Heparin-Based Coacervate of FGF2 Improves Dermal Regeneration

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Heparin-Based Coacervate of FGF2 Improves Dermal Regeneration by Asserting a Synergistic Role with Cell Proliferation and Endogenous Facilitated VEGF for Cutaneous Wound Healing Jiang Wu, Jingjing Ye, Jingjing Zhu, Zecong Xiao, Chaochao He, Hongxue Shi, Yadong Wang, Cai Lin, Hongyu Zhang, Yingzheng Zhao, Xiaobing Fu, Hong Chen, Xiaokun Li, Lin Li, Jie Zheng, and Jian Xiao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00398 • Publication Date (Web): 19 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016

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Heparin-Based Coacervate of FGF2 Improves Dermal Regeneration by Asserting a Synergistic Role with Cell Proliferation and Endogenous Facilitated VEGF for Cutaneous Wound Healing Jiang Wu1#, Jingjing Ye1#, Jingjing Zhu1, Zecong Xiao1, Chaochao He1, Hongxue Shi1, Yadong Wang2, Cai Lin3, Hongyu Zhang1, Yingzheng Zhao1, Xiaobing Fu4, Hong Chen5, Xiaokun Li1, Lin Li1*, Jie Zheng1,5*, and Jian Xiao1*

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School of Pharmaceutical Sciences Key Laboratory of Biotechnology and Pharmaceutical Engineering Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China 2

Department of Bioengineering and the McGowan Institute for Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15219, USA

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The First Affiliate Hospital Wenzhou Medical University, Wenzhou, 325035, China 4

Wound Healing and Cell Biology Laboratory Institute of Basic Medical Science Chinese PLA General Hospital, Beijing 1008553, China 5

Department of Chemical and Biomolecular Engineering The University of Akron, Akron, Ohio 44325, USA

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These authors contribute equally to this work.

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Corresponding authors: Jian Xiao, [email protected]; Lin Li, [email protected]; Jie Zheng, [email protected]

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Abstract Effective wound healing requires complicate, coordinated interactions and responses at protein, cellular, and tissue levels involving growth factor expression, cell proliferation, wound closure, granulation tissue formation, and vascularization. In this study, we develop a heparin-based coacervate consisting of PEAD as a storage matrix, heparin as a bridge, and FGF2 as a cargo (namely heparin-FGF2@PEAD) for wound healing. First, in vitro characterization demonstrates the loading efficiency and control release of FGF2 from the heparin-FGF2@PEAD coacervate. The following in vivo studies examine the wound healing efficiency of the heparin-FGF2@PEAD coacervate upon delivering FGF2 to full-thickness excisional skin wounds in vivo, in comparison with the other three control groups with saline, heparin@PEAD as vehicle, and free FGF2. Collective in vivo data show that controlled release of FGF2 to the wounds by the coacervate significantly accelerates the wound healing by promoting cell proliferation, stimulating the secretion of vascular endothelial growth factor (VEGF) for re-epithelization, collagen deposition, and granulation tissue formation, and enhancing the expression of platelet endothelial cell adhesion molecule (CD31) and alpha-smooth muscle actin (α-SMA) for blood vessel maturation. In parallel, no obvious wound healing effect is found for the control, vehicle, and free FGF2 groups, indicating an importance role of the coavervate in wound healing process. This work designs a suitable delivery system that can protect and release FGF2 in a sustained and controlled manner, which provides a promising therapeutic potential for topical treatment of the wounds. Key words: Fibroblast growth factor-2 (FGF2); Heparin; Control release; Vascular endothelial cells; Wound healing

