Biomimetic Elastomeric Polypeptide-Based Nanofibrous Matrix for

Oct 12, 2018 - ... exudate (Figure 6A), indicating that ampicillin (a commercial antimicrobial agent) exhibited poor antibacterial activity against MR...
2 downloads 0 Views 2MB Size
Subscriber access provided by University of Sunderland

Article

Biomimetic Elastomeric Polypeptide-Based Nanofibrous Matrix for Overcoming Multidrug-Resistant Bacteria and Enhancing Full-Thickness Wound Healing/Skin Regeneration Yuewei Xi, Juan Ge, Yi Guo, Bo Lei, and Peter X Ma ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01152 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Biomimetic Elastomeric Polypeptide-Based Nanofibrous Matrix for Overcoming Multidrug-Resistant Bacteria and Enhancing Full-Thickness Wound Healing/Skin Regeneration Yuewei Xi a, #, Juan Ge a, #, Yi Guo a, Bo Lei a, b, c*, Peter X Ma d, e, f a Frontier

bKey

Institute of Science and Technology, Xi’an Jiaotong University, Xi’an 710054, China

Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology,

Xi'an Jiaotong University, Xi'an 710000, China c

Instrument Analysis Center, Xi’an Jiaotong University, Xi’an 710054, China

d Department

of Biomedical Engineering, University of Michigan, Ann Arbor 48109, USA

e Macromolecular

f Department

Science and Engineering Center, University of Michigan, Ann Arbor 48109, USA

of Materials Science and Engineering, University of Michigan, Ann Arbor 48109, USA

* Corresponding author: Bo Lei, [email protected]

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Overcoming the multidrug-resistant (MDR) bacterial infection is a challenge and urgently needed in wound healing. Few wound dressings possess the capacity to treat MDR bacterial infections and enhance wound healing. Herein, we develop an elastomeric, photoluminescent and antibacterial hybrid polypeptide-based nanofibrous matrix as a multifunctional platform to inhibit the MDR bacteria and enhance wound healing. The hybrid nanofibrous matrix was composed of poly(citrate)-ε-poly-lysine (PCE) and poly-caprolactone (PCL). The PCL-PCE hybrid nanofibrous matrix showed a biomimetic elastomeric behavior, robust antibacterial activity including killing MDR bacteria capacity, excellent biocompatibility. PCL-PCE nanofibrous system can efficiently prevent the MDR bacteria-derived wound infection and significantly enhance the complete skin-thickness wound healing and skin regeneration in a mouse model. PCL-PCE hybrid nanofibrous matrix might become a competitive multifunctional dressing for bacteria-infected wound healing and skin regeneration. KEYWORDS: antibacterial activity, multifunctional nanofibrous matrix, polypeptide, wound infection, wound healing

ACS Paragon Plus Environment

Page 2 of 34

Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

The chronic skin wound resulted from severe bacterial infection has become a main medical threat in past several years.1 Especially, the multidrug-resistant bacteria (MDR)-derived wound infection usually resulted in the severe chronic cutaneous wounds which are difficult to be healed.2 Few drugs are efficient to treat the MDR-associated wound infection. Conventional antibacterial biomaterials including inorganic nanomaterials (silver, zinc, copper) and organic molecules (quaternary ammonium salts, alkylated polyethylenimine) could be used to treat MDR bacteria.3-5 However, the cytocompatibility and hemocompatibility for these antibacterial biomaterials are not satisfied in wound healing.6 To effectively treat MDR infection and enhance wound repair, it is urgent to develop multifunctional bioactive dressing with biocompatibility, effective against MDR infection, biomimetic structure and mechanical properties. Anti-microbial polypeptides (AMPs) such as ε-poly-lysine (EPL) could target the plasma membrane of pathogen cells, which have shown promising ways to reduce the likelihood of drug resistance development.7 As a natural cationic polypeptide and safe polymer (FDA approved in USA), EPL was secreted from Streptomyces albulus. EPL showed excellent biocompatibility and antibacterial activity including MDR.8 Previous studies showed that the antibacterial activity of EPL works through affecting the cell surface structure and physiological metabolism of bacteria.9,10 The cell membranes of bacterial were easily destructed due to the neutralized interaction between EPL (positive charge) and bacterial cell membrane (negative charges). EPL-based biomaterials have shown their promising applications in drug/gene delivery and tissue regeneration.11-13 EPL-based dressing should be effect for treating MDR-derived infection and improving cutaneous wound healing.

