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Efficiency and safety of #-CD-(D3)7 as an siRNA carrier for decreasing MMP-9 expression and improving wound healing in diabetic rats Na Li, Hengcong Luo, Meng Ren, Li-Ming Zhang, Wei Wang, Cheng Lin Pan, Liqun Yang, Guojuan Lao, Junjie Deng, Kaijin Mai, Kan Sun, Chuan Yang, and Li Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 2017
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ACS Applied Materials & Interfaces
Efficiency and safety of β -CD-(D3)7 as an siRNA carrier for decreasing MMP-9 expression and improving wound healing in diabetic rats
Na Li, 1 Heng-Cong Luo, 1,4 Meng Ren, 1 Li-Ming Zhang, 2,3 Wei Wang,1Cheng-Lin Pan,2,3 Li-Qun Yang,3 Guo-Juan Lao,1 Jun-Jie Deng2,3 ,Kai-jin Mai2,3 ,Kan Sun, 1 Chuan Yang,1* Li Yan1* 1
Depatement of Endocrinology, Sun Yat-sen Memorial Hospital, Guangdong
Provincal Key Laboratory of Malignant Tumor Epigenetics and Gene Reguation` Medical Research Center, Sun Yat-sen University, Guangzhou 510120, China 2
School of Materials Science and Engineering, Sun Yat-sen University, Guangzhou 510275, China
3
Department of Polymer and Material Science, School of Chemistry, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Guangdong Provincial Key Laboratory for High Performance Polymer-based Composites, Sun Yat-Sen University, Guangzhou 510275, China 4
Now working in the Third Affiliated Hospital of Guangzhou Medical University,
Guangzhou 510150, China
AUTHOR EMAIL ADDRESS:
[email protected] Na Li and Heng-Cong Luo contributed equally to this work ACS Paragon Plus Environment
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* Co-coressponding authors *Correspondence to Prof. Li Yan and Prof. Chuan Yang Phone: 86-20-81332286 Fax: 86-20-81332404
Email addresses Li Yan:
[email protected] Chuan Yang:
[email protected] Abstract
Over-expression of MMP-9 is critical for diabetic chronic wounds involved in the refractory wound healing process. We aimed to develop a strategy through RNAi to decrease MMP-9 expression and improving diabetic wound healing. We had explored β-CD-(D3)7 as a gene carrier to take siRNA effectively interfere with MMP-9 expression. It has been proved that β-CD-(D3)7 could be used as an effective siRNA delivery system. In this study we want to know exactly about the efficiency and safety of β-CD-(D3)7/MMP-9 siRNA for improving wound healing in diabetic rats. β-CD-(D3)7/MMP-9 siRNA treated animals show lower level of MMP-9 expression which induced to faster wound-close rates. Histological evaluation indicates that β-CD-(D3)7/MMP-9 siRNA significantly increases the content of collagen around the injured tissues. The numbers of neutrophilic ganulocytes was significantly decreased through treatment of β-CD-(D3)7/MMP-9 siRNA. In vivo fluorescence imaging assessment shows that β-CD-(D3)7/MMP-9 siRNA couldn’t cause organ damage and organ accumulation. The results suggest that β-CD-(D3)7/MMP-9 siRNA might be developed as a novel topical agent for the diabetic wounds treatment.
Keywords: β-CD-(D3)7; siRNA; matrix metalloproteinase-9; diabetic wound healing; RNA interference
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1. Introduction
Diabetic foot ulcer is one of the severe complications of diabetes and can cause great harm. Diabetic foot ulcer is correlated with the disordered expression of matrix metalloproteinase (MMPs) of the wound.
1
MMPs are a type of Zn2+-containing
endogenous proteolytic enzymes, which are included in the whole process of wound healing and closely related with wound inflammation, recovery of cell regeneration and migration, and the secretion and effects of growth factors, re-epithelization, the production of ECM, angiogenesis and wound remodeling. Studies have found that the level of matrix metalloproteinase (MMPs) increase significantly in the tissue and effusion of refractory ulcers, and matrix metalloproteinase 9 (MMP9) presented the highest level and activity.
2-7
In the tissue of diabetic foot ulcers, the level of MMP9
can be 10-14 times higher than the wound of non-diabetic patient. The previous studies of our team also have found the MMP9 level in the skin tissues of diabetic rats was over 3 times higher than that of normal rats.8 Over expression of MMP9 can lead to excessive degradation of extracellular matrix, leading to slower wound healing.9 Meanwhile, the inhibition of the high expression of local MMP9 could improve wound healing. However, there hasn’t been any specific inhibitor of MMP9, while tissue inhibitor of metalloproteinase (TIMP), the MMPs inhibitor, is very expensive and not suitable for routine clinical use. Therefore, the application of RNA interference (RNAi) is one of the alternative methods to inhibit the overexpression of MMP-9 of local wound. The application of RNAi in the inhibition of target gene expression so as to down-regulate the related protein expression level accordingly, thus is a promising means for the diagnosis and treatment of diseases.