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1. INTRODUCTION Human skin - the largest living organ in humans – not only serves as a the first physical barrier to protect the underneath organs from damage, but also possesses many biological functions including immunological surveillance and self-healing1. Any serious damage of skin integrity (e.g. burns, lacerations, and diabetic wounds) will cause severe adverse effects on bacterial infection, the loss of blood and electrolyte, and tissue failure2. Skin wound healing is a highly complicated and coordinated process involving several distinct but overlapping stages. In general, it begins with hemostasis, followed by the prevalent inflammation, then leads to another stage for cell migration and proliferation, extracellular matrix deposition, angiogenesis, and tissue formation and remodeling3. Most of severe skin wounds (i.e. nonhealing wounds) are very difficult to heal spontaneously due to the lack of scaffold to guide cell growth and promote the angiogenesis of endogenous growth factors4-7. Emerging evidence reveals that a number of growth factors play important but different roles in skin wound healing, including fibroblast growth factor (FGFs), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) families8, 9,. Among different endogenous growth factors, a number of in vivo studies of FGF2 (16-18.5kDa) have shown that FGF2 is able to stimulate the proliferation and migration of a wide variety of cells (fibroblasts, endothelial cells, and kerotinocytes) at the wound area, accelerate acute wound closure, and promote tissue regeneration and angiogenesis in the process of wound healing6, 8, 10. Moreover, FGF2 can cross-talk with different neurotrophins and growth factors to promote wound healing. For instance, FGF2 interacts with α-smooth muscle actin (α-SMA) and increased its expression to form the connective tissue with the treatment of surgical periodontal defects in diabetic rats11. FGF2 also regulates the release of transforming growth factor β1 (TGF-β1) to promote re-epithelialization at the early stage12. Co-application of FGF2 and VEGF synergistically improves extracellular matrix deposition and neovascularization at wound sites in the diabetic mouse model13. In line with these studies, our previous work14 also showed that regularly administered FGF2 every the other day (1 ml to each wound, 1 µg / ml, dissolved in 0.9% w/v saline) can accelerate the preformed healing wounds in the rat cutaneous wound model. The FGF2-improved wound healing is attributed to combinatorial effects by regulating inflammation response, stimulating fibroblast growth, and enhancing collagen deposition. All these studies above have demonstrated that FGF2 regulates many aspects of wound healing15, 16. On the hand other, like most of growth factors in clinical trials, FGF2 often requires high-dosages and frequent treatments (every the other day, even every day) to retain positive wound healing effects due to its short half-life caused by diffusion and susceptibility to enzymatic degradation17-19. Moreover, FGF2 is highly vulnerable to temperature and pH, and it degrades rapidly if surrounding temperature is above 40 °C or pH is less than 520. Despite biological importance of FGF2, it is also equally 3


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important to design a suitable delivery system that protect and release FGF2 in a sustained and controlled manner21-23. A number of particle-based delivery systems (hydrogels, nanogels, nano/microparticles) have been developed for encapsulation and controlled release of FGF2, but these delivery systems often suffer from low loading efficiency, high denaturation rate, and poor controlled-release24. To overcome these limits above, herein we developed a coacervate delivery system, which integrates poly(ethylene argininylaspartate digylceride) (PEAD) matrix (Fig. 1) with heparin and FGF2 together. In this delivery system, heparin is used to specifically bind to FGF2 to stabilize and prolong FGF2 half-life25, meanwhile PEAD matrix is designed to conjugate the heparin via nonspecific electrostatic interactions between the negatively charged heparins and the positively charged PEAD matrix26-28. The specific heparin-FGF2 binding also helps to prevent large burst release while providing tunable release profiles by attenuating diffusional release through transient interactions with the delivery PEAD matrix. The release of FGF2 from the heparin-FGF2@PEAD delivery system and its wound healing effect was examined in C57BL/6 mice. Additionally, we examined the biological roles of FGF2 in wound healing in mice model. 2. EXPERIMENTAL SECTION Preparation of FGF2 coacevate delivery system. The synthesis of PEAD was in two steps as previous described26. In brief, PEAD and heparin was each dissolved in 0.9 % saline at 10 mg/ml and used a 0.22 µm filter membrane to sterilize. To prepare the delivery vehicle, FGF2 (the Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, China) was first mixed with 10 µl heparin, and then 50 µl PEAD was added (the heparin and PEAD mass ratio was 1:5) under constant stirring at room temperature. The mixed solution immediately turned cloudy to form the delivery vehicle. FGF2 loading efficiency by western blotting. To prepare FGF2-coacervates, 5 µg or 10 µg of FGF2 were mixed with 100 µg of heparin and 500 µg of PEAD at room temperature, separately. Upon mixing, 5 µg or 10 µg FGF2 were loaded into the coacervates, then the resulting FGF2-loaded coacervates were centrifuged at 12,100 rpm for 10 min. Both supernatant and the precipitate of FGF2-coacervates after centrifugation and FGF2-coacervate without centrifugation were mixed with the 5× loading buffer and denatured at 100 oC for 10 min, separated on 12% polyacrylamide gels, and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated in TBS containing 5% skim milk for 90 min and incubated with primary antibodies at 4 oC overnight. The membranes were washed for 7 min for three times with TBS containing 0.05% Tween-20. Then, the membranes were incubated with second antibody for 1 h at room temperature and washed with TBST as usual. FGF2 loading efficiency was detected with rabbit anti-human FGF-2 antibody (sc-79, 1:300, Santa Cruz Biotech, CA, USA) and then visualized using goat anti-rabbit horseradish peroxidase-conjugated antibody (AB22151, 1:10000, Bioworld, Shanghai, China). The signals were then detected using western blotting 4


