(353b) miR233-Loaded Immunomodulatory Adhesives for the Treatment of Chronic Wounds

miR233-loaded Immunomodulatory Adhesives for
the Treatment of Chronic Wounds

Bahram Saleh¹, Harkiranpreet
Dhaliwal², Ehsan Shirzaei Sani¹,
Roberto Portillo-Lara^{1, 3}, Mansoor Amiji²,
Nasim Annabi^{1, 4, 5}

¹Department
of Chemical Engineering, Northeastern University, Boston, MA, USA

²Department
of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA

³Tecnologico
de Monterrey, Escuela de Ingeniería
y Ciencias, Ave. Eugenio Garza Sada
2501, Monterrey, México, 6484

⁴Biomaterials
Innovation Research Center, Brigham and Women’s Hospital, Harvard Medical
School, Boston, MA, USA

⁵Harvard-MIT
Division of Health Sciences and Technology, Massachusetts Institute of
Technology, Cambridge, MA, USA

Statement of Purpose: Diabetic wounds are characterized by impaired
vascularization, uncontrolled inflammation, and bacterial infection.
Conventional wound dressings suffer from inappropriate mechanical and physical
properties, and the inability to control inflammation. In particular, chronic
inflammation in diabetic wounds is mainly attributed to classic proinflammatory (M1) macrophages via local secretion of
cytokines, chemokines, and proteolytic enzymes. In contrast, alternative
anti-inflammatory (M2) macrophages promote tissue repair by producing
anti-inflammatory cytokines. miRNAs are small non-coding RNAs of 18-25
nucleotides that have been reported to regulate macrophage polarization and
function. Among the known miRNAs, miR223 is a potent regulator of inflammatory
responses, as it suppresses M1 responses and induces the M2 responses. Our
group previously developed a naturally derived antibacterial adhesive hydrogel
for wound healing ^{ADDIN EN.CITE  ADDIN EN.CITE.DATA 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. Here, we incorporated nanoparticles (NPs)
(average particle size: 115.0 ± 10.2 nm) containing miR223 inside these
adhesive hydrogels to be delivered topically and control inflammation at the
wound site. The adhesive hydrogel can be sprayed on the wound, and photocrosslinked using a visible light in a few seconds.

Methods:
CD44-targeting hyaluronic acid NPs and gelatin methacryloyl
(GelMA) were synthesized as described previously
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,
ADDIN EN.CITE
<EndNote><Cite><Author>Nichol</Author><Year>2010</Year><RecNum>12</RecNum><DisplayText><style db-id="vp22tvdxeews50et9zlp25rfv5az20seaspw"
timestamp="1508804066">12</key></foreign-keys><ref-type
name="Journal
Article">17</ref-type><contributors><authors><author>Nichol,
J. W.</author><author>Koshy, S. T.</author><author>Bae,
H.</author><author>Hwang, C. M.</author><author>Yamanlar,
S.</author><author>Khademhosseini,
A.</author></authors></contributors><auth-address>Center
for Biomedical Engineering, Department of Medicine, Brigham and
Women&apos;s Hospital, Harvard Medical School, 65 Landsdowne Street,
Cambridge, MA 02139,
USA.</auth-address><titles><title>Cell-laden microengineered
gelatin methacrylate
hydrogels</title><secondary-title>Biomaterials</secondary-title></titles><periodical><full-title>Biomaterials</full-title></periodical><pages>5536-44</pages><volume>31</volume><number>21</number><keywords><keyword>Biocompatible
Materials/chemistry</keyword><keyword>Cell
Adhesion</keyword><keyword>Cell
Movement</keyword><keyword>Cell Proliferation</keyword><keyword>Cells,
Cultured</keyword><keyword>Gelatin/*chemistry</keyword><keyword>Humans</keyword><keyword>Hydrogels/*chemistry</keyword><keyword>Materials
Testing</keyword><keyword>Methacrylates/*chemistry</keyword><keyword>Microfluidics/instrumentation</keyword><keyword>Molecular
Structure</keyword><keyword>Surface Properties</keyword><keyword>Tissue
Engineering</keyword><keyword>Tissue
Scaffolds</keyword></keywords><dates><year>2010</year><pub-dates><date>Jul</date></pub-dates></dates><isbn>1878-5905
(Electronic)&#xD;0142-9612
(Linking)</isbn><accession-num>20417964</accession-num><urls><related-urls><url>https://www.ncbi.nlm.nih.gov/pubmed/20417964</url></related-urls></urls>...3}. Different concentrations of NPs were mixed
with GelMA precursors, containing triethanolamine
and N-vinylcaprolactam. Eosin Y disodium salt was
dissolved separately in phosphate buffer saline and used as the photoinitiator. The NPs/GelMA
precursor solution was then mixed with Eosin Y and photopolymerized.
The adhesive properties of hydrogels were assessed using standardized tests
(e.g., ASTM F2392-04), and compared to commercially available tissue adhesives
such as Evicel and Coseal.
The release rate of miRNAs from the hydrogels was quantified using Quant-iT™ RiboGreen™ RNA Assay Kit. For
RNA uptake studies, J774A.1 macrophages were cultured
in 12 well cell culture dishes. Next, hydrogels were formed on the membrane of transwell inserts, and the inserts were placed into the
wells containing the J774A.1 cells. The miR223 transfection was assessed by
Taqman1 miRNA assay using qRT-PCR. U6 RNA (snRNA) was
used as a housekeeping gene. Once we confirmed the ability of the
immunomodulatory GelMA hydrogels to direct macrophage
re-polarization in vitro, we aimed to evaluate their efficacy to
modulate inflammatory responses and wound healing in vivo, using a
murine model of excisional wounds. Animals were divided into four groups:  i) Dulbecco's
phosphate-buffered saline (DPBS), ii) pristine GelMA
hydrogels, iii) NPs/negative miR233-laden GelMA
hydrogels (GelMA/NPs/negative miR223) and iv)
NPs/miR223-laden GelMA hydrogels (GelMA/NPs/miR223).

