close

Вход

Забыли?

вход по аккаунту

?

adhm.201700695

код для вставкиСкачать
PROGRESS REPORT
Regenerative Medicine
www.advhealthmat.de
Scaffold-Based microRNA Therapies in Regenerative
Medicine and Cancer
Caroline M. Curtin, Irene Mencía Castaño, and Fergal J. O’Brien*
with surgical connotations, which brings
together Biology, Pharmacy, Materials
Science and Bioengineering principles,
and has emerged as a very significant
field with a growing global market valued
at $13.41 Billion in 2016 and estimated to
reach $38.70 Billion by 2021.[2] Traditional
RM approaches based on allografting and
autografting are currently regarded as the
gold standard procedures to repair a wide
number of tissues. However, limitations
such as the requirement for additional
surgical procedures, limited donor tissue,
donor-site morbidity, and chronic pain,
the shortage in tissue/organ availability in
proportion to the increased aging population, complications related to the immune
system, or risk of disease transmission
from donor to patient are prominent
motivators for the development of new
and improved RM approaches.[3] Typically,
biomaterials (scaffolds), cells and external
stimuli are the three major components
applied in RM to provide tissue or organ
grafting alternatives.[4] Scaffolds are classically introduced to provide a template for
cell infiltration and tissue regeneration.
Overall, these three major components can be used individually
or combined, with a big research focus currently placed on different variants of biomaterial-based approaches (Figure 1).
The growing knowledge of molecular and cellular processes
underpinning tissue regeneration allows the exploration of
innovative external stimuli to provide more advanced RM therapies. In addition, an emerging field very much interlinked with
cancer research has started to translate the concept of biomaterial scaffolds from their typical use in RM to in vitro tools
for complex microenvironment modeling; much of this vision
has exciting applications in cancer research. The specific focus
of this Progress Report will be on the use of biomaterial scaffolds which incorporate microRNA therapeutics as an external
stimulus to enhance cell-mediated tissue repair or to serve as
antitumoral treatments. Critical obstacles in the clinical translation of these technologies, including formulation chemistry
and delivery methods are discussed below.
microRNA-based therapies are an advantageous strategy with applications
in both regenerative medicine (RM) and cancer treatments. microRNAs
(miRNAs) are an evolutionary conserved class of small RNA molecules that
modulate up to one third of the human nonprotein coding genome. Thus,
synthetic miRNA activators and inhibitors hold immense potential to finely
balance gene expression and reestablish tissue health. Ongoing industrysponsored clinical trials inspire a new miRNA therapeutics era, but progress
largely relies on the development of safe and efficient delivery systems. The
emerging application of biomaterial scaffolds for this purpose offers spatiotemporal control and circumvents biological and mechanical barriers that
impede successful miRNA delivery. The nascent research in scaffold-mediated miRNA therapies translates know-how learnt from studies in antitumoral
and genetic disorders as well as work on plasmid (p)DNA/siRNA delivery to
expand the miRNA therapies arena. In this progress report, the state of the
art methods of regulating miRNAs are reviewed. Relevant miRNA delivery
vectors and scaffold systems applied to-date for RM and cancer treatment
applications are discussed, as well as the challenges involved in their design.
Overall, this progress report demonstrates the opportunity that exists for
the application of miRNA-activated scaffolds in the future of RM and cancer
treatments.
1. Regenerative Medicine: An Overview
The field of “regenerative medicine” (RM) was conceptually
developed to aid the body’s natural capacity to self-repair, a
capacity which is impaired with age and in cases of disease or
injury.[1] RM is a vast and multifaceted area of medicine, often
Dr. C. M. Curtin, Dr. I. Mencía Castaño, Prof. F. J. O’Brien
Tissue Engineering Research Group
Department of Anatomy
Royal College of Surgeons in Ireland (RCSI)
123 St. Stephens Green, Dublin 2, Ireland
E-mail: fjobrien@rcsi.ie
Dr. C. M. Curtin, Dr. I. Mencía Castaño, Dr. F. J. O’Brien
Trinity Centre for Bioengineering
Trinity College Dublin (TCD)
Dublin 2, Ireland
Dr. C. M. Curtin, Dr. I. Mencía Castaño, Dr. F. J. O’Brien
Advanced Materials and Bioengineering Research (AMBER) Centre
RCSI & TCD
Dublin 2, Ireland
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.201700695.
DOI: 10.1002/adhm.201700695
Adv. Healthcare Mater. 2017, 1700695
2. MicroRNA Biology: An Overview
miRNAs are an evolutionary conserved class of noncoding
single-stranded RNA molecules naturally occurring across all
1700695 (1 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
biological kingdoms; in animals, the foundation member of
this class of RNAs is lin-4 RNA, discovered in 1993 by Ambros
and co-workers.[5] The realization that lin-4 produced two small
RNAs -61 and 22 nucleotides (nt) long, which bind to the lin-14
messenger (m)RNA 3′ UTR region to repress its translation,
was associated with its role in Caenorhabditis elegans larval
development.[6] Followed by seminal work on the let-7 gene and
homologs, we progressively gained understanding of the biogenesis and the mechanism of action of miRNAs[7] (Figure 2).
The first phase of miRNAs biogenesis consists of transcription, either directly from miRNA genes—sometimes coding
for miRNA clusters—or from processed introns of proteincoding genes, rendering long primary transcripts termed primiRNAs.[8] Next, during the maturation phase in the nucleus,
Drosha cleaves the pri-miRNA into a 60–70 nt stem loop precursor miRNA (pre-miRNA) with ≈2 nt 3′ overhangs,[9] which
is transported to the cytoplasm by Ran-GTP and Exportin-5.[10]
Once in the cytoplasm Dicer slices off the stem loop, producing
an imperfect and short-lived miRNA:miRNA*duplex,[11] from
which the ≈22 nt long mature single strand miRNA parts for
assembly into the RNA-induced silencing complex (RISC)
during the final step of the process, while the more unstable
miRNA* strand typically degrades.[12] This miRNA-multiprotein
complex, known as miRISC, is responsible for the mechanism of action of miRNAs, highly similar to the functionality
of small interfering (si)RNA; eventually miRISC interacts,
through base-pair complementarity binding, with the untranslated (UTR) region of the target mRNA. Although the 3′ UTR
region is most prone, 5′ UTR regions are also capable of interacting with miRISC.[13] While perfect sequence complementarity is associated with mRNA degradation,[14] partial binding
leads to translation repression and allows a multitargeting
effect characteristic of miRNAs; one miRNA can silence up
to 100 mRNA targets, and the mRNA coding for the production of one particular protein is susceptible to binding with a
multitude of miRNAs.[15,16] To-date up to one third of the protein-coding genome is reportedly subject to miRNA modulation,[16] and there are over 2500 human miRNAs indexed,[17]
with increasing numbers of studies continuously unraveling
their biological effects at an accelerated pace. Some examples
of miRNA function involve (i) the regulation of developmental
stages of a cell, (ii) lineage commitment, (iii) differentiation,
(iv) proliferation and (v) apoptosis, as well as (vi) inflammation, immune response events and related diseases including
asthma and cystic fibrosis, (vii) tumor formation, and (viii)
the progression of viral infections.[18,19] Thus, by harnessing
miRNA-directed gene regulation, numerous molecular therapeutic pathways may be addressed concurrently.
3. Types of miRNA Therapeutics and Exogenous
miRNA Regulation
In order to exploit the therapeutic potential of miRNAs, the
endogenous mechanism may be modified in two opposing
directions using exogenous miRNA regulators, that is, synthetic
molecules which either mimic or repress the function of endogenous miRNAs.[20] Specifically, mimicking the endogenous
Adv. Healthcare Mater. 2017, 1700695
Caroline M. Curtin obtained
her B.Sc. from the National
University of Ireland, Galway
(NUIG) in 2004 followed
by her Ph.D. in 2010 from
the Regenerative Medicine
Institute at NUIG. She was
recruited as a postdoctoral
Researcher to tissue engineering research group
(TERG) in the Royal College
of Surgeons in Ireland (RCSI)
in 2010 and is currently employed as a Lecturer in Anatomy
and Principal Investigator in the TERG. Her research
focuses on development of gene-activated scaffold systems
for tissue engineering and cancer treatment applications.
Irene Mencía Castaño graduated as BPharm and MSc
in Biochemistry, Molecular
Biology, and Biomedicine
from the Universidad
Complutense de Madrid,
Spain, following which she
was recruited as a BioAT
PhD Scholar to the TERG in
RCSI in 2012. She obtained
her Ph.D. from RCSI in 2015
and is currently employed
as a postdoctoral Researcher in the TERG. Her research
focusses on scaffold-based RNA therapeutics for tissue
repair, with specific interests in the pharmaceutic formulation aspects.
Fergal J. O’Brien is Chair of
Bioengineering & Regenerative
Medicine and Head of the
TERG in RCSI and a PI and
Deputy Director of AMBER.
He is a leading innovator in
the development of advanced
biomaterials for regenerative
medicine. He is a member
of the World Council of
Biomechanics and has served
as Biomaterials Topic Chair
for the Orthopaedic Research Society and as an EU Council
Member of TERMIS. His research focuses on the development and clinical translation of scaffold-based therapeutics
for tissue engineering, with a major focus on functionalizing
these scaffolds as systems to deliver biomedicines.
miRNAs enhances the suppression of target protein synthesis
by degrading the mRNA or inhibiting the protein translation;
on the contrary, inhibiting miRNAs, or preventing their activity
inside the cells, putatively leads to an increase in mRNA and
1700695 (2 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Figure 1. Overview of strategies applied in scaffold-based RM approaches. a) Direct implantation of cell-free scaffold (gray). b) Cells are harvested,
expanded and cultured on scaffolds in vitro, then cell-seeded constructs (green) are implanted at the defect site. c) Direct implantation of cell-free
scaffold bio-activated with proteins, growth factors (GFs), genes, miRNAs, or small chemical drugs (orange), for localized delivery of these external
bio-stimuli to promote tissue regeneration. d) Cells are harvested, expanded in culture, combined with external bio-stimuli and cultured in vitro using
scaffolds, then constructs (blue) are implanted at a defect site.
protein expression[21] (Figure 3). In this way, an additional benefit of miRNA therapy is the potential to respectively switch
off or on gene expression as required by the intended application. Numerous synthetic variants have thus been developed
to pursue miRNA therapeutics, where sequence design and
chemical modifications have made a range of options available
with high binding affinity, enzymatic stability and improved
cellular uptake. These include: (i) hydroxyl group conjugations
in the ribose 2′ position to enhance hydrophobicity, such as
Exiqon’s proprietary locked-nucleic acid technology (LNA) developed by Braasch and Corey,[22] (ii) functionalizations of the phosphate backbone, which introduce enzymatic resistance and have
been extended to a hybrid peptide nucleic acid (PNA) variant by
Adv. Healthcare Mater. 2017, 1700695
Fabani and co-workers,[23] or (iii) morpholino substitutions for
the ribose, which also improve binding affinity (Table 1). Modifications of the ribose ring and phosphate backbone are often
combined within single nts, and significant efforts are concentrated in optimizing the balance between multiply-modified and
unmodified units composing the oligonucleotides.
3.1. miRNA Mimicking
Two main strategies may be considered within the miRNA
mimicking line of therapy: delivery of single or double-stranded
antisense oligonucleotides known as miR-mimics, and delivery
1700695 (3 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Figure 2. miRNA biogenesis and function. (1) Transcription of miRNA genes to primary miRNA (pri-miRNA) is followed by (2) loop-processing by the
Drosha/DGCR8 complex; this generates a precursor (pre)-miRNA that is then (3) transported into the cytoplasm by Exportin-5/Ran-GTP. (4) Dicer/
TRBP cleaves the hairpin releasing the miRNA:miRNA∗ duplex. (5) The mature miRNA separates to form the miRISC complex with the Argonaute
(AGO) protein core, while the miRNA∗ strand degrades. (6) Target mRNAs interact with miRISC complexes, leading to (7) mRNA degradation or
translational repression amongst other complex regulatory functions.
of DNA coding for miRNA precursors, generally pre-miRs
(Figure 3a). Double-stranded miR-mimics can have up to 1000
fold higher potency than the single-stranded counterparts; this
is because the additional passenger (sense) strand supports
more chemical modifications, which are limited for the guide
(antisense) strand in order to form the miRISC complex.[24] The
alternative delivery of DNA-encoded pre-miRs is perhaps burdened by the need for nuclear access and the increased risks
of miRNA biogenesis saturation, although it seems particularly
appealing for long-term effects and incorporation into viral
delivery vectors.
3.2. miRNA Inhibition
In the line of miRNA inhibition therapy, three different
strategies are available: antisense modified oligonucleotides
(AMOs)—also known as anti-miRs or antagomiRs, miRNA
masks or “blockmiRs”—miRNA sponges and small molecule
inhibitors such as dihydropteridine ATP analogs and some
diazobenzenes[25] (Figure 3b). AntagomiRs are the first type of
miRNA inhibitors to demonstrate effectiveness in mammals;[26]
they work by binding with high affinity to individual miRNAs—
due to their chemical modifications—so that the interaction
with the target mRNA is prevented. AntagomiRs can thus affect
the multiple mRNA targets downstream of the single miRNA
inhibited. Distinctively, blockmiRs are ≈15 nt antisense strands
that occupy the target mRNA binding sites, again blocking
Adv. Healthcare Mater. 2017, 1700695
the interaction with miRISC, but allowing protein translation;
blockmiRs feature more predictable pharmacodynamics than
antagomiRs in exchange for no ability to multitarget. miRNA
sponges, often introduced as plasmid (p)DNA vectors, are long
single stranded RNAs that offer yet another mechanism of
action: they compete with target mRNAs for interacting with
a miRNA, ultimately sequestering several miRNA copies at a
time and abrogating protein silencing as a result.[27]
4. Delivery of microRNA Therapeutics
in Regenerative Medicine
The landscape of miRNA therapeutics has progressed at a dramatically fast pace in under 20 years, leading to the first clinical
trials on liver-targeted therapies with a miR-122 inhibitor and a
miR-34 mimic.[28] Such advances assert the belief in projecting
this strategy to become a bench to patient bedside reality.[29]
Specifically in the field of RM, pharmaceutical research and
development (R&D) pipelines are currently exploring miRNAs
to treat fibrosis (miR-21), inflammation (miR-155), atherosclerosis (miR-33), heart failure (miR-208), cardiac repair (miR-15)
and neoangiogenesis (miR-92a).[20] The high interest of RM in
the area of miRNA therapy stems from its great promise to overcome the well documented limitations of direct administration
of growth factors (GF).[30] These limitations include the need
for high dosing due to short half-life and poor systemic distribution, and the associated high costs.[31] In contrast, miRNA
1700695 (4 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
that share.[35,36] miRNA delivery systems for
RM purposes generally combine delivery vectors and 3D biomaterial scaffolds in order
to achieve efficiently sustained therapeutic
effects in a localized manner. The characteristics and functions of both components,
detailed below, generally translate across
pDNA, siRNA, and miRNA delivery systems.
