virus; Ad: Adenovirus; AMO: Anti-miRNA
mesothelioma and non-small-cell lung cancer (NSCLC) in oligonucleotide; ASO: Antisense oligonucleotide; AuNP: Gold nanoparticle;
BBB: Blood-brain barrier; BMSCs: Bone marrow mesenchymal stem cells;
a Phase I study. MRG-106 (Clinical Trials.gov Identifier: CELF2: Elav-like family member 2; CLP: Cecal ligation and puncture; CPP: Cell-
NCT02580552), an anti-miRNA-155 LNA-modified anti- penetrating peptide; CRC: Colorectal cancer; CXCL12: Chemokine (C-X-C
sense inhibitor, was employed for patients with cutaneous motif) ligand-12; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane;
DSDAP: 1,2-distearoyl-3- dimethylammonium-propane; DSPC: 1,2-distearoyl-
T cell lymphoma and mycosis fungoides in a Phase II sn-glycero-phosphatidylcholine; DSPE: 1,2-distearoyl-sn-glycero-3-phosphoryl
potentially registrational clinical trial. MRG-201 (Clinical ethanolamine; EV: Extracellular vesicle; FGFR3: Fibroblast growth factor
Trials. gov Identifier: NCT02603224), a miRNA-29 mimic receptor 3; GalNAc: N-acetylgalactosamine; GC: Gastric cancer; G-
LMWC: Galactosylated low molecular weight chitosan; GO: Graphene oxide;
with a cholesterol-conjugated miRNA duplex, was used for HA: Hyaluronic acid; hBMSC: Human bone marrow mesenchymal stem cell;
patients with scleroderma, and the initiation of a Phase II HBV: Hepatitis B Virus; HCC: Hepatocellular carcinoma; HD Ad: Helper-
clinical trial was announced. RG-125/AZD4076 (Clinical dependent Ad; HD AdV: Helper-dependent adenoviral vector; HDL: High-
density lipoprotein; hMSC: Human bone marrow-derived mesenchymal stem
Trials.gov Identifier: NCT02612662), an anti-miRNA-103/ cell; HP: Hyperbranched polymer; HUVEC: Human umbilical vein endothelial
107 conjugated with N-acetylgalactosamine (GalNAc), was cell; IL-24: Interleukin-24; ITR: Inverted terminal repeat; LNA: Locked nucleic
used in patients with non-alcoholic fatty liver and type 2 acid oligonucleotide; LTR: Long terminal repeat; LVs: Lentivirus; MEF: Mouse
embryonic fibroblast; miRISC: miRNA-induced silencing complex;
diabetes in a Phase I/II trial. Because extracellular miRNA miRNAs: microRNAs; MMLV: Moloney murine leukemia virus; MMP2: Matrix
can be easily separated from patient biological fluids, it is metalloproteinase-2; MP: Microparticle; MSNs: Mesoporous silica
an ideal biomarker candidate for the diagnosis and progno- nanoparticles; MV: Microvesicle; NSCLC: Non-small-cell lung cancer;
NSG: NOD scid gamma; PDX: Patient-derived xenograft; PEG: Polyethylene
sis of disease. For example, miRNA7™ is the first approved glycol; PEG2000: 1,2-distearoylphosphatidylethanolamine- methoxy-
commercially available kit for liver cancer diagnosis via the polyethylene glycol; PEI: Polyethylenimine; PEI-PLL: Poly (L-lysine)-modified
detection of 7 miRNAs. PEI; PLGA: Poly(lactide-co-glycolide; PMOs: Phosphorodiamidate morpholino
oligomers; PNAs: Peptide nucleic acids; PNF: Primary nonfunction; pri-
miRNA: Primary miRNA; PTX: Paclitaxel; RES: Reticuloendothelial system;
Conclusions RV: Retrovirus; SAE: Serious adverse event; sAPPβ: Soluble β-amyloid precur-
Numerous miRNA-based delivery systems have been sor protein; SBMA: Spinal and bulbar muscular atrophy; siFas: siRNA against
Fas receptor; SPC: Soybean phosphatidylcholine; TNBS: 2,4,6-trinitrobenzene
constructed and used to obtain favorable effects in appli- sulfonic acid; Treg: T cell regulatory; VEC: Vascular endothelial cell;
cation. Current research on miRNA-based therapeutics VEGF: Vascular endothelial growth factor
depends mainly on the ability of delivery cargos to pro-
tect oligonucleotides from serum RNase degradation, to Acknowledgements
Not applicable.
improve targeting ability and to enhance therapeutic ef-
fects without triggering immune-related adverse effects. Funding
In most studies, intravenous injection or local treatment This work was supported by the National Natural Science Foundation of
was the main method of administration for in vivo China (31571458, 31771550, 31400671, J1103512, J1210026 and 31870821).
miRNA delivery. Very few studies have used oral admin-
Availability of data and materials
istration for miRNA-based delivery.
Not applicable.
Chemical modification and vehicle complexation have
been explored to stabilize RNAs, but RNA stability does Authors’ contributions
not increase cellular uptake and escape. Both viral and YF performed the literature analysis and wrote the paper. ZH and JC
nonviral vectors have disadvantages, including immuno- supervised the analysis and corrected the manuscript. All authors read and
approved the final manuscript.
genicity and low oligonucleotide-loading capacity. When
administered systemically, such nanocargos can be easily Ethics approval and consent to participate
retained in the liver and spleen and quickly eliminated Not applicable.
Fu et al. ExRNA (2019) 1:24 Page 12 of 14

Consent for publication tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;
Not applicable. 10(12):1470–U209.
21. Jung HJ, Suh Y. Circulating miRNAs in ageing and ageing-related diseases.
Competing interests J Genet Genomics. 2014;41(9):465–72.
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kingdom regulation by microRNA. Cell Res. 2012;22(1):107–26.
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