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Electrospinning porous nanofibrous matrix could mimic the extracellular matrix structure in skin, provide high large surface for cells, and enhance the permeation of nutrients and oxygen.14 Thus, nanofibrous scaffolds and nanomaterials with different components including various biopolymers (chitosan, poly(ε-caprolactone), gelatin, etc.) have been developed for wound healing and tissue regeneration.15-20 Native human skin tissue possesses elastomeric mechanical properties and optimized biological functions.21 However, most of current biodegradable polymer nanofibrous scaffolds in wound healing showed brittle or plastic mechanical behavior or low bioactivity.22,23 Recent years, poly(citric acid)-based (PC) elastomers have presented promising application in tissue engineering and drug/gene therapy, because of the biomimetic viscoelastic properties, good biocompatibility and angiogenic capacity, facile synthesis and low cost, and biocompatible degradation products.24-27 However, because of the intrinsical disadvantages such as low molecular weight, pure PC polymer solution is difficult to form nanofibrous structure through electrospinning technology.28 Therefore, the biomimetic elastomeric PC-based nanofibrous scaffolds are usually fabricated through blending with other natural or synthetic biomedical polymers.29 Particularly, poly(ε-caprolactone) (PCL) could be a suitable electrospinning polymer matrix, due to its high toughness. The electrospinning nanofibrous scaffolds composed of PCL and PC probably showed biomimetic elastomeric behavior, high toughness and enhanced biocompatibility. Here, we reported a biomimetic elastomeric multifunctional PCL-PC-co-ε-poly-lysine (PCE) nanofibrous matrix for overcoming MDR infection and wound healing. PCE hybrid nanofibrous matrix possesses skin-biomimetic elastomeric mechanical behavior, optimized hydrophilicity, robust antibacterial activity including MDR bacteria, excellent biocompatibility. The physicochemical structure and multifunctional properties in vitro/in vivo, wound healing capacity in vivo of PCE nanofibrous matrix was investigated in detail.

ACS Paragon Plus Environment

Page 4 of 34

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

RESULTS AND DISCUSSION Physicochemical Structure Characterizations of PCL-PCE Nanofibers. Figure 1 exhibits the synthesis, surface morphology, nanostructure, and chemical structure of PCL-PCE nanofiber matrix. The PCE was synthesized by the grafting reaction between PC and EPL (Figure 1A), and the chemical structure of PCE was identified through the 1H NMR analysis (Figure S1), the representative peak at 8.57 ppm (-CONH-) indicated the successful synthesis of PCE. Under the optimized conditions, the uniform porous nanofibrous structure with smooth surface was fabricated successfully (Figure 1B). The nanofiber diameters of pure PCL and PCL-PCE hybrids were obtained as 212±40 nm (PCL); 240±54 nm (PCL-10%PCE); 304±69 nm (PCL-30%PCE); 450±100 nm (PCL-50%PCE), respectively (Figure 1C). The incorporation of PCE probably decreased the electrospinning capacity of PCL, and significantly increased the nanofiber diameter of PCL. The FTIR analysis indicated the chemical structure of PCL-PCE hybrid nanofibers, as shown in Figure 1D. Methylene absorption peaks were seen at 2949 cm-1 and 2865 cm-1, carbonyl stretching was seen at 1727 cm-1, and -C-O-C- absorption peaks were seen at 1293 cm-1 and 1240 cm-1 in all samples. The PCL-PCE showed two peaks (1650 cm-1, 1540 cm-1) which were assigned to the amide bonds in PCE polymer. Hydrophilicity, Biomimetic Mechanical Properties and Photoluminescence. The controlled hydration ability for biomaterials was very important in wound healing process. Figure 2 shows hydrophilicity and water absorption measurement of PCL-PCE hybrid nanofibrous matrix. The contact angle of Pure PCL nanofiber mats was 130° ± 3° and the angle significantly decreased as the addition of PCE (Figure 2A,B). Specially, PCL-30%PCE nanofibers matrix had a contact angle of 85° ± 2° and changed to be 41° ± 1° in 30 seconds. The decreased contact angle for the PCL-PCE nanofibers was probably because of the incorporation of EPL (hydrophilic). Additionally, it was realized that with the addition of PCE the water absorption increased