4.1. Vectors for microRNA Delivery
A range of vectors, typically classified as viral
or nonviral, may be employed to aid cellular
entry of miRNAs and subsequently facilitate
their transport from the lysosomes into the
cell cytoplasm, hence preventing the phenomenon of lysosomal degradation.[37,38]
The numerous options available within both
categories have been reviewed extensively
in recent publications,[39,40] yet there is still
no consensus in an optimal system.[38] This
section focuses on miRNA delivery vectors
which have been considered for the development of scaffold-based miRNA delivery systems, their design and characteristics.
4.1.1. Viral Vectors
By their very nature, viruses are excellent at
gaining cellular and nuclear entry; they characteristically achieve high transfection levels
and thus are at the forefront of the clinical
advances in the broad field of gene therapy.
This includes their use in 67% of registered
gene therapy clinical trials,[41] and in the
market-approved GlaxoSmithKline’s Strimvelis and MolMed’s Zalmoxis treatments.[42]
Viral miRNA delivery may serve to stably
modify endogenous miRNA expression; a
Figure 3. Types of miRNA modulators. a) miR-mimicking by pDNA encoding for miRNA precursors, single or double stranded oligonucleotides serves to enhance the formation of miRISC valuable goal to treat chronic degenerative
diseases within the RM arena, such as osteocomplex alongside endogenous miRISC, ultimately leading to decreased levels of messenger
(m)RNA or protein of the target present in the cell. b) miRNA inhibition by Pdna/RNA sponges arthritis. Among the distinct classes, retrovior antagomiRs (antimiRs) impedes the entry of the mature miRNA to form miRISC and abroruses are highly efficient at entering dividing
gates downstream interaction with the target mRNA; “BlockmiRs” conceal the binding sites cells, while lentiviruses, adenoviruses (ADV),
of mRNA directly preventing its entry in miRISC. The three approaches direct to ultimately
and adeno-associated viruses (AAV) can enter
enhanced mRNA and protein levels of the target in the cell.
both dividing and nondividing cells.[43] Retroviral vectors are associated with high risks of
genome integration at undesired locations, an often fatal sidetherapy offers the ability to transiently regulate cellular protein
effect known as insertional mutagenesis. In comparison with
production at low doses, featuring an extended half-life over
retroviruses, lesser insertional mutagenesis risks are reported
GF and pDNA: the cytoplasmic half-life of numerous miRNAs
with the use of lentiviruses,[44] thus perhaps encouraging recent
spans from over 24h to several days[32] while the maximum
documented for pDNA is 5h.[33] However, the delivery of naked
developments with lentiviral engineering of cells prior to seeding
onto biomaterial scaffolds.[45–47] ADVs and AAVs delivering
miRNA has demonstrated limited success, and the development of safe and efficient delivery systems is widely regarded
miRNAs are less explored in biomaterial-based RM approaches,
as the critical progress-limiting factor;[34] in fact, 25% of the US
although their use in traditional in vitro transductions with
miRNA-expressing cassettes has been discussed in other
miRNA therapeutics patents relate to RM applications, while
reviews.[48] While both vectors are dependent on helper viruses,
R&D in miRNA delivery systems is responsible for ≈30% of
Adv. Healthcare Mater. 2017, 1700695
1700695 (5 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Table 1. Effects of chemical design of miRNA therapeutics.
Type of modification
Chemical structure
Function
Properties conferred
Ribose methylation: 2′O-Me
• Enhance hydrophobicity
• Increase duplex melting temperature
↑ Cellular uptake
Ribose fluoridation: 2′F
• Enhance hydrophobicity
• Increase duplex melting temperature
↑ Cellular uptake
↑ Stability
Conformational ribose locking: LNA
• Ribose ring structurally locked in C3′-endo
conformation
• Enhance hydrophobicity
• Improved target binding affinity
↑ Cellular uptake
↑ Bioactivity
Phosphate backbone methylation: P-Me
• Improved enzymatic resistance
• Enhance hydrophobicity
• Increase duplex melting temperature
↑ Cellular uptake
Phosphate backbone thyolation: PS
• Introduces phosphorothioate linkages
• Improved enzymatic resistance
• Enhanced binding to plasma proteins
• Improved pharmacokinetics
↑ Stability
↑ Biodistribution time,
slowed excretion
Morpholino-ribose substitution: PMO
• Introduces phosphorodiamidate linkages
• Removes negative charge
• Improved target binding affinity
↑ Cellular uptake
↑ Bioactivity
↑ Stability
Adv. Healthcare Mater. 2017, 1700695
1700695 (6 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Table 1. Continued.
Type of modification
Chemical structure
• N-(2-aminoethyl)-glycine units substitute the
phosphate backbone
• Removes negative charge
• Improved enzymatic resistance
Peptide-phosphate backbone substitution: PNA
AAVs may shortly garner more attention in the field: their
characteristic small size, their lack of pathogenicity in humans
and their reduced immunogenicity in comparison with ADVs
places them in the spotlight of forthcoming research in miRNA
delivery.[43] Baculoviruses (BV) are similarly nonpathogenic to
humans (they target insects naturally), with the additional safety
advantages of not undergoing chromosomal integration or replication in mammalian cells. BVs are gradually garnering attention as miRNA delivery vectors,[49] with a panel of osteogenic
miRNAs screened for therapeutic effects in vitro, and reported
beneficial bone healing effects when assessed as a cotreatment
with bone morphogenetic protein-2 (BMP2) in a nude mice calvarial defect model.[50] Many disadvantages continue to surround
the use of viral vectors however including immunogenicity and
biodistribution concerns, high production costs and complexity
for high-mass production. A nascent alternative are virus-like
particles (VLPs), a recombinant version of viral capsids recently
explored to deliver miRNAs for the treatment of systemic lupus
erythematosus.[34,51] VLPs resemble the cell tropism and natural
intracellular trafficking capacities of the virus while being noninfectious, and thus hold significant promise for the clinical
advancement of the field in the coming years.
4.1.2. Nonviral Vectors
Typically, nonviral vectors allow the transfer of large cargoes
with varying efficiencies depending on the cell type targeted,
and result in temporary transfection effects.[38,39,52] This temporality can be advantageous for numerous RM applications,
where time-span limit of the therapeutic is of utmost importance, and a maintained effect after tissue regeneration may be
highly detrimental. Additionally, high-mass production of nonviral methodologies tends to be less complex and costly than
that of viral vectors.[52] Several cationic polymers are commercially exploited for miRNA delivery at a research level, while
innovations in the nanotechnology arena increasingly continue
to introduce interesting alternatives.
Cationic lipids are among the most commonly utilized nonviral vectors for gene therapy applications. The use of commercial cationic lipids in biomaterial-based approaches to
deliver miRNA therapeutics is exemplified by the widespread
use of Lipofectamine 2000[53–56] along with Lipofectamine
RNAiMAX[57] and SiPORT NeoFx.[58] Generally, cationic
Adv. Healthcare Mater. 2017, 1700695
Function
Properties conferred
↑ Cellular uptake
↑ Stability
lipids complex with the miRNA cassettes through electrostatic
interaction, and the overall positive charge of the complex dictates the cellular uptake, although neutrally charged liposomes
can also encapsulate the cargo within their core. Complexes
formed by encapsulation of the miRNA cargo, as opposed to
charge adsorption, are not limited to lipid-based vectors and
have also shown effective delivery profiles in RM applications.
The structural versatility of lipid-based vectors is of key value
for adapting their use to different cargos as well as to target
distinct cell types.[59] However, this is counterbalanced by their
reported cytotoxic and proinflammatory effects, some of which
have been associated with their ability to trigger the generation
of intracellular vacuoles.[60]
Polyethyleneimine (PEI) derivatives have also been extensively studied as miRNA delivery systems,[19,61,62] and specifically PEI–poly(ethylene glycol) (PEG) nanoparticles (NP) have
recently demonstrated interesting profiles when encapsulated
within poly(lactic-co-glycolic acid) (PLGA) microspheres in a
poly(l-lactic acid) (PLLA) scaffold[63] or as part of a calcium
chloride cross-linked alginate hydrogel system.[64] PEI is watersoluble and easily attracts negatively charged ions to aid the
polyplex cargo escape the endolysosomal compartment in what
is known as “the proton sponge effect.”[65] The extent of this
effect is dependent on a parameter termed N/P ratio, that is,
the proportion of moles of nitrogen in PEI to moles of phosphate in the cargo. While elucidating the optimal N/P ratio for
a target cell type can render PEI a remarkably efficient miRNA
delivery vector, evidence of significant cell damage derived from
their use remains a considerable drawback in terms of clinical
application.[66] Poly(β-amino esters) (PβAE) are yet another
class of polymers with negligible cytotoxicity and amenable
to gene delivery via the proton sponge effect in a range of cell
types, including hMSCs, hADSCs, human embryonic stem cellderived cells (hESCds), macrophages, HEK-293, U-87 MG and
lung cancer cells.[67–69] Interestingly, PβAEs have demonstrated
30–60% transfection efficiency when delivering pDNA,[68] and
up to 70% silencing following siRNA delivery,[70] with one study
also assessing short hairpin (sh)RNA delivery.[69] Despite the
tunability of these materials, which may specifically allow for
kinetically controlled biodegradation,[71] PβAEs have yet to be
explored as miRNA carriers in scaffold-based delivery platforms.
A diblock copolymer made of dimethylaminoethyl methacrylate has also shown promise for the delivery of siRNA
in hydrogel-based systems. These nanoparticles have
1700695 (7 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
demonstrated significant siRNA delivery and gene knockdown
in a number of studies, which is accredited to the ability of
these NPs to protect and allow sufficient cytosolic accumulation of siRNA.[72] While they have also been used when combined with polylactic acid (PLA) PLA forming amphiphilic starshaped polymers to deliver miRNA-21 and doxorubicin to treat
glioma,[73] as of yet, they have not been used to deliver miRNA
on scaffold-based platforms but appear to hold great potential
for such applications.
Other nonviral synthetic vectors that generally feature low
cytotoxicity have garnered significant attention in pDNA and
siRNA delivery, such as dendrimers,[40] cyclodextrins,[74,75] and
cell penetrating peptides (CPP).[76,77] However, all of these are
to-date minimally explored in RM applications of miRNA therapeutics. Of relevance, polyamidoamine (PAMAM) dendrimers
were recently applied to a dextran-based hydrogel to deliver
miR-205 mimics and antagomiR-221 for breast cancer treatment applications.[78] This work demonstrated a macropinocytosis-mediated uptake mechanism for the dendrimer-miRNA
complexes, with substantially improved efficiency in comparison to the original PAMAM formulations reported.[79] Similarly, cyclodextrins undergo unspecific (clathrin independent)
uptake[80] but also serve to improve water-solubility and biodistribution of the cargo due to their cyclic oligosaccharide
nature.[81] In the case of CPPs, uptake may occur either through
transitory micelle formation, endocytosis in its four variants,
or direct penetration driven by penetratin or TAT (GRKKRRQRRR) domains,[82] which explains their documented high
transfection efficiencies.[76] Overall, dendrimers, cyclodextrins,
and CPPs offer extensive versatility in their structure and associated phys-chem properties, ultimately allowing the formation of the complexes to occur through charge adsorption or
physical encapsulation; research in adapting and optimizing
these vectors for miRNA delivery will undoubtedly impact the
application of miRNA therapeutics in RM.
Natural derived nonviral vectors are also being increasingly
considered for miRNA-RM therapeutics due to their high biocompatibility features: chitosan, exosomes, and inorganic NPs
belong to this category. Chitosan is a cationic polysaccharide
first introduced to gene delivery in the late 1990s[83] that can
be adapted to different nucleic acid cargoes by modifying the
chain architecture and/or the acetylation and polymerization
degree.[84] Thus, while 80% deacetylated chitosan showed low
efficiencies transfecting pDNA, fully deacetylated chitosan
demonstrated remarkable functionality of the eluted siRNA
along a 5 d time-course in a human lung carcinoma cell
line (H1299).[84] More recently, shortening the chain length
has provided improved efficiencies (≈45%) in the transfection of rat mesenchymal stem cells with pDNA,[85] and similarly, highly depolymerized chitosan delivering miR-145 to
human breast adenocarcinoma (MCF-7) cells achieved ≈50%
target silencing.[86] Exosomes play a critical role in paracrine
signaling and intercellular miRNA trafficking guided by the
tissue-specific proteins found on their surface.[87] Early reports
have linked exosomes, and their miRNA content, with several tissue regeneration processes, such aspects could be soon
recapitulated in RM approaches to deliver exogenous miRNA
modulators,[88] and their assessment for this purpose has
indeed begun to focus on siRNA delivery.[89]
Adv. Healthcare Mater. 2017, 1700695
Several types of inorganic NPs based on gold, silver, iron, and
calcium phosphates (CaP) offer innovative aspects in the journey
toward commercially applicable nonviral miRNA delivery vectors. Cysteamine-Au-PEG NPs optimized for miRNA reporters
across neuroblastoma and ovarian cancer cell lines built on the
beneficial amphipilicity and surface area of these materials.[90]
Qureshi et al. developed an interesting photocleavable-Ag nanoparticle system, which showed improved delivery of miR-148b
mimic to human adipose-derived stem cells (hASCs) over the
commercial transfection reagent TurboFect;[91] the preclinical in
vivo evaluation of a therapeutic incorporating this system onto
Matrigel and polycaprolactone (PCL) scaffolds is described in
further detail in Section 5.1.[92] Fe3O4 paramagnetic NPs were
skillfully combined with PEI to deliver miR-335 to human mesenchymal stem cells (MSC), with intended application in the
treatment of cardiovascular diseases, in work carried out by
Schade et al.[62] The range of 80% uptake efficiency reported in
this work was in exchange for acceptable dead cell levels -below
20%, with the added possibility of magnet-controlled tissue targeting following administration of this potential therapeutic
system. CaPs are an often overlooked option in gene therapy,
disregarded for their traditionally low transfection efficiencies
delivering pDNA and their instability in solution.[93] However, research carried out by our lab has optimized CaPs for
this purpose, using a formulation of the hydroxyapatite mineral phase as nonaggregating nanoparticles (nHA) previously
developed in-house.[94] These NPs were originally investigated
to develop a collagen-nHA composite scaffold with improved
mechanical properties;[94] this material was then adapted to
surpass the pDNA transfection efficiency of a commercial CaP
transfection kit in hMSCs,[95] and was further tailored to deliver
both reporter miR-mimics and antagomiRs to the same cell
type, with a single low dose maintaining a silencing efficiency
of greater than 90% over 1 week.[96] Although little is known
regarding the uptake mechanisms of CaP-nucleic acid complexes, their ability to form precipitates on the cell surface and
undergo clathrin and dynamin-dependent endocytosis has been
documented,[37] and their pH-dependent solubility allows complex dissociation to commence within intracellular compartments.[97] Considering the ease of production, cost effectiveness
and high biocompatibility of CaPs,[98] together with the newly
improved efficiency of these nHA particles to deliver miRNA
therapeutics, the proposed system has demonstrated a substantial potential for the derivation of new therapeutics in this field.