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detection reagent and the results were further analyzed by Image Lab (Bio-Rad, Hangzhou, China). Heparin@PEAD complex by scanning electron microscopy. The scanning electron microscopy (SEM) samples were prepared by mixing 500 µg PEAD with 100 µg heparin to form the complex. The samples were rapidly frozen by liquid nitrogen, lyophilized, fixed on the aluminum stub, and sputtered with gold. Use of the liquid nitrogen to rapidly freeze the sample can greatly minimize the phase separation effect on the morphology of the sample. The surface of solid was then viewed by scanning electron microscope (10kV) (Hitachi, Tokyo, Japan). Release of FGF2 from FGF2-coacervate. The controlled delivery system was formed by 500 ng of recombinant human FGF2 to 10 µl heparin and 50 µl PEAD. The solution was mixed and centrifuged for 10 min at 12100 g. The supernatant was removed and 500 µl 0.9% saline was added to the FGF2-coacervate. At days 0, 4, 7, 10, 17, the supernatant was collected for analysis and added with fresh saline. The samples were stored at −80 oC. The released FGF2 was analyzed by FGF2 enzyme-linked immunosorbent assay Kit (ELISA, Westang system, Shanghai, China). The amount of released FGF2 was calculated between the former and final concentrations. The FGF2 release system was carried out at 37 oC. This trend of the release profile was maintained up to the end of the experiment. Mice model. 6-7 weeks male C57BL/6 mice were obtain from Laboratory Animals Center of Wenzhou Medical University. The experiment animals were conducted with adherence to the National Institutes of Health Guide Concerning the Care and Use of Laboratory Animals. All animal experiments were carried out with the guidelines approved by the Animal Experimentation Ethics Committee of Wenzhou Medical University, Wenzhou, China. Mice were maintained on a standard diet and water was provided freely available. Temperature (23±2 oC), humidity (35−60%), and photoperiod (12 h light and 12 h darkness cycle) were kept constant. Preparation of mice cutaneous wound healing model and analysis. Healthy C57BL/6 mice were randomly divided into four groups (n=7). Group 1 were given saline as control. Group 2 were given heparin@PEAD as vehicle. Group 3 were given free FGF2. Group 4 were given heparin-FGF2@PEAD. The treatment groups containing FGF2 were applied to the wound as a 10 µl solution. In brief, animals were anaesthetized with intraperitoneal injection of 4% chloral hydrate (0.01 ml/g) and prepared for wounding under aseptic conditions. Mice were positioned on a form panel and the hair on the dorsum was shaved with an electric clipper. Depilatory creams was used to clear up the residual hair. Two silicone-splinted (the external diameter of 16 mm, the internal diameter of 8 mm and 0.5 mm-thickness) were fixed on wound using 6-0 nylon suture (Lingqiao, Ningbo, China). The use of silicone rings is of importance to reduce skin contraction upon 5


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wounding29. A 6 mm round skin biopsy punch (Acuderm® inc., Ft Lauderdale, FL, USA) was used to create two full-thickness cutaneous wounds on the either side of the dorsal midline. To prevent cross-contamination, the two wounds on each mouse received the same treatment. The experiment was single-dose topical administration on the healing of induced mice skin wounds. The appropriate dressing material was placed on the wounds. Mice were applied a Tegaderm™ transparent dressing (3M Health Care, Brookings, USA) to prevent from infection and wrapped in a thin layer of self-adhesive bandages (MDS, Shanghai, China) to deter chewing of the splints. In order to calculate the wound closure rate based on wound areas, photographs of wounds were taken immediately after surgery at different time points. These bandages only gently held the mice and didn’t limit the motion of the mice. Each mouse was fed separately with food and water. The bandage was removed and the size of the wounds was photographed from 7 to 17 days through the transparent dressing. Wound area was measured by Image-Pro plus to trace the wound margin, and all of the measured data were compared with post-wounding. Since the fixed splints and wraps sometimes will be destroyed by mouse, new splints and wraps should be provided to replace the damaged ones as soon as possible to keep the wounds in a consistent way. Their wounds were harvested for histological analyses on the 7th day, four wounds per group, and remaining animals were sacrificed at the day of terminal biopsy (on the 17th day). Wounds and surrounding were excised for histological evaluation. The wound closure rate was calculated as follows30:

Histological analysis. After anesthesia, wound tissue from surrounding was excised, maintained in cold 4% paraformaldehyde in 0.01M phosphate buffered saline (PBS, PH=7.4) overnight, embedded in paraffin. Sections of 5-µm thickness were cut with a microtomes (LEICA RM2235, Germany) and mounted in Poly-L-Lysine-coated, stored at room temperature. Four wounds were analysed at each time point and only the sections from the wounded center were used for analysis. Skin sections were stained with Hematoxylin and Eosin (H&E) (Beyotime Institute of Biotechnology, China) for morphological evaluation and with Masson’s trichrome staining (Beyotime) for assessment of collagen content following the manufacturer’s protocols. The rest of skin was stored at −80 oC for other tests. Immunohistochemical staining. Sections were dewaxed and hydrated, and were pretreated with 3% H2O2 and 80% carbinol for 15 min to quench endogenous peroxidase activity. After washing by PBS, the sections were heated to antigen recovery, permeabilized with 0.5% Triton X-100 and blocked nonspecific antibody binding in 5% bovine serum albumin (BSA) (Beyotime) in PBS for 45 mins at room temperature. Subsequently, primary antibodies diluted in PBS containing 1% BSA were used including rabbit polyclonal anti-wide spectrum cytokeratin (ab9377, 1:75, Abcam), mouse monoclonal anti-PCNA (sc25280, 1:200, Santa Cruz Biotech, CA, 6