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

significantly, due to the enhanced hydrophilicity of PCL-PCE matrix. These results suggested that our PCL-PCE matrix may have enhanced ability to screen extravasate and nutrient transport during the wound healing. Human soft tissues, including skin, skeletal muscle, show representative viscoelastomeric and strong mechanical properties. Typically, the human skin tissue in the age of 7 months to 3 years possesses a tensile Young’s modulus about 28.42 MPa.30 Conventional biodegradable biomedical polymers such as PCL and PLA have the high tensile modulus compared to skin tissue while they are not elastomeric biomaterials.31,32 Figure 3 shows the mechanical properties of PCL-PCE hybrid nanofibrous matrix. In Figure 3B the tensile modulus of PCL-10%PCE (26.66 ± 2.17 MPa), PCL-30%PCE (25.07 ± 3.32 MPa) and PCL-50%PCE (21.43 ± 0.27 MPa) was significantly lower than that of pure PCL nanofiber mats (46.29 ± 2.16 MPa), suggesting that PCE increased the PCL-PCE nanofiber mats stiffness. In contrast, in Figure 3C the elongation at break for PCL-30%PCE (197 ± 4%) and PCL-50%PCE (225 ± 13%) were more than 30% higher than pure PCL nanofibers (149 ± 7%),which were comparable to human skin (200 ± 15.6%) as reported.21 Moreover, in the range of 50%, PCL-PCE nanofibers presented better antifatigue strength-elongation behavior (Figure S2). Taken together, these data suggest PCL-PCE nanofibers are mechanically more appropriate than pure PCL nanofiber mats to withstand the physiological stains dressed on skin grafts. The intrinsical photoluminescent ability of PCL-PCE nanofibers was investigated for potential bioimaging application.33 The PCL-PCE nanofiber mats exhibited an excitation wavelength of 365 nm (as shown in Figure S3). Under that excitation at 365 nm, compared to PCL nanofiber mats, the improved fluorescent emission for PCL-PCE nanofiber mats was observed, and with the increase of PCE contents, the fluorescence intensity increased obviously (Figures S3B,C). The in vivo test showed that from 0 day to 9 days, as compared to PCL

ACS Paragon Plus Environment

Page 6 of 34

Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

nanofiber mats (left on the mouse), PCL-30%PCE nanofiber mats covering the wound (right on the mouse) showed a strong photoluminescent and bioimaging ability (Figure S3D). After 12 days, the wound covered with PCL-PCE nanofibers has healed and fluorescence disappeared, indicating that no material residual in the mouse (Figure S3D). Overall, these results indicated that PCL-PCE nanofibers provide intrinsic fluorescence, which enabled non-invasive monitoring of the wound dressing change in vivo. Inherent Antibacterial Properties against Normal and MDR Bacteria. Microbial infections may prevent wound repair process, and antimicrobial wound dressing can promote healing process by reducing the inflammatory response at the wound site.34,35 The antibacterial ability of PCL-PCE nanofibers was evaluated against S. aureus, E. faecalis, E. coli, P. aeruginosa and MRSA (MDR Gram-positive bacterium) in vitro (Figure 4 and Figure S4). After contact with nanofiber mats for 2 h, more than 99.99% of S. aureus, E. faecalis, E. coli, P. aeruginosa and MRSA were killed in PCL-30%PCE and PCL-50%PCE group (Figures 4A,B and Figure S4A,B), indicating their excellent antibacterial activity for normal and MDR bacteria. PCL-10%PCE showed a 69.5% kill ratio for S. aureus, a 51.7% kill ratio for E. faecalis, a 69.5% kill ratio for E. coli, a 78.4% kill ratio for P. aeruginosa and 71.7% ratio for MRSA while pure PCL nanofibers showed no obvious antibacterial effect (Figures 4A,B and Figure S4A,B). These results indicated that the antibacterial property of PCL-PCE nanofiber mats should be originated from PCE, PCL-PCE with high PCE content demonstrated the increased antibacterial ability against normal and MDR bacteria. After incubation with PCL-PCE for 2 h, the morphology of E. coli, S. aureus and MRSA cells was observed to determine the antibacterial process (Figure 5). The PCL nanofibers treated S.aureus, E. coli and MRSA cells possess the regular cellular morphology and no obvious damage was observed. However, after contact with PCL-30%PCE nanofibers for 2 h, the significant morphological changes occur on the S. aureus, E. coli and MRSA. Apparently, amounts of S. aureus, E. coli