4.2. Scaffolds for microRNA Delivery
In the late 1990s, the idea of “gene-activated matrixes” emerged;
the first report documented collagen-based scaffolds delivering
β-galactosidase pDNA to significantly promote bone formation.[99,100] This concept has now more recently also been translated to miRNA delivery. The primary role of 3D biomaterial
scaffolds is to act as templates for cell attachment and tissue
repair; however their use to locally deliver therapeutics has
become increasingly appealing to target a specific tissue more
safely.[36,101] The scaffolds 3D microenvironment can provide
physical protection from degradation and prevent the rapid
clearance of small complexes from the target site; without this
1700695 (8 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Figure 4. Distinction between types of scaffold-based miRNA delivery. a) In situ or “cell-free” miRNA activated scaffolds (orange glow) are obtained by
adding the exogenous miRNA complexes (with the select delivery vector) into the biomaterial; a host cell population then comes in contact with this
platform following in vivo implantation, taking up the complexes and becoming transfected (orange glow). b) Ex situ or “cell-based” miRNA activated
scaffolds result from pretransfecting a cell population in vitro with the miRNA complexes, then collecting and seeding the derived cells onto the biomaterial. The cell-activated scaffold is then ready for in vivo implantation.
protection, the effective action time-frames may be too short to
achieve therapeutic efficacy.[102] Thus, it is becoming well-established that the combination of the aforementioned miRNA
delivery vectors together with biomaterial scaffolds will prolong the time-frames of localized delivery,[99,103,104] a favorable
prospect for RM applications. Of note, functional differences
have been reported across monolayer and scaffold transfection experiments, being a testament to the effect of the 3D
microenvironment. Thus the necessity to carry out thorough in
vitro characterization of the scaffold-based miRNA therapeutic
system should not be overlooked.
Scaffolds can facilitate “cell-free” or “cell-mediated” miRNA
delivery, also termed in situ or ex situ delivery, respectively
(Figure 4). In situ transfection indicates that miRNAs exist
within the scaffold structure prior to the addition of cells; these
scaffolds are implanted “cell-free” in the tissue void, where they
will interact with the resident and homing cells to regenerate
such defects.[105] The scaffold acts as a depot for the complexes
and then infiltrating cells establish contact with these along the
structure of the scaffolds to internalize them and become transfected. On the contrary, ex situ transfection consists in first
administering the miRNA treatment to a controlled cell population, which is then mixed with or seeded onto the relevant scaffold. This second strategy may require an additional period of
in vitro culture before implantation in the defect site; ultimately
this cell population is the only one modified by the miRNA
treatment and thus responsible for the therapeutic outcome
upon implantation. At present, in situ miRNA delivery systems remain undeveloped in comparison with pDNA-activated
Adv. Healthcare Mater. 2017, 1700695
scaffolds, and ex situ miRNA delivery counts for a higher
number of reports.[102,106] Whether one of the routes may be
better suited for clinical translation remains to be elucidated,
although in situ transfecting scaffolds hold greater potential to
exist as “off-the-shelf” products.[107]
Many distinct scaffold types have been tuned to serve
the purpose of miRNA delivery, including several hydrogels,[58,92,106,108–114] electrospun fibers,[63,115–118] and more
prolifically porous or spongy scaffolds.[46,47,50,54,55,96,112,119–130]
Before reviewing the details of their applications, summarized in Table 2 and described further in the next section, we
will discuss the key characteristics making these materials
amenable to miRNA delivery. First very general qualities must
be fulfilled: biocompatibility, bioresorbability and ease of fabrication.[103] Additionally, ease of sterilization, stability and
long-term storage, are highly important from the perspective of clinical applicability.[131] High porosity, tissue anastomosis and mechanical strength are relevant aspects for orthopedic applications; however, favorable rheological properties,
including responsiveness to different physical stimuli and a
good viscoelastic component are the main factors to optimize
in the case of hydrogel formulations.[132] Overall, the scaffold architecture must allow the ingrowth of neovascularization for the successful application of nearly every biomaterial
in RM, and recently, the impact of material nanotopography
is also gaining recognition.[133] In order to specifically adapt
any scaffold to miRNA delivery, the material must retain the
miRNA complexes while facilitating their sufficient exposure
to the infiltrating cells.[134] Reciprocally, the presence of miRNA
1700695 (9 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Table 2. Summary of reports of scaffold-based miRNA delivery in RM.
Scaffold type
Vector type
Therapeutic
End-point analysis result
In vivo model/cell type
Ref.
Appl.
Gelatin-coated PLGA
scaffold disc
Baculovirus
ASCs, BMP-2+
miR-148b mimic
transduced
12 weeks: complete bridging: bone
area = 94%, BV = 89%, BD = 95%
4 mm critical-size calvarial defect in nude mice
[50]
Bone
Gelatin-coated PLGA
scaffold disc
Baculovirus
ASCs, BMP-2+
miR-148b mimic
transduced
12 weeks: filling 83% defect area,
75% BV, bone density 89% of original
4 mm critical-size
calvarial bone defects in
nude mice
[122,123]
Gelatin- Spongostan
scaffold
Baculovirus
OVX-BM-MSCs transduced with miR-214 or
miR-140 sponges
Bone healing, bone density, trab. no,
trab. thickness and trab. space: further synergy by BMP2 co-expression
Osteoporotic rat femoral
metaphysis defect model
[130]
Poly(glycerol sebacate) porous scaffold
discs
Lentivirus
BM-MSCs, miR-31
inhibitor transduced
8 weeks postoperatively: significant
improvement in healing vs control
groups (near full bridging)
8 mm calvarial rat defect
(12-w.o. male Fischer
344)
[46]
β-TCP scaffold
Lentivirus
ASCs or BM-MSCs,
miR-31 inhibitor
transduced
Bone regeneration in 8 weeks in rat
model and in 16 weeks in canine,
Runx2-Satb2 loop mechanism
5 mm rat calvarial defect,
10 mm canine orbital
bone defect
[120,121]
Poly(sebacoyl diglyceride) (PSED) scaffold
Lentivirus
BM-MSCs, miR-135 mimic
or inhibitor transduced
8 weeks: respectively enhanced or
decreased newly formed bone and
trabecular number
8 mm critical-sized rat
cranial defects
[47]
PSED scaffold
Lentivirus
BM-MSCs transduced
with miR-125b inhibitor
8 weeks: enhanced SMAD4, Runx2
and Osterix expression in newly
formed bone
8 mm critical-sized rat
cranial defects
[127]
Demineralized bone
matrix
Lentivirus
BM-MSCs transduced
with miR125b inhibitor
51% increased BMD, 79% increase
in Tb.N; histologically enhanced new
bone generation and bone maturity
Bilateral segmental
femoral defects in
athymic mice
[128]
Nanofibrous PLLA
scaffold
HP-PEI-PEG
26a mimic (cell-free in
vivo)
Bone regeneration in 8 weeks in both
healthy and osteoporotic mice
5 mm critical-sized mice
calvarial bone defect
[63]
HyStem-HP and
Hya/gelatine/PEG
hydrogels
siPORT NeoFxTM
hBM-MSCs, miR-26a
mimic transduced
Histomorphometry at 8 weeks (ectopic
model): increased bone formation vs
control; micro-CT analysis at12W (calvarial defect model) full bridging
3 and 5 mm mice calvarial
defect (nude C57BL6J),
mouse (m) or hBM-MSCs
[58]
HA/TCP scaffold disc
Lipofectamine
2000
rat ASCs, miR-26a
mimic transduced
Histomorphometry at 12 weeks:
increased new bone forming area vs
control groups
3.5 mm diameter rat
cortical tibial defect
[55]
Porous collagen-nHA
scaffold
nHA particles
GAPDH targeting
miR-mimic, antagomiR-16
Silencing functionality of ≈20% and
88.4%, respectively
hMSC, 2D and
3D delivery profile
characterization
[96]
Porous collagen-nHA
scaffold
nHA particles
antagomiR-133a-3p
Enhanced Runx2 and osteocalcin
expression, increased alkaline phosphatase activity and Ca2+ deposition
hMSC, 2D and 3D assessment of therapeutic
osteogenesis
[124]
Matrigel or PCL
scaffold
PC-SilverNP
Pre-miR-148b transduced
ASCs
4 and 12 weeks: healing improved
32.53 ± 8.3%
4 mm critical-sized
mouse calvarial defect
[92]
EXg 3D scaffold
Naked
miR-2861
miR-2861 and RUNX2 overexpression
Human periodontal ligament (hPDL) SCs
[126]
Atelocollagen
Naked
miR-222 inhibitor
8 weeks: positive radiographic, µCT
and histological evaluation
Rat refractory fracture
model
[113]
Naked- nucleofection
miR-148b mimic + miR489 inhibitor
66% increase alkaline phosphatase
activity and 4-fold increase in Ca2+
deposition (day 10) over untreated
control
hBM-MSC, 2D and 3D
assessment of therapeutic osteogenesis
[106]
Alginate hydrogel +
osteochondral biopsy
RNAiMAX-
BM-MSCs transduced 2X
with miR-221inhibitor
12 weeks: cartilage repair with
absence of CollX
Subcutaneously implant
in nude mice
[112]
Cartilage
Fibrin hydrogel
siPORT
NeoFxTM
antagomiR-133a and
anatagomiR-696
Enhanced expression of PGC-1a, and
contractile force
2 weeks myotubes culture
[111]
Muscle
PEG-norbornene
hydrogel
Adv. Healthcare Mater. 2017, 1700695
1700695 (10 of 22)
Bone
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Table 2. Continued.
Vector type
Therapeutic
End-point analysis result
In vivo model/cell type
Ref.
Appl.
REDV peptidetrimethyl
chitosan-PEG
miR-126
8 weeks: in vitro down-regulation of
SPRED-1, improved endothelialization in vivo
Rabbit 1 cm left common
carotid artery resection
[116]
Cardiovascular
Fibrinogen, Matrigel
and thrombin
hydrogel
Dharmafect
miR-1+ miR-133+ miR208+ miR-499
MMP-dependent mechanism of successful reprogramming
Direct reprogramming
of fibroblasts into
cardiomyocytes
[110]
4S-StarPEG collagen
scaffold (gel)
Naked
miR-29b
4 weeks: reduced wound contraction,
improved coll III/I ratio, higher
MMP-8: TIMP-1 ratio
Full thickness rat skin
excisional wounds
[108,109]
2 and 5 weeks following MI: myocardial function maintenance at
Mice ischemia reperfusion model
4 weeks: reduced hyperplasia and
inhibited SMCs proliferation
In rat vein neointimal
hyperplasia model
[114]
OPC remyelination
[117]
NSCs self-renewal and
differentiation control
[129]
Scaffold type
PEG-PCL-gelatin
electrospun fibers
Glycosan HyStem
hydrogel
Lentivirus
miR-26a mimics
TransIT-TKO
miR-219 and miR-338
Collagen spongebased scaffold
n/a
miR-20
Aminopropyl-silica
nanofibers
n/a
Let-7
Pluronic F-127 gel +
vein segments
Electrospun PCL fiber
scaffold
Unraveling miR signature
[118]
complexes in the scaffold structure must not negatively affect
its mechanical and structural features or deter cellular retention and attachment to the scaffold.[135]
Depending on the nature of their components, biomaterials are typically classified as synthetic, natural, ceramics, or
composites, that is, combinations of these. Certain materials
are more applicable for a fabrication technique or a type of formulation. For example, PEG is a synthetic polymer frequently
employed in the preparation of hydrogels, such as the commercial HyStem-HP utilized by Li et al. to deliver miR-26a
mimics,[58] or the PEG-norbornene system for human bone
marrow derived (BM)-MSCs nucleofected with a dual miR-148b
mimic + miR-489 inhibitor combination.[106] An in situ forming
PEG-hydrogel has also been the supporting biomaterial of an
UV-triggered “on-demand” miR-20a release system.[136,137]
A rare synthetic polymer for RM applications, pluronic, has
recently aided the in vivo lentiviral delivery of miR-26a mimics
for cardiovascular applications;[114] this is an amphiphilic triblock copolymer that self-assembles in situ producing a micellated gel. Characteristically, synthetic polymers offer extensive
possibilities to tailor their mechanical properties and degradation rates by altering chain lengths, as well as proportions of
the mixture components. Although several of these materials
hold Food and Drug Administration approval for RM purposes,
in the case of synthetic polymers biodegradable through esterhydrolysis, their acidic degradation by-products can exert an
inhibitory effect on tissue formation.[138] To diminish these
undesired effects, it is frequently combined with natural polymers such as hyaluronic acid (HyA), as in the commercial
HyStem-HP and Glycosan HyStem. Other natural polymers
included in miRNA eluting hydrogels are alginate,[112] silk
fibrin,[111] fibrinogen, thrombin, and Matrigel.[92,110] Of note,
hydrogels are the first biomaterials used successfully for the
Adv. Healthcare Mater. 2017, 1700695
Neurological
delivery of naked miRNA therapeutics,[106,108,109] that is, without
the addition of a delivery vector. While this strategy simplifies
the fabrication of the final miRNA-activated scaffolds, it may
also accelerate the release of the cargo, leading to undesired
bolus effects.
Electrospinning is a distinct fabrication method that produces
nanofibrous scaffolds typically using material sources such as
gelatin,[115] synthetic PLLA,[63] PCL,[92,117] or PEG–PCL copolymers.[116] Interestingly, most of the nonviral, nonlipid vectors
tested for 3D-miRNA delivery have utilized biomaterials fabricated using this method: Qureshi’s photocleavable silver NPs,[92]
the commercial Transit-TKO transfection reagent,[115,117,139]
an innovative peptic sequence REDV-trimethyl chitosan-PEG
complex,[116] and hyperbranched (HP) PEI–PEG polyplexes.[63]
Moreover, with the exception of the Ag-NPs, all the remaining
systems have been developed as in situ “cell-free” miRNA transfecting platforms, underscoring great clinical potential and versatility of this fabrication method. Finally, spongy porous scaffolds account for the largest evidence in miRNA delivery: 50%
of these are ceramic-based or ceramic-composite materials, the
other half is represented by a mix of compositions. Ceramics
are inorganic crystalline structures with characteristically high
melting points, hardness and brittleness, among which CaPs
have been widely studied. CaPs exist in forms of different stoichiometric proportion of calcium to phosphate; this includes
brushite, tricalcium phosphate (TCP), and hydroxyapatite (HA),
the mineral component found in teeth and bone.[140] β-TCP scaffolds were used by Deng et al. in preclinical studies of lentiviral
miR-31 inhibition,[45,120] and HA/TCP scaffolds were utilized,
in combination with Lipofectamine 2000, by Eskildsen et al.
to deliver miR-138 inhibitors in vitro,[54] and more recently by
Wang and co-workers to deliver miR-26a mimics in vivo.[55]
Similarly, nHA/PCL and carboxy-methyl-cellulose/zinc-doped
1700695 (11 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
nHA composite scaffolds have been combined with Lipofectamine 2000 and X-treme Gene transfection reagent respectively.[56,125] The common denominator of this body of work is
the minimal intervention of the scaffold in the delivery of the
miRNA, as the cells are transfected ex situ prior to scaffold
seeding. Notably, in the work carried out in our lab by Mencía
Castaño et al. the porous collagen-nHA scaffolds take active
part in the in situ delivery of both miR-mimics and antagomiRs to hMSCs.[96,124] These reports demonstrate the differences between the 3D miRNA-activated system in comparison
to monolayer: in the in situ transfection the scaffold retains the
miRNAs within its 3D structure, relatively limiting the exposed
surface area of the complexes to the infiltrating cells while prolonging the onsite presence of these complexes.