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USA), rabbit polyclonal anti-VEGF (sc-152, 1:200, Santa Cruz Biotech, CA, USA), rabbit polyclonal anti-collagen III (ab7778, 1:1000, Abcam), rabbit polyclonal anti-TGF-β1 (ab92486, 1:500, Abcam) incubated at 4 oC overnight. Followed by goat anti-mouse or goat anti-rabbit HRP-conjugated secondary antibodies, incubating for 2 h at room temperature and the reaction was stopped with DAB chromogen kit (ZSGB-BIO, Beijing, China) then counterstained with hematoxylin. The result images were acquired using Nikon positive position microscope (Nikon, 80i, Tokyo, Japan). The positive numbers of PCNA, VEGF were counted and quantified by optical density through Image-Pro plus. Immunohistochemistry for these markers were performed simultaneously in all wound samples as well as negative controls with 1% BSA. Immunofluorescent Staining. 17-day skin sections were stained with rabbit polyclonal anti-CD31 (ab28364, 1:200, Abcam), mouse monlclonal anti-alpha smooth muscle actin (α-SMA) (ab7817, 1:100, Abcam) followed by goat anti-rabbit IgG Alexa Fluor® 647 (ab150083, 1:1500, Abcam) and goat anti-mouse IgG Alexa Fluor® 488 (ab150113, 1:1500, Abcam), respectively, and stained with DAPI (Beyotime). The fluorescent images were taken by Nikon confocal laser microscope (Nikon, A1 PLUS, Tokyo, Japan). The number of CD31 (endothelia cell) or α-SMA (mural cell) in the tissue was counted and confirmed by DAPI-positive nuclei. The diameter of blood vessels were measured and averaged by several randomly selected vessels using Image-Pro plus software. The value was divided by the area of the field and measured by NIS Elements Version 3.2 software (Nikon, Tokyo, Japan). All of the illustrations were assembled and processed digitally. Statistical analysis. All data were expressed as mean±standard deviations (SD). Statistical differences were performed using one-way analysis of variance (ANOVA) followed by Tukey’s test with GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). For all tests, * p value＜0.05, ** p value ＜0.01, *** p value ＜ 0.001. 3. RESULTS AND DISCUSSION 3.1. The characterizations, loading efficiency, and release kinetics of FGF2-coacervate In Fig. 1, cationic PEAD contains two positively charged functional groups of amino and guanidine groups under physiological conditions, enabling PEAD to interact with heparin strongly. Fig. 2A shows the preparation process of FGF2-coacervate (i.e. heparin-FGF2@PEAD). It can be seen that both PEAD and heparin-FGF2 had excellent solubility in aqueous solution, as shown as transparent solutions. Upon mixing PEAD solution with heparin-FGF2 solution, the transparent solution became a milky suspension solution, and after 12 h, the heparin-FGF2@PEAD complex precipitated down to the bottom (as indicated by a red arrow in Fig. 2A). These data indicate the formation of the heparin-FGF2@PEAD complex. To confirm the formation of heparin@PEAD via electrostatic interactions, 7