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and MRSA were damaged and the cell membranes are no longer intact. The results of the bacterial morphology observation can support the antibacterial mechanism that EPL could induce the destruction of cell membrane.9 To demonstrate the activity against MDR bacteria in vivo of the PCL-PCE nanofibers, the MRSA-infected wound model were created. The macroscopic photos of the wounds with various dressings at 3 days were taken and the bacteria from the infected tissues were cultured (Figure 6). After MRSA bacterial infection and different treatment of 3 days, the wound treated with PBS, 3M TegadermTM film pure PCL and ampicillin became inflamed and had a light yellow exudate (Figure 6A), indicating that ampicillin (a commercial antimicrobial agent) exhibited poor antibacterial activity against MRSA infections. Additionally, LB agar plates derived from tissues suggested that the wound treated with PBS, 3M TegadermTM film pure PCL and ampicillin exhibited high bacterial growth, while the wound covered with PCL-30%PCE and PCE film showed a strong antibacterial activity against MRSA (Figures 6B,C). The antibacterial property of PCL-PCE nanofiber mats should be originated from PCE polymer. Cytotoxicity and Hemocompatibility Investigation. Figure 7 exhibits the cytotoxicity and hemocompatibility of PCL-PCE nanofibrous matrix. After culture 5 days, the lots of green live fibroblasts (L929) were on the surface of PCL-PCE and only few dead cells (red) was seen (Figure 7A). The cells numbers on PCL-10%PCE (b) and PCL-30%PCE (c) were significantly higher than PCL control. The Alamar blue test further indicated that PCL-10%PCE and PCL-30%PCE nanofibrous matrix significantly enhanced the cells viability and proliferation after 5 days incubation, compared with PCL nanofibers (Figure 7B). The blood compatibility of the PCL-PCE nanofibers was further investigated by testing the hemolytic activity (Figures 7C,D). After incubation with pure PCL (b), PCL-10%PCE (c) and PCL-30%PCE (d), the RBCs showed a similar morphology with PBS group (a) and no obvious hemolysis was observed (Figure 7C). However, after

ACS Paragon Plus Environment

Page 8 of 34

Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

incubation with PCL-50%PCE group (e), the RBCs presented some degrees of damages and hemolysis, compared with Triton-X100 group (f). The further calculation confirmed that PCL-10%PCE and PCL-30%PCE group only resulted in the hemolysis ratio below 5% which was in the range of hemocompatibility. These results confirmed that PCL-10%PCE and PCL-30%PCE nanofiber mats demonstrated the excellent cytocompatibility and blood compatibility. Cutaneous Wound Healing Evaluation In Vivo. Based on the results of mechanical properties and biocompatibility, the developed PCL-PCE nanofiber mats (PCL-10%PCE and PCL-30%PCE) were employed to treat full-thickness cutaneous wound healing in vivo, pure PCL nanofiber mats and the 3M TegadermTM film (as a commercial dressing) was employed as the control (Figure 8-11). The macroscopic wound healing process was observed after covering the various samples for 3, 7, 10 and 14 days (Figure 8). On day 3, the exudate in wound was absorbed for PCL-10%PCE and PCL-30%PCE group, as compared to untreated, 3M and PCL group (Figure 8A). On day 7, a decreased wound size was seen with the PCL-PCE nanofiber dressings relatively to pure PCL nanofiber mats and 3M TegadermTM film. After treatment for 10 days, significant wound closure occurred in the PCL-30%PCE group, in comparison with the pure PCL and 3M TegadermTM film treated groups. After 14 days, wound treated with PCL-PCE nanofiber dressings became smooth with newborn epidermal tissue, while wound treated with pure PCL nanofiber mats and 3M TegadermTM film showed a red and uneven scar. Meanwhile, it is noteworthy that PCL-PCE nanofiber mats easily adhere to the wound without the need for a bioadhesive, which may have benefit for protecting wound healing. The further calculation confirmed that PCL-30%PCE group demonstrated the best wound healing ability including significantly decreased wound size and wound closure time, as compared to other groups (Figures 8B,C). The wound healing process was further evaluated through the histological examination (Figure 9-11). On