Taken together, significant research has explored the miRNA
delivery potential of a wide range of material types and fabrication methods in combination with either viral or nonviral
delivery vectors. Although the field of scaffold-based miRNA
delivery is nowadays at a nascent juncture, these reports reflect
the vast landscape of options available to derivate advanced
materials tailored to the specific needs of each particular RM
application, whose therapeutic potential is illustrated in the
next section.
5. MicroRNA-Scaffold Based Therapies
for Tissue Repair
The vast potential of miRNA therapies for tissue repair and RM
has been extensively reviewed in the last few years in either
general terms[25,141] or with more focused applications in mind
such as bone/orthopedics,[104,142] angiogenesis,[143] the cardiovascular system,[144] or skin,[145] but the area of scaffold-based
miRNA delivery in RM is still relatively in its infancy. To date,
reports on scaffold-mediated miRNA delivery have focused
more on specific areas of regenerative medicine including osteogenesis and bone repair as well as the cardiovascular arena
(Table 2), but this progress report aims to give a broader overview encompassing a variety of areas of RM.
5.1. Osteogenesis and Bone Repair
The majority of available reports for scaffold-based miRNA
delivery in the osteogenesis and bone repair field focus on the
application of miRNA 26a.[58,63,146] Li et al.[58] documented the
first report in 2013 using BM-MSCs, HyStem-HP hydrogel, and
miR-26a mimic. They demonstrated almost full repair of the
defect 12 weeks postimplantation and significantly enhanced
vascularization confirming that miR-26a had the capacity
to regulate endogenous angiogenic-osteogenic coupling for
enhanced bone regeneration. Wang et al.[146] employed an alternative approach with ASCs, miR-26a mimic and Lipofectamine
2000 on porous HA scaffolds. Following 12 weeks implantation, histological assessment showed markedly enhanced bone
formation. The final and most eloquent report went a few
steps further and delivered HP-PEI-miR-26a polyplexes on a
nanofibrous PLLA scaffold without an external cell source in
contrast to the earlier reports[58,146] and of great importance,
Adv. Healthcare Mater. 2017, 1700695
mechanistically identified the bone regeneration target gene for
miR-26a for the first time.[63] To further support the spatiotemporal release of the miRNA, the miRNA-loaded polyplexes were
also encapsulated in biodegradable PLGA microspheres which
were then immobilized onto the scaffolds. Importantly, these
two-stage, cell-free, miR-26a loaded scaffolds demonstrated significantly enhanced bone regeneration in critical-sized calvarial
bone defects in both healthy and osteoporotic mice after only
8 weeks. miR-26a has previously been shown to regulate suppression of connective tissue growth factor,[147] angiogenesis—
through bone morphogenetic protein 2 (BMP2)/drosophila
mothers against decapentaplegic protein (SMAD) signaling
-in endothelial cells,[148] and atherosclerosis via a EphA2/p38
MAPK/vascular endothelial growth factor (VEGF) pathway.[149]
Work by Luzi et al.[150] has however pointed at a complex role
for the miR-26a::SMAD1 match in human ASCs osteogenesis.
Finally, Zhang et al.[63] importantly demonstrated that miR-26a
regulates osteoblast function through targeting glycogen synthase kinase-3beta (GSK-3beta), a gene known to improve bone
mass and increase the mineral apposition rate and bone mineral density (BMD) when inhibited in OVX mice.[151]
Deng et al.[46,120,121] have focused on the assessment of scaffold-mediated miR-31 delivery in various different cells, scaffolds and defect models. Their first study on the evaluation
of ASCs combined with lentivirus-miR-31 loaded β-TCP scaffolds[120] showed that miR-31-knockdown ASCs significantly
improved bone regeneration in vivo. This work highlighted a
Runx2/Satb2/miR-31 regulatory loop activated by BMP to play
a vital function in ASC osteogenesis and bone regeneration.
Having previously reported that knockdown of endogenous
miR-31 in vitro significantly improved the osteogenic capacity
of BM-MSCs,[152] the group went on to assess the therapeutic
potential of lentivirus-miR-31-modified BM-MSCs cultured on
poly(glycerol sebacate) scaffolds.[46] Results demonstrated that
miR-31 inhibition could successfully enhance bone repair in
the defect area. Their final miR-31-based study assessed the
role of lentivirus-miR-31-inhibitor modified BM-MSCs seeded
on β-TCP scaffolds in repairing orbital bone defects in a canine
model for 16 weeks.[121] The results of this study confirmed their
previous reports with downregulation of miR-31 demonstrating
a significant role in osteogenesis and bone regeneration.
miR-148b mimic has also been highlighted in a number of
scaffold-based studies. Mariner et al.[123] first described its application in combination with miR-489 inhibitor to accelerate in
vitro osteogenic differentiation of BM-MSCs. Cells were transfected to coexpress both the mimic and inhibitor using nucleofection before being photoencapsulated on PEG-norbornene
scaffolds. Results showed a significant enhancement of alkaline phosphatase (ALP) activity, calcium deposition and osteogenic gene expression. An alternative method involving ASCs
transduced with BVs for codelivery of BMP2 pDNA and miR148b on a gelatin-coated PLGA scaffold was investigated to
determine the effect on osteogenic differentiation.[153] The BV
hybrid used, composed of flippase recombinase (FLPo) and Frtflanking cassette, has previously shown to substantially prolong
transgene expression.[122] Having assessed the osteogenic effect
of a number of miRNAs in vitro, miR-148b was chosen as the
frontrunner and demonstrated enhanced in vitro osteogenesis
when cotransduced with BMP2. Scaffolds containing ASCs
1700695 (12 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
coexpressing BMP-2/miR-148b were implanted into criticalsize mouse calvarial bone defects and showed accelerated bone
healing and remodeling after 12 weeks, filling ≈94% of the bone
defect area and ≈89% of the bone defect volume. Following on
from this study, Li et al.[154] focused on elucidation of the molecular target for 148b as it had not been previously identified. This
study revealed miR-148b directly targets NOG, which negatively
regulates BMP2-induced osteogenesis and bone formation. The
authors recapitulated the codelivery of BMP2 and miR-148b[153]
utilizing an alternative Cre/loxP-based BV vector for stronger
transgene expression and reduced cytotoxicity,[122] in an attempt
to improve the calvarial bone healing. These scaffolds demonstrated superior bone regeneration and restoration of the bone
density to 89% of the original, indicating the potential of this
alternative scaffold system. Qureshi et al.[92] also assessed miR148b delivery from scaffolds for enhanced bone repair. Photocrosslinked-miR-148b was conjugated to silver NPs (PC-miR148b-SNP) to allow for greater temporal control and improve
bone repair of a critical-sized mouse calvarial defect. PC-miR148b-SNP conjugates combined with ASCs were loaded on PCL
scaffolds and efficiently regulated ASC osteogenic differentiation and improved calvarial defect closure by ≈32%. Ultimately
the results demonstrated the translational potential of the photoactivated miRNA delivery system to enhance ASC osteogenic
differentiation.
Recent work from our Tissue Engineering Research Group
(TERG) in Dublin has concentrated on nHA particle-based
delivery of miR-mimics and miR-inhibitors from porous
collagen-nHA scaffolds paying particular attention to bone
repair.[96,124] Studies on scaffold-based gene delivery in the lab
originally began with the delivery of pDNA including BMP2[95]
and combinations of BMP2 and VEGF[155] utilizing the nHA
particles[94] and coll-nHA scaffolds[94,156] developed in-house
specifically for bone repair. Having demonstrated the dramatically enhanced therapeutic potential of such systems not
only for bone but also cartilage and nerve repair, we also progressed to investigating the utility of alternative gene delivery
vectors including PEI,[85,155,157] chitosan,[40,158] Lipofectamine
2000,[75] and cyclodextrin,[75,159] different genetic cargo such
as pDNA,[85,95,155,157] miRNA,[96,124] and siRNA,[75,159] and
various scaffold types including collagen alone,[85,95] collagennHA,[75,95,96,124,155,159] collagen-HA[85] and collagen hyaluronic
acid (coll-HyA)[85] to tailor our systems depending on the
final application required.[158] Delivery of the miR-mimics and
miR-inhibitors from porous collagen-nHA scaffolds yielded
very promising results demonstrating silencing functionality
of ≈20% and 88.4%, respectively. The antagomiR-functionality levels achieved in our “coll-nHA-nanomiR” system were
improved over miR-mimic silencing, although 20% silencing
can still trigger therapeutic improvements in vivo,[108] providing
evidence suggestive of the potential benefit of the TERG system.
A more recent study functionalized the collagen-nHA scaffolds
with miR-133a inhibitor, delivered using nonviral nHA particles, to enhance hMSC osteogenesis, building on the validated
direct targeting of miR-133a-(3p) over Runx2, a master transcription factor of osteogenesis.[160] Results comprehensively
demonstrated increased gene expression of Runx2 and osteocalcin, in addition to enhanced alkaline phosphatase activity and
calcium deposition, showing improved therapeutic potential of
Adv. Healthcare Mater. 2017, 1700695
the scaffold previously optimized for bone repair applications
(Figure 5). In vivo assessment of this system (data not yet published) has further demonstrated the benefits of the application
of these coll-nHA-nanomiR scaffolds.
The Alsberg lab has recently focused their attention on
the effect of miR-20a to guide enhanced stem cell osteogenesis.[136,137] Nguyen et al.[137] developed in situ forming PEG
hydrogels for controlled, localized and sustained delivery of
siRNA (siNoggin) and miR-20a, complexed to PEI, to BM-MSCs.
Extended delivery of siNoggin cotransfected with miR-20a from
this polymeric scaffold allowed enhanced osteogenic differentiation of encapsulated stem cells and extended regulation of
cell behavior. Following on from this study, Huynh et al.[136]
developed the first on demand dual-crosslinked in situ forming
photodegradable PEG-based hydrogel to actively control
the release of unmodified/chemically modified siNoggin or
miR-20a complexed to PEI via UV irradiation to induce the
enhanced osteogenic differentiation of BM-MSCs. This study
presents an ideal system for “on-demand” release of genetic
materials via external UV application that holds promise for
tissue regeneration.
Gelatin nanofibers have been used for the localized transient
delivery of miR-29a inhibitor to increase extracellular matrix
(ECM) deposition as the miR-29 family are known to negatively
regulate ECM synthesis, targeting collagens, and osteonectin/
SPARC.[115] miR-29a inhibitor, complexed with TKO transfection agent, and loaded on these electrospun gelatin nanofibers
demonstrated continuous release of miR-29a inhibitor while
cultured MC3T3-E1 cells produced more osteonectin, among
other markers. BMSCs from transgenic pOBCol3.6 cyan
reporter mice, grown on nanofibers loaded with miR-29a inhibitor also showed elevated Col3.6 cyan expression demonstrating
successful transfection and enhanced collagen production. Lentivirus has been used to deliver miR-135 to ASCs for assessment of osteogenesis and bone repair in vitro and in vivo.[47]
For in vivo assessment, transduced ASCs were combined with
poly(sebacoyl diglyceride) (PSeD) scaffolds and implanted in
critical-sized rat calvarial defects. Results correlated with in vitro
assessment and showed that miR-135 mimic treatment resulted
in significant new bone formation with elevated BMD, increased
trabeculae number (Tb.N) and enhanced mineralization
staining. In contrast, miR-135 inhibitor treatment attenuated
these processes suggesting that miR-135 positively regulates in
vitro and in vivo ASC osteogenesis and bone regeneration.
Lipofectamine 2000-miR-221-inhibitor transfected ASCs
seeded on a nHA/PCL scaffold has also been assessed for
osteogenic enhancement.[56] This in vitro study demonstrated
significantly enhanced Runx2, osteocalcin and ALP activity. Interestingly, although gene expression demonstrated significantly
enhanced Runx2 and osteocalcin on miR-221-inhibitor transfected ASCs in monolayer, no significance was obtained in ALP
activity compared to control ASCs in osteogenic media. miR-15b
has been tested in combination with a carboxymethyl cellulose-nHA based scaffold to determine its effect on osteogenic
differentiation using mouse(m) MSCs transfected with
miRNA-15b using X-treme Gene transfection reagent.[125]
Individually, media from the scaffold and miR-15b treated
mMSCs were able to promote osteoblast differentiation and when these treatments were combined there was
1700695 (13 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
Figure 5. NanoantagomiR-133a(-3p) loaded scaffolds demonstrate enhanced hMSC osteogenesis. a,b) Runx2 and ALP mRNA expression was upregulated in the nanoantagomiR-133a-3p loaded scaffolds at day 3 and 7, respectively. Mean + standard deviation, n ≥ 4, *p < 0.05. c) Calcium content was
increased by day 14 and maintained after 28 d compared to the control groups. Mean + standard deviation, n = 3, **p < 0.001, #p < 0.001 compared
to all other groups. d) Alizarin red staining demonstrated calcium deposits and e) OCN immunofluorescence staining (green; blue = DAPI stained
nuclei) showed increased protein expression in the nanoantagomiR-133a loaded scaffolds after 14 and 28 d, scale bar = 50 µm. Adapted with permission.[124] Copyright 2016, the authors. Published under CC-BY 4.0 license.
an additive effect on promotion of osteoblast differentiation showing that this system may harbor some utility for
increasing mMSC osteogenesis in vitro. miR-2861 has
been shown to be increase the osteogenic commitment of
human periodontal ligament stem cells (hPDLSCs) grown
on a 3D scaffold and demonstrated the successful overexpression of miR-2861 and RUNX2 in hPDLSCs cultured in
presence of the scaffold under osteogenic and standard
conditions.[126] Xie et al.[127] showed that miR-146a negatively
regulates osteogenesis and bone regeneration of ASCs both in
vitro and in vivo. Overexpressing miR-146a using lentivirus suppressed ASC osteogenesis while inhibition of miR-146a greatly
promoted this process in vitro, corresponding with modulation
of miR-146a’s direct target, SMAD4, an important coactivator
in the BMP signaling pathway. Lentivirus-miR-146a mimic/
inhibitor transduced ASCs were then combined with PSeD scaffolds and implanted in critical-sized rat cranial defects. Treatment with miR-146a inhibitor greatly enhanced ASC-mediated
bone regeneration with higher expression levels of SMAD4,
Runx2, and Osterix detected in newly formed bone, thus outlining the important role for miR-146 in bone regeneration.