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cross-sectional images of heparin@PEAD samples were investigated using SEM. As shown in Fig. 2B, the cross-section area of heparin@PEAD reflects their interior morphologies, which mainly consist of ribbon-like structures in globular domains. These ribbon-like domains further confirmed that this coacervate would provide superb accommodation for the loaded FGF2. In Fig. 2C, the loading efficiency of FGF2 into the FGF2-coacervate was measured by western blot. The amounts of FGF2 in the supernatant and in the settled coacervate after centrifugation were compared to the total amount of FGF2 in the loading solution. For the higher amount of growth factors (10 µg), the loading amounts of FGF2 in the supernatant and in the loading solution were almost same, and the loss of FGF2 was negligible after the incubation with PEAD matrix, indicating ~93% loading efficiency because of the large excess of heparin relative to FGF2. Then we utilized the FGF2-coacervate to evaluate the release of FGF2 from heparin-FGF2@PEAD. Fig. 2D shows the cumulative release profile of FGF2 from the FGF2-coacervate into saline over 17 days, and the FGF2 release was determined using ELISA. The FGF2 release behavior exhibited a typical two-phase release profile: an initial burst release during the first 24 h and a slow sustained release during the rest of the time course (day 2–day 17). The burst release percentage of FGF2 from supernatant was 13.2±0.7 % during initial 24 h. After 24 h, the release percentage of FGF2 was slowly and gradually increased to a steady plateau of 56.1±3.4 % at day 17. The competency of the FGF2 release from heparin-FGF2@PEAD indicates a diffusion-controlled release at the initial phase (24 h) and a combined release behavior from the degradation, dissolution, and/or erosion of coacervates in the second phase (2–17 days). It has been reported that a sustained release of FGF2 for at least over 5 days is required to achieve regenerative in rat spinal cord injury model31. However, due to both positively charged nature of PEAD and FGF, there is no direct interactions between PEAD and FGF. Instead, the negatively charged heparin was used as a bridge to associate both positively charged PEAD and FGF2 together through its heparin domain in FGF2 and its negative-positive charge interactions between heparin and PEAD. Strong binding of FGF2 to heparin-PEAD may modulate its release from the heparin containing polymeric matrix, resulting in ~40% unreleased FGF2. All these results above only support that our designed FGF2-coacervate is workable in vitro, but do not necessarily mimic in vivo scenario, where enzymatic degradation of FGF2-coacervate is anticipated. Next, we systematically performed a series of in vivo tests on the FGF2-coacervate to obtain different aspects of the wound healing capacity of the FGF2-coacervate in C57BL/6 mice model. 3.2. FGF2-coacervate enhances wound closure in mice To evaluate the role of FGF2-coacervate in the skin wound healing process, we created full-thickness cutaneous wounds in C57BL/6 mice, followed by the examination of the wound healing process when treating the wounds with saline as control, heparin@PEAD as delivery vehicle, free FGF2, and FGF2-coacervate, respectively. Fig. 3A shows sequential photographs of the four types of treated 8


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wounds on day 7, 10, 14, and 17, respectively. It can be seen that the saline-treated and heparin@PEAD-treated wounds displayed similar wound closure rates of 14.5±4.1% and 14.2±7.0%. The wounds treated with both free FGF2 and FGF2-coacervate accelerated wound closure at day 7 and 14, but the wounds treated with the FGF2-coacervate recovered much faster with better skin appearance than other groups. In particularly, after day 17 the heparin-FGF2@PEAD-treated wounds were completely healed and almost scar-less, while FGF2-treated wounds still retained large residual wound areas. Quantitatively, Fig. 3B compares the wound closure rates for the four treated wounds. Consistent with visual inspection of wound healing in Fig. 3A, the rate of wound closure in heparin-FGF2@PEAD-treated mice was higher than that of wound closure in other three group mice at all treatment times (days 7, 10, 14, and 17). At day 17, the final wound closure rates were 98.2±2.6% for heparin-FGF2@PEAD-treated group, 78.2±6.7% for FGF2-treated group, 76.4±10.8% for heparin@PEAD treated group, and 72.9±4.6 % for control group, respectively. The wounds were still covered with eschar. The FGF2-treated wounds showed comparable wound closure rates to the two control groups, but 20% lower than heparin-FGF2@PEAD-treated wounds, indicating that sustained release of FGF2 from the FGF2-coacervate has a positive effect on re-epithelialization. 3.3. FGF2-coacervate induces more granulation tissue formation Mice from each group were euthanized on days 7 and 17 post-treatment for clinical observation and histological analysis. Fig. 4 shows representative images of HE-stained (Hematoxylin and Eosin-stained) histological wound sites for four groups. In the control and vehicle groups, even at day 17, the wounds were still not fully closed. In the heparin-FGF2@PEAD-treated group, the regenerated skin was translucent and thin. The peripheral region was well integrated into the surrounding native skin tissue and there was complete appendage regeneration, as indicated by the formation of many hair follicles and blood vessels. In the other three groups, however, no obvious skin appendage regeneration was observed. In parallel, Fig.5 shows the histological collagen deposition in the dermis of regenerated skin in four groups at day 7 and 17 using masson trichrome staining (MTS). At day 7, MTS-stained sections of wounds treated with the heparin-FGF2@PEAD revealed the abundant granulation tissues formation with increased cellularity. This observation was similar with the HE-stained results in Fig. 4. However, for the control, vehicle, and free FGF2 groups, the gross appearance of granulation tissues were almost indistinguishable, suggesting that the delivery vehicle and free FGF2 had no apparently positive effect on wound sites (Fig. 5A-D). At day 17 post-wounding, when more than 90% of wound closure was reached in the heparin-FGF2@PEAD group, the accelerated granulation tissue formation effect became even more pronounced. When compared to the untreated defects (Fig. 5E), extensive collagen deposition and thick wavy collagen fibers were observed in the wounds (Fig. 5H). Moreover, the underlying collagen fibers of heparin-FGF2@PEAD treated group (Fig. 5H, S1A, S1B) were well organized and morphologically similar 9