10
Currently, researches on RNA
interference (RNAi) are mainly focused on tumor diseases, infectious diseases and genetic disorders,11-13 while studies on the employment of RNAi technique in the treatment of refractory ulcers is not currently reported.The highlight research topic on
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RNAi is the development of novel non-viral vector system to mediate siRNA into cells.
14-15
In previous experiments, our research team have successfully synthesized
star-branched cationic polymer β-CD-(D3)7, which consists of β-Cyclodextrin and third-generation poly (ami-doamine) (PAMAM). 16 Also, in cellular studies, it has been proved that β-CD-(D3)7 is with low-toxicity and can effectively carry siRNA into cells with an RNAi effect. In preliminary animal studies, the injection of β-CD-(D3)7/MMP9 compound in the skin of local wound of diabetic rats could improve the wound healing process.
17
This study aimed to conduct an overall
evaluation of the efficiency and safety of β-CD-(D3)7/MMP9 compound in the wound healing process of skin ulcers in diabetic rats.
2. Materials and methods
2.1. Materials
β-CD-(D3)7 was synthesized in our previous work
16
(Fig.1) Lipofectamine® 2000
was bought from Thermo Fisher Scientific (Waltham, MA, USA).Opti-MEM medium was
bought
from
GIBCO(USA).MMP-9
siRNA
were
purchased
from
GenePharm(China).MMP-9siRNA(Rat):5’-GGGCUUAGAUCAUUCUUCATT-3; antisense:3’-UGAAGAAUGAUUAAGCCCAG-5;S-D rats that weighted 230–250 g were acquired from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). 2.2. Preparation of β-CD-(D3)7/ MMP-9siRNA complexes
β-CD-(D3)7/MMP-9siRNA complexes were formed using MMP-9 siRNA at a concentration of 0.22 µg/50 µL in Opti-MEM medium (Gibco,Grand Island).The N/P ratio of complexes was screened in our previous study.17 The N/P ratio=10 was the optimal ratio used in the following experiment. The mixture underwent gentle vortex and incubated for 30 min at room temperature and used as soon as prepared. ACS Paragon Plus Environment
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Lipofectamine2000/MMP-9siRNA complexes were formed according to the manufacturer's instruction.
2.3. Establishing streptozotocin-induced diabetes rats
Weight of S-D rats were scope from 230–250 g. Diabetes was induced through 50mg/kg streptozotocin’s (STZ,sigma, USA) intra-abdominal injection after 12 hours without provision. After 48 h, if glucose concentrations were 16.7 mmol/L or higher in heparinized tail vein blood (measured by Glucometer) were thought to be diabetic. All rats were kept for 4 weeks with appropriate rat diet. As demanded to keep blood glucose levels higher than 16.7 mmol/L and to prevent acute crisis, STZ-treated rats were given everyday injections of 1-3.0 units of insulin. The body weight and blood glucose were measured weekly.
2.4. Diabetes rats’ skin wound model
2 ml blood from the tail vein was obtained. Then a full-thickness skin wound in the diabetic rats’ model was created through standard method in our previous publication. 8
The rats were separated into six groups at random. The rats receiving MMP-9 siRNA
gene therapy were given intradermal injections of the naked siRNA (5.5ug) or complexed with β-CD-(D3)7 or lipofectamine2000 (n =12 animals per group) every 2 days. The negative control groups were treated with Opti-MEM, β-CD-(D3)7 or lipofectamine2000 (n=12 animals per group). Each wound site was then digitally photographed every 2 day, and wound regions were decided on photos using Image J software. Changes in wound regions over time were presented as the percentage of the original wound regions.
2.5 Efficiency ofβ β-CD-(D3)7as an siRNA carrier for promoting wound
healing in diabetic rats
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2.5.1 Wound closure analysis
Wound areas of each group were digitally photographed every 2 day and were determined on photographs using Image J software. Wound closure’s percentage was estimated as original wound size's percentage: [(initial area – final area)/initial area] ×100%.
2.5.2 Detection the expression of MMP-9 mRNA, keratin14 mRNA and keratin5 mRNA in re-epithelialized tissues
Wound re-epithelialization was decided by assessing keratin 5 and 14 expression levels. The MMP-9 mRNA levels were examined by RT-PCR after different treatment. Quantitative real-time PCR was executed adopting a StepOnePlusTM Real-Time PCR System (TAKARA, Japan). Target genes' expression levels were determined using the comparative Ct approach through normalizing the target gene to the endogenous reference (GAPDH). 2.5.3. Histological analysis
Wound tissue specimens were fixed in 4% paraformaldehyde and were incubated overnight at 4°C. Four consecutive 4μm sections were collected for each slide and were then dewaxed to water, followed by staining with hematoxylin and eosin or Masson’s trichrome staining and visualized by light microscopy. H&E staining was used to observe the numbers of neutrophilic ganulocytes and Masson’s trichrome staining was used to observe the content of collagen in different groups. 2.6 Safety ofβ β-CD-(D3)7as an siRNA carrier for promoting wound
healing in diabetic rats
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2.6.1 Survival rates of different groups
We calculated the survival rates at the end of study. The survival rate=the remaining rats/12*100%.