Lentiviral-based miR-125b down regulation has demonstrated
an important role in the process of BMSC osteogenic differentiation and in vivo bone repair by targeting bone morphogenetic protein type 1b receptor (BMPR1b).[128] Demineralized
bone matrices (DBMs) preseeded with the miR-125b inhibitor
Adv. Healthcare Mater. 2017, 1700695
transduced BMSCs displayed improved repair capacity of the
defects. This was highlighted by an increase in BMD by 51%, an
increase in Tb.N by 79% and enhanced histological staining for
new bone generation and bone maturity. Having demonstrated
the inhibition of miR-222 promoted osteogenic and chondrogenic differentiation in vitro, miR-222 has been mixed with atelocollagen and administered in a rat refractory fracture model for in
vivo bone healing assessment.[113] Bone union at the fracture site
and enhanced capillary density was achieved in miR-222 inhibitor treated groups 8 weeks postimplantation. Overall, this study
successfully demonstrated the effect of miR-222 knockdown on
acceleration of bone healing by enhancing osteogenesis, chondrogenesis, and angiogenesis. Osteoporotic bone defects have
also been healed by BV-engineered OVX-BM-MSCs expressing
miRNA sponges carrying miR-214 either alone or in combination
with BMP2.[130] This assessment was carried out by implanting
gelatin Spongostan scaffolds preseeded with transduced OVXBM-MSCs. Coexpression of BMP2 plasmid and miR-214 sponges
in OVX-BM-MSCs enhanced synergistic healing. These results
present a new approach for osteoporotic bone defect treatment.
5.2. Cartilage Repair
There remains a major unmet clinical need for the development of repair strategies for damaged cartilage. Even repair of
1700695 (14 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
small cartilage defects is hampered for reasons including the
avascular composition of cartilage tissue, low cell density and
slow diffusion of nutrients among others, but it is believed that
development of tissue-engineered RM strategies comprised of a
cell source such as MSCs, a biomaterial scaffold/hydrogel and
external signals such as the inclusion of GFs/genes/miRNAs
may hold particular promise. While substantial efforts elucidating miRNAs involved in cartilage development, disease and
repair have been reported to-date, perhaps surprisingly there
has only been one report describing scaffold-based miRNA
delivery; thus cartilage miRNA research is still very much in
its infancy.
Lolli et al.[112] recently published a body of work showing
silencing of antichondrogenic miR-221 in hMSCs promotes
cartilage repair in vivo. This group previously demonstrated
that silencing of the antichondrogenic regulator miR-221
resulted in enhanced chondrogenic marker expression in
monolayer BM-MSCs cultured without TGF-β.[159] This study
was validated here both in pellet culture and additionally in
vivo.[161] This study was validated here both in 3D pellet culture and additionally in vivo.[112] Lipofectamine RNAiMAXmiR-221 inhibitor was used to transfect monolayer BM-MSCs
and showed higher than 95% miR-221 knockdown before
resuspension in alginate. This alginate-cell suspension was
then added to simulated osteochondral defects and implanted
subcutaneously in nude mice. miR-221 inhibition significantly promoted in vivo cartilage repair after 12 weeks and
deposition of the hypertrophic marker, collagen type X, was
completely absent. This study opens new possibilities for
the use of scaffold-based miRNA delivery in cartilage tissue
engineering.
5.3. Muscle Repair
In order to use 3D engineered human skeletal muscle tissue
for translational studies to treat muscular diseases or injuries,
and as in vitro preclinical models for drug testing, differentiation and functional behavior of muscle cells within the microenvironment must be possible. One such strategy involves
the application of miRNA delivery but reports to-date remain
limited.
Recently, Cheng et al.[111] have been able to stimulate contraction of engineered 3D myobundles consisting of a fibrin
hydrogel and human skeletal myoblasts (HSkM). The study
examined the effects of siPORT NeoFxTM inhibition of miR133a and miR-696 on myogenic differentiation and function
in 2D and 3D cultures of engineered myotubes when cultured
under static conditions for 2 weeks. Overall, combined inhibition of miR-133a and miR-696 augmented differentiation,
promoted expression of PGC-1a, and amplified the contractile
force in 3D engineered myobundles validating 2D results.
5.4. Cardiovascular Tissue Repair
Repair of damaged myocardium for cardiovascular applications
is limited due to the low proliferative rate of cardiomyocytes
in adults[162] while insufficient angiogenesis has implications
Adv. Healthcare Mater. 2017, 1700695
in vascular disease. Scaffold-based miRNA delivery for cardiovascular-related tissue has garnered a reasonable amount of
interest in recent years as it can limit the risk of undesired offtarget miRNA effects on multiple mRNAs in several different
tissues and is currently a growing area of research as documented here.
Zhou et al.[116] developed an electrospun fibrous membrane
delivery system and target delivery carriers of miR-126 to vascular endothelial cells (VECs) in the local vascular environment.
The scaffold consisted of a bilayer poly(ethylene glycol)-b-poly(llactide-co-e-caprolactone) (PELCL), PCL, and gelatin. The innermost layer of PELCL was loaded with complexes in REDV peptide-modified trimethyl chitosan-PEG and was responsible for
regulating the response of VECs. Results showed that released
miR-126 controlled VEC proliferation by down-regulation of
SPRED-1 gene expression in vitro and improved endothelialization was found in vivo. Overall results suggest a useful platform for the localized delivery of miRNAs in vascular tissue
engineering.
Tissue-engineered hydrogels have also been utilized in a
study to enhance the direct reprogramming of fibroblasts into
cardiomyocytes using miRNAs.[110] As direct reprogramming
of cardiac fibroblasts in vitro is rather modest, a 3D hydrogel
was used to enhance Dharmafect-miR combo (miR-1, miR-133,
miR-208, miR-499) reprogramming of neonatal fibroblasts via
a matrix metalloproteinase (MMP)-dependent mechanism in a
more effective manner than 2D cell culture.
miR-29b has been the subject of a couple of papers related
to wound healing and myocardial infarction.[108,109] Firstly it
was shown to ameliorate ECM remodeling when delivered
from a collagen-based scaffold. Primary fibroblasts seeded on
scaffolds loaded with naked miR-29b demonstrated reduced
collagen type I and collagen type III mRNA expression of ≈40%
and ≈15% respectively following 2 weeks of in vitro culture.
The scaffolds were used to treat full thickness rat skin excisional wounds in vivo and showed reduced wound contraction,
a 25% increase in granulation tissue, increased collagen type
III/I ratios and improved MMP-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio when the scaffolds were functionalized with miR-29b in a dose-dependent manner (0.5 or 5 µg
miR-29b). As myocardial infarction (MI) results in debilitating
remodeling of the myocardial ECM, Monaghan et al.[109] performed a proof-of-principle study to modify aggressive myocardial remodeling by injecting a HyA-based hydrogel delivering
exogenous miR-29b. Myocardial ischemia/reperfusion was
performed on C57BL/6 mice for 45 min after the miRNA-activated hydrogel, composed of thiolated-HyA cross-linked with
PEG-diacrylate (Glycosan HyStem) and naked exogenous miR29b, was injected into the border zone of the infarcted myocardium. This resulted in an ≈10% increase in ejection fraction,
the maintenance of myocardial function following MI with
significantly decreased elastin, newly deposited collagen fibers,
increased vascularity of the myocardial scar and significantly
altered ECM-specific biochemical signals found at the border
zone of the infarct providing evidence for the use of injectable
biomaterials and exogenous miRNAs as a local treatment for
ischemic tissue to modulate tissue remodeling.
Another study undertook local lentiviral miRNA-26a delivery
in a rat vein neointimal hyperplasia graft model.[114] MiR-26a
1700695 (15 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
has been shown to target MAPK6 to inhibit vascular smooth
muscle cell (VSMC) proliferation making it a potential therapeutic target for autologous vein graft diseases. Vein segments
were immersed in lentivirus solution (LV3-miR-26a or LV3NC) and additional lentivirus solution was also preloaded into
Pluronic F-127 gel and gently painted around the grafted vein
segments. Delivery of LV3-miR-26a to the jugular vein inhibited graft-induced neointimal formation by up to 30%, resulted
in decreases in the intima-to-media ratio and the number of
MAPK6-expressing cells was also markedly decreased in the
LV3-miR-26a-transduced jugular veins. Thus, this study underscores yet another therapeutic function for this popular miR26a in RM applications.
In a similar theme of research, miR-221 sponge therapy was
utilized to attenuate neointimal hyperplasia and enhance vein
graft blood flow.[163] ADVs encoding miR-221 sponge (Ad-miR221-SP) were utilized to prevent VSMC proliferation in vitro
and neointimal formation in vivo. The same F-127 pluronic gel
was utilized to locally deliver Ad-miR-221-SP to the vein graft
walls in a rat model. miR-221 sponge gene transfer reduced cell
proliferation, neointimal area, thickness, and neointima/media
ratio in vein grafts versus controls. Improved hemodynamics
were detected in vein grafts and finally p27 (Kip1) was identified as a possible target gene of miR-221 in vein grafts. Overall
results offer this miR-sponge therapy as a promising method to
prevent vein graft failure.
5.5. Neurological Tissue Repair
To date, the strategies available for the treatment of majority of
neurological pathologies remain suboptimal but scaffold-based
miRNA delivery techniques offer new potential for the field
of neural tissue engineering. Here, we document the reports
available up to now in this research field.
The collective effects of scaffold topography and sustained
gene silencing of miR-219 and miR-338 on oligodendroglial
precursor cell (OPC) development have been assessed.[117] The
miRNAs, complexed with TransIT-TKO, were integrated onto
electrospun PCL fiber scaffolds with various fiber diameters
and orientations. Efficient knockdown of differentiation inhibitory factors was achieved irrespective of fiber size or orientation. Overall the study presented a promising method for
directing OPC maturation in neural tissue engineering and
controlling remyelination in the central nervous system. The
authors published a similar paper assessing the same miRNAs
to enhance OPC differentiation and maturation.[139] Overall, the
results of this study demonstrated the ability of PCL nanofibers
to provide topographical cues and miRNA reverse transfection
to direct OPC differentiation.
Recently, an aligned collagen-PCL-co-ethylethylene phosphate
nanofibrous hydrogel scaffold has been utilized to nonvirally
deliver miR-222-micellar NPs to direct axon regeneration in
spinal cord injury treatment.[164] In vivo analysis was carried out
on a rat spinal cord model to assess the efficacy of this hydrogel
system. Results revealed aligned axon regeneration 1 week
postinjury with no excessive inflammatory response or scar
tissue formation. Results thus demonstrated the potential of
this hydrogel system for neural tissue engineering applications.
Adv. Healthcare Mater. 2017, 1700695
Cui et al.[129] utilized a 3D collagen sponge-based scaffold to
culture neural stem cells (NSCs) and PA-1 cells for different
purposes. Though exogenous miRNA was not delivered on the
scaffolds, 3D culture was utilized to demonstrate downregulation of several miRNAs, including miR-20, whose target genes
are known to regulate self-renewal and differentiation of stem
cells in 3D cultured PA-1 cells. NSC differentiation was also
restricted in 3D scaffolds in comparison to 2D monolayer cell
culture. Essentially, the study suggests 3D cell culturing might
offer a system to demonstrate the regulatory mechanism of
cell modulators, which are challenging to uncover in conventional 2D cell cultures. Mercado et al.[118] used a similar strategy
using aminopropyl-silica nanofibers for the analysis of differentially expressed mRNAs and miRNAs of NSC differentiated
on these nanofibers. The nanomaterial promoted the enhanced
expression of let-7 miRNAs, which exhibit critical functions
in NSC differentiation. Though these studies do not involve
scaffold-based delivery of miRNAs, they highlight the utility of
3D scaffold-based systems for determination of more realistic
responses compared to or in addition to 2D and point to a new
line of research in the arena of neural tissue engineering.
5.6. Other Tissue Types: Cancer Treatments
While there is a large volume of research on the potential applications of miRNAs in cancer development and its treatment, it
is clear that selective and targeted treatment to tumor cells alone
needs increased research efforts. In direct and stark contrast to
the typical systemic delivery of most conventional chemotherapeutic drugs, scaffold-based miRNA delivery systems surface as
a highly appealing strategy. Furthermore, in recent years much
interest has started to grow on the use of 3D scaffolds as a
tool to study, in vitro, complex processes within a more in vivo
representative microenvironment. This research avenue holds
much potential to significantly improve the knowledge on targets of antitumoral treatments. As a result, there has been an
increase of late on the development of safe, effective scaffoldbased miRNA delivery systems explored in several cancer types,
in the form of both proof-of-concept and more complex detailed
studies, as documented herein.
A dipeptide Gly-Ala linked with biphenyl-substituted tetrazole (Tet-GA) gel for 3D culture has been described for miR-122
mimic delivery into 3D HepG2 cultured cells as a proof-ofconcept study.[165] miR-122 is a liver-specific miRNA that is
often reduced in hepatocellular carcinoma. Results indicated
that the miRNAs were able to inhibit target gene expression
demonstrating the first indication of 3D miRNA delivery into
live cells facilitated by a Tet-GA gel. Work performed more
recently by the same group focused on the use of a supramolecular hydrogel formed by spiropyran conjugated galactose
(MCI-Gal gel) for photocontrolled release of miR-122 mimic
and target-mediated delivery of the miRNA into the same
HepG2 cells cultured on the gel.[166] Visible light irradiation
was used to accelerate the release of the encapsulated miRNA
while the incorporation of galactose targeting ligand was able to
improve the miRNA delivery efficiency through ligand–receptor
interaction. Encapsulation of miRNA in the gel matrix was
proven to have no adverse effect on the biological function of
1700695 (16 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
the miRNA. This supramolecular hydrogel therefore provides
a possibility of investigating miRNA function in targeted cell
lines using a spatiotemporal approach.