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to normal dermal skin (Fig. S1C). Consequently, both Fig. 4 & Fig. 5 shown the expression of the newly formed blood vessels and skin appendage, indicating that the heparin-FGF2@PEAD group has a significantly higher blood vessel area and blood vessel numbers in the healing wound area than the other three groups, suggesting that the FGF2-coacervate enables to recruit and proliferate cells to form blood vessels in the wounded area. In Fig. 6, the wounds were stained with the wide spectrum cytokeratin to evaluate re-epithelialization. Consistent with the HE and Masson results, the appendage regeneration of skin wounds were not observed in both control and the vehicle groups at 17 day post-surgery. Although the number of skin appendages increased from day 7 to day 17 post-surgery for both FGF2 and FGF2-coacervate groups, the FGF2-coacervate-treated wounds showed the much higher number of skin appendages and the thicker epithelial layers than the FGF2-treated wounds. Additionally, a close-up visualization (Fig. 6, 400X) also revealed the regeneration of hair follicles in the wounds treated with the FGF2-coacervate, demonstrating the accelerating effect of FGF2-coacervate on the wound re-epithelization in vivo. These in vivo data also had a good match to the results of wound closure rates. Taken together, the FGF2-coacervate was demonstrated to exhibit the improved healing effect on re-epithelialization, granulation tissue formation, and collagen deposition, due to its sustaining release of FGF2. 3.4. FGF2-coacervate promotes keratinocytes and fibroblasts proliferation Proliferating cell nuclear antigen (PCNA) was further used as a stained marker to evaluate cell proliferation as shown in Fig. 7. When treating with the FGF2-coacervate, cells around the wounds, such as keratinocytes, fibroblasts and others, proliferated very obviously, as compared to cells treated with no or vehicle and FGF2. Specifically, at day 7, the proliferation of cells in the epithelium and in the dermal with the treated of FGF2-coacervate were ~2.0 and 4.6 times higher than that of cells in the other three groups where most cells were inactive (Fig. 7B-C), indicating that the FGF2-coacervate possesses more active ability to stimulate cell proliferation at the wound sites. We performed further quantification of specific florescence staining of α-SMA to detect the density changes of activated fibroblast (the presence of myofibroblast) between different treated groups32, 33. Using anti-α-SMA antibody to represent the formation of myofibroblast revealed that α-SMA-positive cells in FGF2-Heparin@PEAD group arranged more compactly and orderly than those in other groups (Fig. 8A). Quantitatively, FGF2-Heparin@PEAD group (7.8±1.2%) exhibited the highest density of α-SMA-positive cells as compared to other three groups (3.6±2.0% to 4.2±2.6%,) (Fig. 8B), indicating more myofibroblast were also promoted by FGF2 coacervate leading to the quicker wound healing. Moreover, TGF-β1 is another important growth factors driving angiogenesis and myofibroblast differentiation during granulation tissue formation12, 34. At day 7 post-wounding, the wounds in all groups revealed the increased expression amounts of TGF-β1 (Fig. S2A), and the FGF2-coacervate treated wounds showed the highest 10