Results:
We first synthesized NPs-loaded GelMA adhesive
hydrogels using varying concentrations of NPs. We optimized the mechanical and
physical properties of the engineered adhesives for skin tissue regeneration
(elastic modulus of around 100 kPa, extensibility
more than 40). Our results showed strong adhesion of NPs-loaded GelMA in a burst pressure test. The burst pressure of 5 %(w/w)
NPs in GelMA hydrogels (18.5±0.5 kPa)
was significantly higher than that commercially available adhesives, Evicel (i.e., 1.5 ± 1.0 kPa) and Coseal (1.6 ± 0.1 kPa).  100% cumulative miRNA release was also
obtained after 48 h in vitro for GelMA
hydrogels containing 5.0 %(w/w) NPs. qPCR analysis of miR223 gene expression
levels showed a 1541±340 fold increase in miR223
expression in transfected macrophages at 24 h post incubation, as compared to
controls. Next at day 12 of in vivo studies, the percentage of closure
for wounds treated with GelMA/NPs/miR223 (i.e., 95.8
± 4.3 %) was significantly higher than GelMA/NPs/negative
miR223 (66.7 ± 2.9 %), GelMA (60.8 ± 3.5 %) and DPBS
(44.9 ± 7.9 %). Moreover, our results demonstrated that wounds treated with GelMA/NPs/miR223 showed > 2-fold increase in the
expression of the CD31 marker (expressed on vascular endothelial cells) as well
as 2-fold increase in the thickness of the collagen layer by evaluation of
Masson’s trichrome stained sections of wound biopsies, as compared to GelMA/NPs/negative miR223 treatment at day 12.

Conclusions:
Our results demonstrated that the engineered hydrogels could be used as a
multifunctional adhesive, with the ability to control inflammation and promote
tissue healing. In addition, due to its strong adhesion to skin and rapid
closure of the wound, the engineered adhesive could also prevent bacterial
infection.

References:

 ADDIN EN.REFLIST 1.         Annabi N.
Rana D. Shirzaei Sani E. Portillo-Lara R. Gifford J.
L. Fares M. M. Mithieux S. M. Weiss A. S.
2017;139;229-243.

2.         Ganesh S. Iyer
A. K. Morrissey D. V. Amiji M. M. 2013;34
(13);3489-502.

3.         Nichol J. W Koshy S. T. Bae H. Hwang C.
M. Yamanlar S. Khademhosseini
A. 2010;31 (21);5536-44.

Acknowledgments:
This work was supported by the National Institutes of Health (NIH) under award
number [1R01EB023052; 1R01HL140618]