Conversely, Beavers et al.[167] described a novel system for
the delivery of an inhibitor of miR-122. The authors described
a porous silicon and polymer nanocomposite (PSNP) system
for delivery of PNAs as anti-miRNA therapies describing it as
the first example of an endosomolytic PNA delivery platform
and of in vivo PNA-mediated miR-122 inhibition. PNAntimiR122-loaded PSNPs were injected intravenously into mouse
tail veins. Results showed 46% miR-122 inhibition, equating
to 16 times lower than that reported previously for RNAntagomiR-122,[26] and 2.5 times lower than 2-O-methoxyethyl
phosphorothioate antisense oligos. Efficacy of this inhibition
further resulted in 1.5-fold increase in mRNA levels of validated
downstream targets and ultimately provoked a beneficial 20%
reduction in HDL–cholesterol.[168] This study overcomes both
the crucial systemic and intracellular delivery obstacles facing
PNA by development of the strategic nanocomposite.
miR-34a, a known tumor suppressor gene, has also been
a miRNA of interest in a few scaffold-based cancer-related
miRNA delivery studies.[169,170] PCL scaffolds containing miR34a mimics were fabricated as a “neutral-lipid-emulsion”
delivery system and then implanted in the flank of severe
combined immunodeficiency (SCID) mice.[169] In vivo results
corroborated in vitro experiments where miR-34a mimics confirmed significant anti-proliferative effects, apoptotic activity
and control of gene expression, thus proving evidence of the
antitumoral activity of miR-34a in preclinical scaffold systems
of this disease. Zhou et al.[170] have most recently described a
proof-of-concept study outlining a multifunctional supramolecular hydrogel formed by the conjugate of the peptic sequence
GRGDS with biaryltetrazole (Tet(II)–GRGDS) for the delivery
of miR-34a into encapsulated U87 cells, a human primary
glioblastoma cell line. The presence of biaryltetrazolein in the
hydrogel results in a sensitive photo-response reaction which
allowed photodegradation of the hydrogel for release of the
encapsulated live cells. U87 cells were firstly cotransfected with
Luc-miR-34a plasmid and β-galactosidase plasmid using Lipofectamine2000 before being mixed with the hydrogel. Functionality results showed a significant decrease of ≈80% of luciferase
signals in U87 cells encapsulated in the Tet(II)–GRGDS gel
indicating its potential as a 3D carrier. Shatsberg et al.[171] have
also utilized delivery of miR-34a as a replacement therapy in a
proof-of-concept study, delivered by 6 different polymeric nanogels (NGs), which are based on a polyglycerol-scaffold, with the
view to inhibition of glioblastoma via downregulation of miR34a target oncogenes. U87 cells, as used in the previous study,
were transfected with NG-miR-34a nanopolyplexes and resulted
in significant downregulation of miR-34a target genes, while
treatment of SCID mice with intratumoral injection of human
U87s expressing NG-miR-34a showed dramatic knockdown of
tumor growth. The study outlined the vital characteristics for
the coherent design of polymeric delivery systems for oligonucleotides and this novel therapeutic method may lead the path
for safe, efficient therapies for glioblastoma.
Conde et al.[78] have described a recent study describing
the use of a self-assembled RNA-triple-helix structure containing two miRNAs, a miR-205 mimic and antagomiR-221,
that synergistically abrogate tumors in a triple-negative breast
cancer mouse model (Figure 6). The RNA triplex NPs were
combined with PAMAM dendrimers and dextran aldehyde to
form a dextran–dendrimer–RNA triplex hydrogel that strongly
adheres to tumor tissue. For in vivo assessment, hydrogel scaffolds were loaded with the miRNAs or drugs (doxorubicin,
paclitaxel, and the monoclonal antibody bevacizumab (Avastin)
Figure 6. In vivo miRNA modulation and tumor therapy via PAMAM RNA-triple-helix dextran-based hydrogel scaffolds. a) Single or dual-colored
miR-nanoconjugates forming the RNA-triple-helix nanoparticles within the hydrogel scaffolds demonstrate homogenous distribution throughout its
structure. b) 12 µm cryosection of RNA-triple-helix hydrogel depicting dextran presence (Alexa Fluor 405) and with nanoparticles containing the
dual-colored miRs shown as red spots. c) Presence of the RNA-triple-helix nanoparticles embedded in dextran hydrogels is detected via fluorescence
live imaging when implanted in female SCID mice with triple-negative breast tumor xenograft, n = 5. Adapted with permission.[78] Copyright 2016,
Macmillan Publisher Ltd.
Adv. Healthcare Mater. 2017, 1700695
1700695 (17 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
and implanted for 13 d next to the tumor in the mammary fat
pad of SCID mice. Only the RNA-triple-helix hydrogel scaffolds (carrying miR-205 mimic and antagomiR-221) resulted
in efficient, sustained inhibition of tumor progression, with
≈90% tumor size reduction. The findings suggested that this
may be an effective anticancer platform for local control endogenous miRNA expression in cancer. Many of the same authors
were also involved in another study involving local miR-96 or
miR-182 treatment for targeting Palladin, a gene involved in
the invasive behavior of breast cancer cells, thereby preventing
metastatic breast cancer.[172] An oxidized AuNP-dextran-dendrimer based hydrogel system was used to provide an effective
local, selective, and sustained release of miR-96/miR-182, and
demonstrated prominent suppression of metastasis in a mouse
breast cancer model. By combining the codelivery of miRNAs
with the chemotherapy drug, cisplatin, significant primary
tumor reduction and metastasis inhibition were obtained. This
combined therapy (involving cisplatin and miRNAs) thus suggests a plausible reason for the inclusion of miRNA therapy as
an additive to conventional chemotherapy.
6. Conclusion and Future Outlook
As the immense potential of miRNA therapeutics has exploded,
scaffold-mediated miRNA-based delivery systems have garnered significant attention in recent years. miRNA therapeutics have been widely explored for cancer treatment and
genetic disorders, although their use in RM has somewhat
lagged behind. To date, much of the research has focused on
miRNA profiling and elucidating miRNA involvement in signaling pathways but slowly this work has begun to inspire RM
applications as we have described herein. Unfortunately however, there is currently no real consensus on which direction
is best for the field to proceed as it is still laying its foundations. As can be deduced from Table 2, the nature of scaffolds
used is highly variable, vector type in terms of RM although
variable has had the most reports using lentivirus or baculovirus and the cells types most commonly assessed include
BM-MSCs and ASCs. In terms of miRNA targets, some leaders
have emerged depending on the application being assessed;
miR-26a, miR-31, and miR-148b have set the precedent for the
osteogenesis and bone repair field, miR-29b for the cardiovascular field while miR-122 and miR-34a are the current frontrunners when it comes to miRNA scaffold-based therapies
for cancer treatment. A number of challenges still need to be
fully addressed and resolved however before the true potential
of miRNA scaffold-based therapies is realized including development of the most efficient scaffold-vector delivery system,
biological stability, precise targeting capacity and prolonged
but transient presentation in addition to limitation of offtarget effects. Despite the challenges identified with miRNA
therapeutics, companies like Santaris Pharma, Regulus Therapeutics, MiRagen Therapeutics, and EnGeneIC have recently
embarked in clinical trials, taking up the challenge to translate
miRNA therapies to patient benefit. From these early stage
trials it appears that a new era of clinical translation is on the
horizon, with the focus on the application of miRNA-based
medicine looming to go beyond cancer therapies. In particular,
Adv. Healthcare Mater. 2017, 1700695
personalized medicine, based on the patient’s personal miRNA
signature, may be one area where precise tailoring of scaffoldmediated miRNA therapies can be exploited. To date, the field
of scaffold-mediated RNA delivery resides at a relatively nascent juncture; however, the bulk of reports on siRNA delivery
provide valuable strategies thought to accommodate miRNA
therapeutics almost seamlessly. Ultimately, the development of
efficient and effective scaffold-based miRNA therapies would
appear to offer significant promise though much work still
needs to be completed from a regulatory perspective. In conclusion, we believe scaffold-mediated miRNA delivery will play
a vital role in the future of RM and cancer therapy. With the
constant evolution and rapid identification of new targets on
an almost daily basis, we envisage a spate of new strategies
will drive this field to even further, exciting new levels in the
coming years.
Acknowledgements
C.M.C. and I.M.C. contributed equally to this work. This work was
supported by the European Research Council under the European
Community’s Horizon 2020 Framework Programme/ERC grant
agreement no. 665777.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
cancer, microRNA, miRNA, regenerative medicine, scaffold
Received: June 2, 2017
Revised: August 21, 2017
Published online:
[1] a) W. A. Haseltine, e-biomed: J. Tissue Eng. Regener. Med. 2001,
2, 17; b) D. Sheyn, O. Mizrahi, S. Benjamin, Z. Gazit, G. Pelled,
D. Gazit, Adv. Drug Delivery Rev. 2010, 62, 683.
[2] A. Joshi, MnM, http://www.marketsandmarkets.com/MarketReports/regenerative-medicine-market-65442579.html (accessed:
May 2017).
[3] a) E. M. Younger, M. W. Chapman, J. Orthop. Trauma 1989, 3, 192;
b) J. P. Gleeson, N. A. Plunkett, F. J. O’Brien, Eur. Cell Mater. 2010,
20, 218.
[4] F. J. O’Brien, Mater. Today 2011, 14, 88.
[5] a) M. Lagos-Quintana, R. Rauhut, W. Lendeckel, T. Tuschl,
Science 2001, 294, 853; b) N. C. Lau, L. P. Lim, E. G. Weinstein,
D. P. Bartel, Science 2001, 294, 858; c) R. C. Lee, V. Ambros,
Science 2001, 294, 862.
[6] a) R. C. Lee, R. L. Feinbaum, V. Ambros, Cell 1993, 75, 843;
b) B. Wightman, I. Ha, G. Ruvkun, Cell 1993, 75, 855.
[7] a) B. J. Reinhart, F. J. Slack, M. Basson, A. E. Pasquinelli,
J. C. Bettinger, A. E. Rougvie, H. R. Horvitz, G. Ruvkun, Nature
2000, 403, 901; b) F. J. Slack, M. Basson, Z. Liu, V. Ambros,
H. R. Horvitz, G. Ruvkun, Mol. Cell 2000, 5, 659;
c) A. E. Pasquinelli, B. J. Reinhart, F. Slack, M. Q. Martindale,
M. I. Kuroda, B. Maller, D. C. Hayward, E. E. Ball, B. Degnan,
1700695 (18 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
P. Muller, J. Spring, A. Srinivasan, M. Fishman, J. Finnerty,
J. Corbo, M. Levine, P. Leahy, E. Davidson, G. Ruvkun, Nature
2000, 408, 86.
[8] Y. Lee, K. Jeon, J. T. Lee, S. Kim, V. N. Kim, EMBO J. 2002, 21,
4663.
[9] a) Y. Zeng, B. R. Cullen, RNA 2003, 9, 112; b) E. Basyuk, F. Suavet,
A. Doglio, R. Bordonne, E. Bertrand, Nucleic Acids Res. 2003, 31,
6593; c) Y. Lee, C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee,
P. Provost, O. Radmark, S. Kim, V. N. Kim, Nature 2003, 425, 415.
[10] a) R. Yi, Y. Qin, I. G. Macara, B. R. Cullen, Genes Dev. 2003, 17,
3011; b) E. Lund, S. Guttinger, A. Calado, J. E. Dahlberg, U. Kutay,
Science 2004, 303, 95.
[11] a) A. Grishok, A. E. Pasquinelli, D. Conte, N. Li, S. Parrish, I. Ha,
D. L. Baillie, A. Fire, G. Ruvkun, C. C. Mello, Cell 2001, 106, 23;
b) G. Hutvagner, J. McLachlan, A. E. Pasquinelli, E. Balint,
T. Tuschl, P. D. Zamore, Science 2001, 293, 834; c) C. Z. Chen,
L. Li, H. F. Lodish, D. P. Bartel, Science 2004, 303, 83.
[12] a) A. A. Aravin, M. Lagos-Quintana, A. Yalcin, M. Zavolan,
D. Marks, B. Snyder, T. Gaasterland, J. Meyer, T. Tuschl, Dev.
Cell 2003, 5, 337; b) L. P. Lim, N. C. Lau, E. G. Weinstein,
A. Abdelhakim, S. Yekta, M. W. Rhoades, C. B. Burge,
D. P. Bartel, Genes Dev. 2003, 17, 991; c) A. Khvorova, A. Reynolds,
S. D. Jayasena, Cell 2003, 115, 209; d) D. S. Schwarz, G. Hutvagner,
T. Du, Z. Xu, N. Aronin, P. D. Zamore, Cell 2003, 115, 199.
[13] J. R. Lytle, T. A. Yario, J. A. Steitz, Proc. Natl. Acad. Sci. USA 2007,
104, 9667.
[14] H. Guo, N. T. Ingolia, J. S. Weissman, D. P. Bartel, Nature 2010,
466, 835.
[15] a) A. Jacobsen, J. Silber, G. Harinath, J. T. Huse, N. Schultz,
C. Sander, Nat. Struct. Mol. Biol. 2013, 20, 1325; b) Z. Mourelatos,
J. Dostie, S. Paushkin, A. Sharma, B. Charroux, L. Abel,
J. Rappsilber, M. Mann, G. Dreyfuss, Genes Dev. 2002, 16, 720;
c) D. P. Bartel, Cell 2004, 116, 281.
[16] B. P. Lewis, C. B. Burge, D. P. Bartel, Cell 2005, 120, 15.
[17] Y. C. Lo, Y. H. Chang, B. L. Wei, Y. L. Huang, W. F. Chiou, J. Agric.
Food Chem. 2010, 58, 6643.
[18] a) R. Hu, H. Li, W. Liu, L. Yang, Y. Tan, X. Luo, Expert Opin. Ther.
Targets 2010, 14, 1109; b) W. W. Yau, P. O. Rujitanaroj, L. Lam,
S. Y. Chew, Biomaterials 2012, 33, 2608; c) P. J. McKiernan,
C. M. Greene, Mediators Inflamm 2015, 2015, 529642;
d) I. K. Oglesby, N. G. McElvaney, C. M. Greene, Respir. Res. 2010,
11, 148.
[19] P. McKiernan, O. Cunningham, C. Greene, S.-A. Cryan, Int. J.
Nanomed. 2013, 8, 3907.
[20] E. van Rooij, A. L. Purcell, A. A. Levin, Circ. Res. 2012, 110, 496.
[21] B. C. Bernardo, F. J. Charchar, R. C. Y. Lin, J. R. McMullen, Heart,
Lung Circ. 2012, 21, 131.
[22] a) D. A. Braasch, D. R. Corey, Chem. Biol. 2001, 8, 1;
b) M. Petersen, J. Wengel, Trends Biotechnol. 2003, 21, 74.
[23] M. M. Fabani, M. J. Gait, RNA 2008, 14, 336.
[24] E. van Rooij, S. Kauppinen, EMBO Mol. Med. 2014, 6, 851.
[25] K. R. Beavers, C. E. Nelson, C. L. Duvall, Adv. Drug Delivery Rev.
2015, 88, 123.
[26] J. Krutzfeldt, N. Rajewsky, R. Braich, K. G. Rajeev, T. Tuschl,
M. Manoharan, M. Stoffel, Nature 2005, 438, 685.
[27] a) M. S. Ebert, J. R. Neilson, P. A. Sharp, Nat. Methods 2007, 4,
721; b) M. S. Ebert, P. A. Sharp, RNA 2010, 16, 2043.
[28] a) H. L. Janssen, S. Kauppinen, M. R. Hodges, N. Engl. J. Med.
2013, 369, 878; b) ClinicalTrials.gov, U.S. N. I. H., http://clinicaltrials.gov/show/NCT01829971 (accessed: October 2013).