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TGF-β1 level (Fig. S2B). The enhanced temporal expression of TGF-β1 concentration was driven by FGF2 stimulation from FGF2-coacervate groups and consequently accelerated the granulation tissue formation. But, at day 17, the TGF-β1 concentration was decreased in the FGF2-coacervate treated group (Fig. S2C & Fig. S2D), as compared to the other groups, indicating that the FGF2-coacervate group improved wound healing by releasing FGF2 to induce the higher TGF-β1, stimulating expression of cytokine at first, followed by reducing scar formation by decreasing the TGF-β1. 3.5. FGF2-coacervate facilitates endogenous VEGF expression In addition to FGF2, a number of proangiogenic factors regulating cell division and cell survival, including VEGF, platelet-derived growth factor (PDGF), and tumor necrosis factor (TNF-α)35-37, not only are important for neovascularization38, 39, but also stimulate with each other to assert a synergistic role in promoting angiogenesis40, 41 . At day 7, immunohistochemical staining showed significant expression of VEGF in the FGF2-coacervate-treated group as compared to the other three groups. The high expression of VEGF was observed not only in the wound edges (Fig. 9A), but also in the wound centers (Fig. 9B). This indicates that VEGF and FGF2 play a key synergistic role in wound healing by stimulating endothelial cell proliferation, migration into the wound area, and inducing angiogenesis. But, at day 17, there was no significant difference in VEGF expression between all groups (Fig. 9C). This suggests that FGF2-induced expression of VEGF to promote angiogenesis only occur at the first 7 days, not at the later stage of wound healing. A number of studies reported similar observation that FGF2 stimulated the expression of VEGF at the wound sites, and co-interplay of FGF2 and VEGF resulted in a synergistic impact on skin wound healing42. Moreover, Nogami et al. demonstrated the fluctuation of the expression of VEGF in the wound healing process, revealing that VEGF was activated in the early stage of tissue repair process to induce vascularization43. 3.6. FGF2-coacervate increases vascularization in wound The proper wound healing requires angiogenesis of the newly-generated dermis, a process involving the proliferation and migration of endothelial and mural cells 44, 45. To evaluate neovascularization of the wounds, we quantified the expression of CD31 and α-smooth muscle actin (α-SMA) - the biomarker of endothelial cells and mural cells in blood vessels in skin tissue sections stained with immunofluorescence. In Fig. 10, immunofluorescence detection of CD31 and α-SMA together after 17 days confirmed the number of newly formed and mature blood vessels in the wounds treated with FGF2-coacervate, but there was only a very small amount of vascular vessels and smooth muscle cells found in wounds treated with free FGF2 or saline. In Fig. 10C, blood vessel density as shown was significantly higher in wounds treated with FGF2-coacervate (13.2±3.1%) vs. wounds treated with vehicle (8.2±1.7%) or saline only (6.6±1.6%) or FGF2 only (7.4±1.8%). Moreover, vessel diameter as shown in Fig. 10D was larger in the healing wounds of the FGF2-coacervate group (31.8±8.7 µm), compared with the other groups (13.5±7.7 µm, 9.4±5.0 µm, and 11


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5.3±9.0 µm, respectively). It was also observed the co-location of both mural cells and endothelial cells around the wound treated with FGF2-coacervate, but the wounds from other three groups did not show the detectable cell proliferation. This is also consistent with the previous observation that there were approximately ~3-fold more cells in the wounds of FGF2-coacervate-treated mice. These data indicate that the control release of FGF2 promotes the recruitment of endothelial cells to the wound area and enhances the vessel growth in healing wounds. 4. CONCLUSIONS This work develops a heparin-FGF2@PEAD coacervate to efficiently and safely deliver FGF2 to full-thickness dermal wounds in mice, followed by the investigation the mechanism of action of the controlled release of FGF2 for accelerating wound healing. The coacervate is demonstrated to play a synergistic role in increasing the proliferation and migration of wound area related cells, improving granulation tissue formation and angiogenesis, up-modulating VEGF expression, thus enhancing wound healing. Additionally, we provide valuable data for better understanding the role of the FGF2 in healing full-thickness skin wounds, i.e. the controlled release of FGF2 is important for accelerating skin wound healing as compared to other cytokines. Hence, the FGF2-coacervate delivery system is highly promising for the future treatment of chronic wounds. On the other hand, we should also mention that for this coacervate system, the negatively charged heparin is used as a bridge to associate charged PEAD first, then binds to FGF2 through heparin domain, forming the coacervate. Both ionic and pH environments are very crucial for this coacervate system, especially for different GFs with different isoelectric points. Thus, different GFs delivery systems should be carefully designed using heparin crosslinked hydrogels or modified heparin polymers especially for co-delivery of varied GFs toward chronic wounds. ASSOCIATED CONTENT Supporting Information Figures S1 and S2.

AUTHOR INFORMATION Corresponding Author Jian Xiao, [email protected]; Lin Li, [email protected]; Jie Zheng, [email protected] Author Contributions J. Wu, J. Ye, L. Li, J. Zheng, and J. Xiao designed the experiments. Y. Wang and H. Chen prepared the PEAD materials. J. Wu, J. Ye, J. Zhu, Z. Xiao and C. He performed in vivo experiments. C. Lin, H. Zhang, Y. Zhao, X. Fu, and X. Li performed cell 12


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experiments. J. Wu, J. Ye, H. Shi, H. Zhang, and H. Chen analyzed the data. J. Wu, J. Ye, J. Xiao and J. Zheng wrote the paper. The manuscript was written through the inputs of all the authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT J.W. thanks for the financial support from Zhejiang Provincial Natural Science Foundation of China (LQ15E030003), Wenzhou Science & Technology Bureau of China (Y20140727) and the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences (YKFJ001). J.X. thanks for the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents. This work was also supported by National Natural Science Funding of China (81372112, 81372064, 81302775, 81472165, 81571923), Zhejiang Provincial Natural Science Foundation of China (Y14H150023, Y14H090062, LY13H030008, LY14H150008), State Key Basic Research Development Program (2012CB518105), Zhejiang Provincial Project of Key Group (2010R50042). J.Z. thanks the financial support, in part, from NSF grants (CAREER Award CBET-0952624, CBET-1510099, DMR1607475), Alzheimer Association (2015-NIRG-341372), and National Natural Science Foundation of China (NSFC- 21528601).