2.6.2 Histological analysis
The rats were sacrificed on 0d and 7d after wound formation. Hemotoxylin-eosin staining was used to detect organs’ histological changes including liver, heart and kidney for evaluating the safety ofβ-CD-(D3)7as an siRNA carrier for promoting wound healing in diabetic rats.
2.6.3Evaluation of liver and kidney function
Rats’ vessel blood of each group was saved before and after the experiment for liver and kidney function tests.500μl plasma was used to detect ALT、TBIL、Cr and BUN in our Endocrine laboratory (Siemens biochemistry analyzer)
2.6.4 In vivo fluorescence imaging
400ul
OPti-MEM,
Cy-5
labled-siRNA,
Fluorescein
labled-β-CD-(D3)7,
or
Fluorescein labled-β-CD-(D3)7/Cy-5 labled-siRNA complexes were intradermal injected around the wound (n =5 animals per group) every 2 days. The fluorescence photos were captured at 30min, 24 h, 48 h and 168h after the intradermal injection through in vivo fluorescence imaging system for small animals (Carestream, USA). After carrying the wound's fluorescent images, the rats were sacrificed, inferior vena cava's 4ml blood were kept and the organs (lung, heart, liver, spleen, kidney, and muscle) were kept for isolated organ imaging. Then tissue distribution of fluorescently was calculated. The fluorescence intensity in vivo imaging screens was quantified through the Carestream MI software. ACS Paragon Plus Environment
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2.6.5 Statistical analysis
Quantitative data is expressed as average ± SD and the statistical software program SPSS16.0 was used for calculations. Student's t test (two-tail) was used for statistical analyses and analysis of variance (ANOVA) was used for Multiple Comparisons data, in which Bonferroni was used for the comparison between two groups. Differences were considered significant if the P value was < 0.05.
3. Results
3.1. Wound closure analysis
Wounds
treated
with
β-CD-(D3)7/MMP-9siRNA complexes
showed
faster
wound-close rates compared with other groups. (Fig.2-3) The wound close rate in nanocomplexes group was 46.2 ± 4.31% which had significant differences compared with that of lipofectamine2000/MMP-9siRNA complexes group( 37.9 ± 2.4%).The naked siRNA is more easier to enzymatic degradation at the wound and showed lower gene-silencing effects compared with that of β-CD-(D3)7/MMP-9siRNA complexes group. Some of results about this part had been published in our previous article. 17
3.2. The expression of MMP-9, keratins 5 and 14 in regenerate skin
Compared with non-diabetic skin, the diabetic skin showed increased MMP-9 expression in mRNA. mRNA levels in the diabetic group in the beginning of the formation of wound (0d) increased9.7foldcompared with non-diabetic rats’ skin. The MMP-9 level in each treatment group was increased at 7d compared with the MMP-9 at 0d. It had significantly lower MMP-9 expression after the treatment with β-CD-(D3)7/MMP-9siRNA
complexes
and
lipofectamine2000/MMP-9siRNA
complexes, although it remained higher than that in the skin at 0d. (Fig4A) Keratins 5 and 14 were also studied to assess the regeneration of epithelial. ACS Paragon Plus Environment
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(Fig4B-4C)The transcriptional level of Keratin 5 and 14 in diabetic skin were decreased compared to non-diabetic skin. The Keratin5 or keratin 14 mRNA level after treated with β-CD-(D3)7/MMP-9siRNA complexes at 7th day were higher than that in other groups. But it had no significant differences between groups treated with β-CD-(D3)7/MMP-9siRNA complexes and other diabetic groups.
3.3. Histological analysis about neutrophilic ganulocytes and collagen formation
HE-staining and Masson’s trichrome staining sections from diabetic rats’ skin showed that the epidermis and dermis layer were thin and atrophic, including disorganization of connective tissue fiber bundles, increased space between collagen fiber bundles. Interstitial cells had a rounded, shrunken, crenated appearance in the diabetic skin (Fig5-6). The content of collagen, the infiltration of neutrophilic granulocytes was calculated in both groups at 7th day. (Fig7-8) The numbers of neutrophilic ganulocytes, were significantly decreased around the injured tissues in β-CD-(D3)7/MMP-9siRNA complexes group than in other groups. (P