[29] K. Takeda, Br. J. Pharmacol. 2009, 157, 151.
[30] R. R. Chen, D. J. Mooney, Pharm. Res. 2003, 20, 1103.
[31] J. M. Wozney, V. Rosen, Clin. Orthop. Relat. Res. 1998, http://www.
ncbi.nlm.nih.gov/pubmed/9577407, p. 26.
Adv. Healthcare Mater. 2017, 1700695
[32] M. J. Marzi, F. Ghini, B. Cerruti, S. de Pretis, P. Bonetti,
C. Giacomelli, M. M. Gorski, T. Kress, M. Pelizzola, H. Muller,
B. Amati, F. Nicassio, Genome Res. 2016, 26, 554.
[33] E. E. Vaughan, J. V. DeGiulio, D. A. Dean, Curr. Gene Ther. 2006,
6, 671.
[34] Y. Pan, Y. Zhang, T. Jia, K. Zhang, J. Li, L. Wang, FEBS J. 2012, 279,
1198.
[35] M. Monaghan, A. Pandit, Adv. Drug Delivery Rev. 2011, 63, 197.
[36] S. Y. Chew, Adv Drug Delivery Rev. 2015, 88, 1.
[37] P. Ilina, Z. Hyvonen, M. Saura, K. Sandvig, M. Yliperttula,
M. Ruponen, J. Controlled Release 2012, 163, 385.
[38] P. Midoux, C. Pichon, J. J. Yaouanc, P. A. Jaffres, Br. J. Pharmacol.
2009, 157, 166.
[39] D. W. Pack, A. S. Hoffman, S. Pun, P. S. Stayton, Nat. Rev. Drug
Discovery 2005, 4, 581.
[40] R. M. Raftery, D. P. Walsh, I. Mencía Castaño, A. Heise, G. P. Duffy,
S.-A. Cryan, F. J. O’Brien, Adv. Mater. 2016, 28, 5447.
[41] M. Edelstein, The Journal of Gene Medicine Clinical Trial
site,
http://www.wiley.com//legacy/wileychi/genmed/clinical/
(accessed: May 2017).
[42] a) E. M. A. European Medicines Agency, EMA/CHMP/249031/2016,
2016; b) E. M. A. European Medicines Agency, EMA/454627/2016,
2016.
[43] Y. P. Liu, B. Berkhout, Biochim. Biophys. Acta, Gene Regul. Mech.
2011, 1809, 732.
[44] a)
S. Laufs,
G. Guenechea,
A. Gonzalez-Murillo,
K. Zsuzsanna Nagy, M. Luz Lozano, C. del Val, S. Jonnakuty,
A. Hotz-Wagenblatt, W. Jens Zeller, J. A. Bueren, S. Fruehauf,
J. Gene Med. 2006, 8, 1197; b) E. Montini, D. Cesana, M. Schmidt,
F. Sanvito, C. C. Bartholomae, M. Ranzani, F. Benedicenti,
L. S. Sergi, A. Ambrosi, M. Ponzoni, C. Doglioni, C. Di Serio,
C. von Kalle, L. Naldini, J. Clin. Invest. 2009, 119, 964.
[45] Y. Deng, H. Zhou, D. Zou, Q. Xie, X. Bi, P. Gu, X. Fan, Biomaterials
2013, 34, 6717.
[46] Y. Deng, X. Bi, H. Zhou, Z. You, Y. Wang, P. Gu, X. Fan, Eur. Cell
Mater. 2014, 27, 13.
[47] Q. Xie, Z. Wang, H. Zhou, Z. Yu, Y. Huang, H. Sun, X. Bi, Y. Wang,
W. Shi, P. Gu, X. Fan, Biomaterials 2016, 75, 279.
[48] M. B. Mowa, C. Crowther, P. Arbuthnot, Expert Opin. Drug Delivery
2010, 7, 1373.
[49] C. L. Chen, W. Y. Luo, W. H. Lo, K. J. Lin, L. Y. Sung, Y. S. Shih,
Y. H. Chang, Y. C. Hu, Biotechnol. Bioeng. 2011, 108, 2958.
[50] Y.-H. Liao, Y.-H. Chang, L.-Y. Sung, K.-C. Li, C.-L. Yeh, T.-C. Yen,
S.-M. Hwang, K.-J. Lin, Y.-C. Hua, Biomaterials 2014, 35, 4901.
[51] Y. Pan, T. Jia, Y. Zhang, K. Zhang, R. Zhang, J. Li, L. Wang,
Int. J. Nanomed. 2012, 7, 5957.
[52] J. L. Santos, D. Pandita, J. Rodrigues, A. P. Pego, P. L. Granja,
H. Tomas, Curr. Gene Ther. 2011, 11, 46.
[53] K. Wu, W. Song, L. Zhao, M. Liu, J. Yan, M. O. Andersen,
J. Kjems, S. Gao, Y. Zhang, ACS Appl. Mater. Interfaces 2013, 5,
2733.
[54] T. Eskildsen, H. Taipaleenmaki, J. Stenvang, B. M. Abdallah,
N. Ditzel, A. Y. Nossent, M. Bak, S. Kauppinen, M. Kassem, Proc.
Natl. Acad. Sci. USA 2011, 108, 6139.
[55] Z. Wang, D. Zhang, Z. Hu, J. Cheng, C. Zhuo, X. Fang, Y. Xing,
Mol. Med. Rep. 2015, 12, 3345.
[56] S. Hoseinzadeh, A. Atashi, M. Soleimani, E. Alizadeh,
N. Zarghami, In Vitro Cell Dev. Biol. Anim. 2016, 52, 479.
[57] Y. Chen, J. Gelfond, L. M. McManus, P. K. Shireman, Physiol.
Genomics 2011, 43, 621.
[58] Y. Li, L. Fan, S. Liu, W. Liu, H. Zhang, T. Zhou, D. Wu, P. Yang,
L. Shen, J. Chen, Y. Jin, Biomaterials 2013, 34, 5048.
[59] M. Kulkarni, U. Greiser, T. O’Brien, A. Pandit, Trends Biotechnol.
2010, 28, 28.
1700695 (19 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
[60] a) K. Lappalainen, I. Jaaskelainen, K. Syrjanen, A. Urtti, S. Syrjanen,
Pharm. Res. 1994, 11, 1127; b) H. J. Burger, J. D. Schuetz,
E. G. Schuetz, P. S. Guzelian, Proc. Natl. Acad. Sci. USA 1992, 89,
2145.
[61] a) Y. Zhang, Z. Wang, R. A. Gemeinhart, J. Controlled Release 2013,
172, 962; b) A. Schade, P. Muller, E. Delyagina, N. Voronina,
A. Skorska, C. Lux, G. Steinhoff, R. David, Stem Cells Int. 2014,
2014, 197154.
[62] A. Schade, E. Delyagina, D. Scharfenberg, A. Skorska, C. Lux,
R. David, G. Steinhoff, Int. J. Mol. Sci. 2013, 14, 10710.
[63] X. Zhang, Y. Li, Y. E. Chen, J. Chen, P. X. Ma, Nat. Commun. 2016,
7, 10376.
[64] L. Liu, S. Shu, G. S. Cheung, X. Wei, Biomed. Res. Int. 2016, 2016,
3892685.
[65] J.-P. Behr, CHIMIA Int. J. Chem. 1997, 51, 34.
[66] R. Ghosh, L. C. Singh, J. M. Shohet, P. H. Gunaratne, Biomaterials
2013, 34, 807.
[67] a) E. Vuorimaa, T. M. Ketola, J. J. Green, M. Hanzlikova,
H. Lemmetyinen, R. Langer, D. G. Anderson, A. Urtti,
M. Yliperttula, J. Controlled Release 2011, 154, 171; b) K. Guk,
H. Lim, B. Kim, M. Hong, G. Khang, D. Lee, Int. J. Pharm. 2013,
453, 541; c) D. Jere, C. X. Xu, R. Arote, C. H. Yun, M. H. Cho,
C. S. Cho, Biomaterials 2008, 29, 2535.
[68] F. Yang, J. J. Green, T. Dinio, L. Keung, S. W. Cho, H. Park,
R. Langer, D. G. Anderson, Gene Ther. 2009, 16, 533.
[69] Q. Yin, Y. Gao, Z. Zhang, P. Zhang, Y. Li, J. Controlled Release 2011,
151, 35.
[70] K. Remaut, N. Symens, B. Lucas, J. Demeester, S. C. De Smedt,
J. Controlled Release 2010, 144, 65.
[71] P. Gupta, S. P. Authimoolam, J. Z. Hilt, T. D. Dziubla, Acta Biomater. 2015, 27, 194.
[72] a) Y. Wang, D. W. Malcolm, D. S. W. Benoit, Biomaterials 2017,
139, 127; b) C. E. Nelson, A. J. Kim, E. J. Adolph, M. K. Gupta,
F. Yu, K. M. Hocking, J. M. Davidson, S. A. Guelcher, C. L. Duvall,
Adv. Mater. 2014, 26, 607.
[73] X. Qian, L. Long, Z. Shi, C. Liu, M. Qiu, J. Sheng, P. Pu, X. Yuan,
Y. Ren, C. Kang, Biomaterials 2014, 35, 2322.
[74] K. A. Fitzgerald, M. Malhotra, C. M. Curtin, F. J. O’Brien,
C. M. O’Driscoll, J. Controlled Release 2015, 215, 39.
[75] K. A. Fitzgerald, J. Guo, R. M. Raftery, I. M. Castano, C. M. Curtin,
M. Gooding, R. Darcy, F. J. O’Brien, C. M. O’Driscoll, Int. J. Pharm.
2016, 511, 1058.
[76] Z. Guo, H. Peng, J. Kang, D. Sun, Biomed. Rep. 2016, 4,
528.
[77] B. N. Sathy, D. Olvera, T. Gonzalez-Fernandez, G. M. Cunniffe,
S. Pentlavalli, P. Chambers, O. Jeon, E. Alsberg, H. O. McCarthy,
N. Dunne, T. L. Haut Donahue, D. J. Kelly, J. Mater. Chem. B 2017,
5, 1753.
[78] J. Conde, N. Oliva, M. Atilano, H. S. Song, N. Artzi, Nat. Mater.
2016, 15, 353.
[79] J. Haensler, F. C. Szoka Jr., Bioconjug. Chem. 1993, 4, 85.
[80] A. Diaz-Moscoso, D. Vercauteren, J. Rejman, J. M. Benito,
C. Ortiz Mellet, S. C. De Smedt, J. M. Fernandez, J. Controlled
Release 2010, 143, 318.
[81] J. Wang, Z. Lu, M. G. Wientjes, J. L. Au, AAPS J. 2010, 12,
492.
[82] E. M. McErlean, C. M. McCrudden, H. O. McCarthy, in Gene
Therapy—Principles and Challenges (Ed: D. D. Hashad), InTech,
Rijeka, 2015.
[83] F. C. MacLaughlin, R. J. Mumper, J. Wang, J. M. Tagliaferri, I. Gill,
M. Hinchcliffe, A. P. Rolland, J. Controlled Release 1998, 56,
259.
[84] J. Malmo, H. Sorgard, K. M. Varum, S. P. Strand, J. Controlled
Release 2012, 158, 261.
Adv. Healthcare Mater. 2017, 1700695
[85] R. M. Raftery, E. G. Tierney, C. M. Curtin, S. A. Cryan, F. J. O’Brien,
J. Controlled Release 2015, 210, 84.
[86] B. Santos-Carballal, L. J. Aaldering, M. Ritzefeld, S. Pereira,
N. Sewald, B. M. Moerschbacher, M. Gotte, F. M. Goycoolea, Sci.
Rep. 2015, 5, 13567.
[87] M. Quattrocelli, M. Sampaolesi, Adv. Drug Delivery Rev. 2015, 88,
37.
[88] S. G. Ong, W. H. Lee, K. Kodo, J. C. Wu, Adv. Drug Delivery Rev
2015, 88, 3.
[89] J. P. Armstrong, M. N. Holme, M. M. Stevens, ACS Nano 2017, 11,
69.
[90] R. Ghosh, L. C. Singh, J. M. Shohet, P. H. Gunaratne, Biomaterials
2013, 34, 807.
[91] A. T. Qureshi, W. T. Monroe, V. Dasa, J. M. Gimble, D. J. Hayes,
Biomaterials 2013, 347799.
[92] A. T. Qureshi, A. Doyle, C. Chen, D. Coulon, V. Dasa, F. Del Piero,
B. Levi, W. T. Monroe, J. M. Gimble, D. J. Hayes, Acta Biomater.
2015, 12, 166.
[93] a) S. Bose, S. Tarafder, Acta Biomater. 2012, 8, 1401; b) J. Li,
Y. C. Chen, Y. C. Tseng, L. Huang, J. Controlled Release 2010, 142, 416.
[94] G. M. Cunniffe, F. J. O’Brien, S. Partap, T. J. Levingstone,
K. T. Stanton, G. R. Dickson, J. Biomed. Mater. Res. A 2010, 95, 1142.
[95] C. M. Curtin, G. M. Cunniffe, F. G. Lyons, K. Bessho,
G. R. Dickson, G. P. Duffy, F. J. O’Brien, Adv. Mater. 2012, 24, 749.
[96] I. Mencía Castaño, C. M. Curtin, G. Shaw, J. M. Murphy,
G. P. Duffy, F. J. O’Brien, J. Controlled Release 2015, 200, 42.
[97] S. Neumann, A. Kovtun, I. D. Dietzel, M. Epple, R. Heumann, Biomaterials 2009, 30, 6794.
[98] Q. Wu, D. Chen, M. J. Zuscik, R. J. O’Keefe, R. N. Rosier, J. Bone
Miner. Res. 2008, 23, 552.
[99] J. Bonadio, E. Smiley, P. Patil, S. Goldstein, Nat. Med. 1999,
5, 753.
[100] J. Fang, Y. Y. Zhu, E. Smiley, J. Bonadio, J. P. Rouleau,
S. A. Goldstein, L. K. McCauley, B. L. Davidson, B. J. Roessler,
Proc. Natl. Acad. Sci. USA 1996, 93, 5753.
[101] a) K. Lee, E. A. Silva, D. J. Mooney, J. R. Soc. Interface 2011, 8,
153; b) M. Keeney, J. J. van den Beucken, P. M. van der Kraan,
J. A. Jansen, A. Pandit, Biomaterials 2010, 31, 2893.
[102] M. K. Nguyen, O. Jeon, M. D. Krebs, D. Schapira, E. Alsberg, Biomaterials 2014, 35, 6278.