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Fig. 1. Illustration of Heparin-FGF2@PEAD from preparation to wound healing process. Heparin-FGF2@PEAD enhance the cutaneous skin wound by promoting keratinocyte and fibroblast proliferation, stimulating the secretion of vascular endothelial growth factor (VEGF), and enhancing re-epithelization, granulation tissue formation, collagen deposition, and angiogenesis.

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A

PEAD

heparin-FGF2

12 h

+ heparin-FGF2@PEAD

B

D

C

5 µg S

C

Release (%)

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10 µg L

S

C

L

60

40

20

0 0

5

10 Days

15

Fig. 2. The characterization, loading efficiency, and release kinetics of heparin-FGF2@PEAD. (A) The preparation process of FGF2-coacervate. The red arrow points to the precipitation particles. (B) Scanning electron micrograph of the interior morphology of heparin@PEAD complex at 1000×. Heparin@PEAD complex was largely composed of ribbon-like structures. Scale bars: 500 µm. (C) The loading efficiency of FGF2 into the FGF2-coacervate. (D) The cumulative release profile of FGF2 from heparin-FGF2@PEAD during 17 days.

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B

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Saline Vehicle Free FGF2 FGF2-coacervate

*

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80 ***

60 40 *

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10 Time (d)

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Fig. 3. Wound closure of FGF2 coacervate treated wound closure in mouse. (A) Sequential photographs of four types of treated wounds on day 7, 10, 14, and 17. The units is mm. (B) The wound closure rates for the four types of the treated wounds. *** P < 0.001, ** P < 0.01, * P < 0.05, compared to control group, n=7.

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Fig. 4. Representative images of HE-stained (Hematoxylin and Eosin-stained) histological wound sites for the four groups. The black arrows pointed the skin appendages.Scale bars=500 µm.

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Fig. 5. The histological collagen deposition of masson trichrome staining (MTS). (A) Representative images of MTS histological wound sites for the four groups. The black arrows pointed the skin appendages. Scale bars (A-H)=500 µm, scale bars (i, ii, iii)=200 µm.

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Fig. 6. Immunohistochemical with the cytokeratin in wounds for the four groups. Representative light microscopy images indicates appendage regeneration in the presence of hair follicles within the granulation tissue after treated. Scale bars (low magnification)=500 µm, scale bars (high magnification)=50 µm.

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A

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150 100 50

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er va

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F2

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Fig. 7. Immunohistochemical with the PCNA in wounds for the four groups. The histogram represents the positive cells and optical density of the immunohistochemistry results. The red dotted line is used to separate the epidermis and the dermis. Scale bars=50 µm. *** P < 0.0.001 versus control, ### P < 0.001 versus free FGF2.

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Fig. 8. (A) The presence of α-smooth muscle actin (α-SMA) expression myfibroblast upon post wounding for control, vehicle, free FGF2 and FGF2 coacervate group. (B) The histogram represents the quantitation of myofibroblasts through α-smooth muscle actin (α-SMA) expressions. Scale bars=50 µm.* P < 0.05 versus control, # P < 0.05, versus free FGF2.

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A

100

50

C

on tr

ol

0

FGF2-coacervate

B

##

Ve hi cl e Fr ee FG FG F2 F2 -c oa ce rv at e

Free FGF2

Vehicle

** 150

(VEGF)

Control

The Number of Positive Cells/mm 2

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on tro l

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Fig. 9. Immunohistochemistry of the expression of VEGF (A) at the wound edge at the day 7, (B) at the center of wound at the day 7, and (C) at the healing area at the 22


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day 17. The histogram represents the positive cells and optical density of the immunohistochemistry results (D, E, F). The red dotted line is used to separate the epidermis and the dermis. Scale bars=50 µm. ** P < 0.01 versus control, ## P < 0.001 versus free FGF2, NS means no significant difference between them.

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D **

20 15 10 5

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50 40 30 20 10


ic l

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Vassel Diameter/µm

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Fig. 10. Immunofluorescence detection of CD31 and α-SMA at the wound area on the day 17. (A) Representative confocal images of granulation tissue near the wound margin show endothelial cell (CD31, red) and mural cell (α-smooth muscle actin, green) with DAPI (blue) nuclear staining. Scale bars=200 µm. (B) High magnification revealed by the co-localization of cells (yellow) as potential mature vessels. The 24


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circular vessel-like structures were observed in the wound area. Scale bars =50 µm. (C) The histogram represents the blood vessel density of the immunofluorescence results. (D) The histogram represents the vessel diameter of the blood vessels. ** P < 0.01 versus control, # P < 0.05, ## P < 0.001 versus free FGF2.

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Heparin-Based Coacervate of FGF2 Improves Dermal Regeneration

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