[103] F. J. O’Brien, B. A. Harley, M. A. Waller, I. V. Yannas, L. J. Gibson,
P. J. Prendergast, Technol. Health Care 2007, 15, 3.
[104] M. Sriram, R. Sainitya, V. Kalyanaraman, S. Dhivya,
N. Selvamurugan, Int. J. Biol. Macromol. 2015, 74, 404.
[105] a) G. Pelled, A. Ben-Arav, C. Hock, D. G. Reynolds, C. Yazici,
Y. Zilberman, Z. Gazit, H. Awad, D. Gazit, E. M. Schwarz, Tissue
Eng., Part B 2010, 16, 13; b) N. Kimelman-Bleich, G. Pelled,
Y. Zilberman, I. Kallai, O. Mizrahi, W. Tawackoli, Z. Gazit, D. Gazit,
Mol. Ther. 2011, 19, 53.
[106] P. D. Mariner, E. Johannesen, K. S. Anseth, J. Tissue Eng. Regener.
Med. 2011, 6, 314.
[107] C. H. Evans, Expert Rev. Mol. Med. 2010, 12, e18.
[108] M. G. Monaghan, S. Browne, K. Schenke-Layland, A. Pandit, Mol.
Ther. 2014, 22, 786.
[109] M. G. Monaghan, M. Holeiter, E. Brauchle, S. L. Layland, Y. Lu,
A. Deb, A. Pandit, A. Nsair, K. Schenke-Layland, Tissue Eng., Part A
2017, https://doi.org/10.1089/ten.TEA.2016.0527.
[110] Y. Li, S. Dal-Pra, M. Mirotsou, T. M. Jayawardena,
C. P. Hodgkinson, N. Bursac, V. J. Dzau, Sci. Rep. 2016, 6, 38815.
[111] C. S. Cheng, L. Ran, N. Bursac, W. E. Kraus, G. A. Truskey, Tissue
Eng., Part A 2016, 22, 573.
[112] A. Lolli, R. Narcisi, E. Lambertini, L. Penolazzi, M. Angelozzi,
N. Kops, S. Gasparini, G. J. van Osch, R. Piva, Stem Cells 2016, 34,
1801.
1700695 (20 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
[113] M. Yoshizuka, T. Nakasa, Y. Kawanishi, S. Hachisuka, T. Furuta,
S. Miyaki, N. Adachi, M. Ochi, J. Orthop. Sci. 2016, 21, 852.
[114] J. Tan, L. Yang, C. Liu, Z. Yan, Sci. Rep. 2017, 7, 46602.
[115] E. N. James, A. M. Delany, L. S. Nair, Acta Biomater. 2014, 10,
3571.
[116] F. Zhou, X. Jia, Y. Yang, Q. Yang, C. Gao, S. Hu, Y. Zhao, Y. Fan,
X. Yuan, Acta Biomater. 2016, 43, 303.
[117] H. J. Diao, W. C. Low, Q. R. Lu, S. Y. Chew, Biomaterials 2015, 70,
105.
[118] A. T. Mercado, J. M. Yeh, T. Y. Chin, W. S. Chen, Y. W. Chen-Yang,
C. Y. Chen, J. Biomed. Mater. Res., Part A 2016, 104, 2730.
[119] K. Wu, W. Song, L. Zhao, M. Liu, J. Yan, M. Ø. Andersen, J. Kjems,
S. Gao, Y. Zhang, ACS Appl. Mater. Interfaces 2013, 5, 2733.
[120] Y. Deng, H. Zhou, D. Zou, Q. Xie, X. Bi, P. Gu, X. Fan, Biomaterials
2013, 34, 6717.
[121] Y. Deng, H. Zhou, P. Gu, X. Fan, Invest. Ophthalmol. Vis. Sci. 2014,
55, 6016.
[122] L. Y. Sung, C. L. Chen, S. Y. Lin, S. M. Hwang, C. H. Lu, K. C. Li,
A. S. Lan, Y. C. Hu, Nucleic Acids Res. 2013, 41, e139.
[123] P. D. Mariner, E. Johannesen, K. S. Anseth, J. Tissue Eng. Regener.
Med. 2012, 6, 314.
[124] I. Mencia Castano, C. M. Curtin, G. P. Duffy, F. J. O’Brien, Sci. Rep.
2016, 6, 27941.
[125] S. Vimalraj, S. Saravanan, M. Vairamani, C. Gopalakrishnan,
T. P. Sastry, N. Selvamurugan, Int. J. Biol. Macromol. 2016, 93, 1457.
[126] F. Diomede, I. Merciaro, S. Martinotti, M. F. Cavalcanti, S. Caputi,
E. Mazzon, O. Trubiani, J. Biol. Regul. Homeostatic Agents 2016, 30,
1009.
[127] Q. Xie, W. Wei, J. Ruan, Y. Ding, A. Zhuang, X. Bi, H. Sun, P. Gu,
Z. Wang, X. Fan, Sci. Rep. 2017, 7, 42840.
[128] H. Wang, Z. Xie, T. Hou, Z. Li, K. Huang, J. Gong, W. Zhou,
K. Tang, J. Xu, S. Dong, Cell Physiol. Biochem. 2017, 41, 530.
[129] Y. Cui, Z. Xiao, T. Chen, J. Wei, L. Chen, L. Liu, B. Chen, X. Wang,
X. Li, J. Dai, Stem Cells Dev. 2014, 23, 393.
[130] K. C. Li, Y. H. Chang, C. L. Yeh, Y. C. Hu, Biomaterials 2016, 74,
155.
[131] R. W. Bucholz, Clin. Orthop. Relat. Res. 2002, p. 44, http://www.
ncbi.nlm.nih.gov/pubmed/11937865.
[132] A. Hasan, A. Khattab, M. A. Islam, K. A. Hweij, J. Zeitouny,
R. Waters, M. Sayegh, M. M. Hossain, A. Paul, Adv. Sci. 2015, 2,
1500122.
[133] M. M. Stevens, J. H. George, Science 2005, 310, 1135.
[134] L. Zhang, T. J. Webster, Nano Today 2009, 4, 66.
[135] E. L. Hedberg, C. K. Shih, J. J. Lemoine, M. D. Timmer,
M. A. Liebschner, J. A. Jansen, A. G. Mikos, Biomaterials 2005, 26,
3215.
[136] C. T. Huynh, M. K. Nguyen, M. Naris, G. Y. Tonga, V. M. Rotello,
E. Alsberg, Nanomedicine 2016, 11, 1535.
[137] M. K. Nguyen, O. Jeon, M. D. Krebs, D. Schapira, E. Alsberg, Biomaterials 2014, 35, 6278.
[138] E. Dawes, N. Rushton, Clin. Mater. 1994, 17, 157.
[139] H. J. Diao, W. C. Low, U. Milbreta, Q. R. Lu, S. Y. Chew, J. Controlled Release 2015, 208, 85.
[140] a) S. Pina, J. M. Oliveira, R. L. Reis, Adv. Mater. 2015, 27, 1143;
b) M. Sadat-Shojai, M.-T. Khorasani, E. Dinpanah-Khoshdargi,
A. Jamshidi, Acta Biomater. 2013, 9, 7591.
[141] a) B. Peng, Y. Chen, K. W. Leong, Adv. Drug Delivery Rev. 2015,
88, 108; b) c) M. Gori, M. Trombetta, D. Santini, A. Rainer, Expert
Opin. Biol. Ther. 2015, 15, 1601.
[142] a) M. Ochi, T. Nakasa, G. Kamei, M. A. Usman, E. Mahmoud,
J. Orthop. Sci. 2014, 19, 521; b) C. N. Salinas, K. S. Anseth, J. Dent
Res. 2009, 88, 681.
[143] M. Katoh, Int. J. Mol. Med. 2013, 32, 763.
[144] D. Fan, E. E. Creemers, Z. Kassiri, Circ. Res. 2014, 114, 889.
Adv. Healthcare Mater. 2017, 1700695
[145] K. J. Miller, D. A. Brown, M. M. Ibrahim, T. D. Ramchal,
H. Levinson, Adv. Drug Delivery Rev. 2015, 88, 16.
[146] Z. Wang, D. Zhang, Z. Hu, J. Cheng, C. Zhuo, X. Fang, Y. Xing,
Mol. Med. Rep. 2015, 12, 3345.
[147] K. Kim, J. H. Kim, I. Kim, J. Lee, S. Seong, Y. W. Park, N. Kim, Mol.
Cells 2015, 38, 75.
[148] B. Icli, A. K. Wara, J. Moslehi, X. Sun, E. Plovie, M. Cahill,
J. F. Marchini, A. Schissler, R. F. Padera, J. Shi, H. W. Cheng,
S. Raghuram, Z. Arany, R. Liao, K. Croce, C. MacRae,
M. W. Feinberg, Circ. Res. 2013, 113, 1231.
[149] K. Zuo, K. Zhi, X. Zhang, C. Lu, S. Wang, M. Li, B. He, Cell Physiol.
Biochem. 2015, 35, 477.
[150] E. Luzi, F. Marini, S. C. Sala, I. Tognarini, G. Galli, M. L. Brandi,
J. Bone Miner. Res. 2008, 23, 287.
[151] a) N. H. Kulkarni, J. E. Onyia, Q. Zeng, X. Tian, M. Liu,
D. L. Halladay, C. A. Frolik, T. Engler, T. Wei, A. Kriauciunas,
T. J. Martin, M. Sato, H. U. Bryant, Y. L. Ma, J. Bone Miner. Res.
2006, 21, 910; b) N. H. Kulkarni, T. Wei, A. Kumar, E. R. Dow,
T. R. Stewart, J. Shou, M. N’Cho, D. L. Sterchi, B. D. Gitter,
R. E. Higgs, D. L. Halladay, T. A. Engler, T. J. Martin, H. U. Bryant,
Y. L. Ma, J. E. Onyia, J. Cell Biochem. 2007, 102, 1504.
[152] Y. Deng, S. Wu, H. Zhou, X. Bi, Y. Wang, Y. Hu, P. Gu, X. Fan, Stem
Cells Dev. 2013, 22, 2278.
[153] Y. H. Liao, Y. H. Chang, L. Y. Sung, K. C. Li, C. L. Yeh, T. C. Yen,
S. M. Hwang, K. J. Lin, Y. C. Hu, Biomaterials 2014, 35,
4901.
[154] K. C. Li, S. C. Lo, L. Y. Sung, Y. H. Liao, Y. H. Chang, Y. C. Hu,
J. Tissue Eng. Regener. Med. 2016, https://doi.org/10.1002/term.2208.
[155] C. M. Curtin, E. G. Tierney, K. McSorley, S. A. Cryan, G. P. Duffy,
F. J. O’Brien, Adv. Healthcare Mater. 2015, 4, 223.
[156] G. M. Cunniffe, C. M. Curtin, E. M. Thompson, G. R. Dickson,
F. J. O’Brien, ACS Appl. Mater. Interfaces 2016, 8, 23477.
[157] E. G. Tierney, K. McSorley, C. L. Hastings, S. A. Cryan, T. O’Brien,
M. J. Murphy, F. P. Barry, F. J. O’Brien, G. P. Duffy, J. Controlled
Release 2013, 165, 173.
[158] R. M. Raftery, D. P. Walsh, I. M. Castano, A. Heise, G. P. Duffy,
S. A. Cryan, F. J. O’Brien, Adv. Mater. 2016, 28, 5447.
[159] a) J. C. Evans, M. Malhotra, K. A. Fitzgerald, J. Guo, M. F. Cronin,
C. M. Curtin, F. J. O’Brien, R. Darcy, C. M. O’Driscoll, Mol. Pharmaceutics 2017, 14, 42; b) K. A. Fitzgerald, J. Guo, E. G. Tierney,
C. M. Curtin, M. Malhotra, R. Darcy, F. J. O’Brien, C. M. O’Driscoll,
Biomaterials 2015, 66, 53.
[160] Z. Li, M. Q. Hassan, S. Volinia, A. J. van Wijnen, J. L. Stein,
C. M. Croce, J. B. Lian, G. S. Stein, Proc. Natl. Acad. Sci. USA 2008,
105, 13906.
[161] A. Lolli, E. Lambertini, L. Penolazzi, M. Angelozzi, C. Morganti,
T. Franceschetti, S. Pelucchi, R. Gambari, R. Piva, Stem Cell Rev.
2014, 10, 841.
[162] M. J. van Amerongen, F. B. Engel, J. Cell Mol. Med. 2008, 12,
2233.
[163] X. W. Wang, X. J. He, K. C. Lee, C. Huang, J. B. Hu, R. Zhou,
X. Y. Xiang, B. Feng, Z. Q. Lu, Int. J. Cardiol. 2016, 208,
79.
[164] L. H. Nguyen, M. Gao, J. Lin, W. Wu, J. Wang, S. Y. Chew, Sci. Rep.
2017, 7, 42212.
[165] J. Li, R. Kooger, M. He, X. Xiao, L. Zheng, Y. Zhang, Chem.
Commun. 2014, 50, 3722.
[166] X. Xiao, J. Hu, X. Wang, L. Huang, Y. Chen, W. Wang, J. Li,
Y. Zhang, Chem. Commun. 2016, 52, 12517.
[167] K. R. Beavers, T. A. Werfel, T. Shen, T. E. Kavanaugh, K. V. Kilchrist,
J. W. Mares, J. S. Fain, C. B. Wiese, K. C. Vickers, S. M. Weiss,
C. L. Duvall, Adv. Mater. 2016, 28, 7984.
[168] C. Esau, S. Davis, S. F. Murray, X. X. Yu, S. K. Pandey, M. Pear,
L. Watts, S. L. Booten, M. Graham, R. McKay, A. Subramaniam,
1700695 (21 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
www.advhealthmat.de
S. Propp, B. A. Lollo, S. Freier, C. F. Bennett, S. Bhanot,
B. P. Monia, Cell Metab. 2006, 3, 87.
[169] M. T. Di Martino, E. Leone, N. Amodio, U. Foresta, M. Lionetti,
M. R. Pitari, M. E. Cantafio, A. Gulla, F. Conforti, E. Morelli,
V. Tomaino, M. Rossi, M. Negrini, M. Ferrarini, M. Caraglia,
M. A. Shammas, N. C. Munshi, K. C. Anderson,
A. Neri, P. Tagliaferri, P. Tassone, Clin. Cancer Res. 2012, 18,
6260.
Adv. Healthcare Mater. 2017, 1700695
[170] Z. Zhou, Q. Yi, T. Xia, W. Yin, A. A. Kadi, J. Li, Y. Zhang, Org.
Biomol. Chem. 2017, 15, 2191.
[171] Z. Shatsberg, X. Zhang, P. Ofek, S. Malhotra, A. Krivitsky,
A. Scomparin, G. Tiram, M. Calderon, R. Haag, R. Satchi-Fainaro,
J. Controlled Release 2016, 239, 159.
[172] A. Gilam, J. Conde, D. Weissglas-Volkov, N. Oliva,
E. Friedman, N. Artzi, N. Shomron, Nat. Commun. 2016, 7,
12868.
1700695 (22 of 22)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Документ
Категория
Без категории
Просмотров
0
Размер файла
2 940 Кб
Теги
adhm, 201700695
1/--страниц
Пожаловаться на содержимое документа