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

day 7, the thick epithelialization area was observed in H&E images of PCL-30%PCE group (Figure 9 and 10). On day 10, the reconstructed dermis tissue with new skin appendages such as hair follicle appeared in H&E stained sections in PCL-10%PCE group and PCL-30%PCE group (Figure 9 and 10). The PCL-30%PCE group presented much more new skin appendages than the PCL-10%PCE group (Figure 10). The wound in PCL-10%PCE and PCL-30%PCE group had an enhanced vascularization compared with that treated with 3M TegadermTM film or was untreated on day 7 (Figure S5). Almost complete regeneration of dermis tissue with skin appendages were observed in PCL-30%PCE group on day 14 (Figure 9 and 10). However, the wounds of the untreated group, 3M TegadermTM film group and PCL group did not show the complete regeneration of epidermis and dermis tissue (Figure 10). After skin injury, the thickness of the mouse epidermis change from thin to thick and then gradually became thinner, close to normal skin epidermis thickness (Figure S6). On day 14, the epidermis of PCL-30%PCE group was almost the same as normal skin epidermis, indicating their better regeneration (Figure 11A, Figure S6). The cell density of epidermis in PCL-30%PCE group represented the largest cell density which was very close to normal skin (Figure 11B). After 14 days, PCL-30%PCE group showed the maximum number of hair follicles which was also similar with normal skin tissue (Figure 11C, Figure S7). Additionally, PCL-30%PCE group also showed the thickest dermis tissue (Figure 11D). The Masson’s staining showed that the collagen deposition in the wound region was observed in all groups and PCL-30%PCE treated group showed the most dense collagen deposition (Figure S8). Transforming growth factor-β (TGF-β) is a closely related factor in wound healing.36 On day 3, the gene expressions of TGF-β1 in PCL-30%PCE group was significantly higher than other groups (Figure S9A), and no significant difference was observed at day 7. The formed collagen was also the indicator for wound healing. Here, the collagen level was calculated by calculating hydroxyproline in the regenerated skin tissue. The

ACS Paragon Plus Environment

Page 10 of 34

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

collagen levels continued to increase from day 3 to day 10, and then slightly decreased in all groups on day 14 (Figure S9B). The significantly high collagen level in the PCL-30%PCE nanofiber group was observed in 14 days relative to untreated group, while no big difference between 3M TegadermTM film and the untreated group. VEGF regulates the angiogenesis, re-epithelization and collagen synthesis during cutaneous wound healing. On day 7, PCL-30%PCE group showed significantly higher protein expression of VEGF than other groups (Figure S9C-D). CD31 is a transmembrane protein expressed in early angiogenesis. Positive staining for CD31 could be observed in all groups at 7th day (Figure S10). These results further confirmed that PCL-30%PCE could enhance the neovascularization, the collagen deposition and the final wound healing. The better regeneration of epidermis and dermis tissue is probably due to the antibacterial ability and optimized physicochemical properties of PCL-30%PCE nanofiber mats. The porous PCL-30%PCE nanofiber mats could adhere on the wound surface, absorb the exudate and provide suitable environment for wound healing. The skin-biomimetic elastomeric modulus for PCL-30%PCE probably offers a suitable mechanical signal for tissue formation. The broad-spectrum antibacterial ability for PCL-30%PCE nanofiber mats prevented the wound bacterial infections which often occur and hinder the healing of the wound. Previous studies reported several nanomaterials-based dressing with antibacterial activity for enhanced wound healing. For example, Ag and ZnO-based matrix,37,38 peptide-modified gold nanofibrous structure,39 metal-organic framework-based

hydrogel,40

bioactive

glass/ceramic-based

nanostructure41,42

and

antibacterial

polymers-based dressing43,44 have shown great potential in enhancing wound healing. Compared with reported wound dressing, our PCL-PCE matrix possesses several advantages in wound healing application. Firstly, the polypeptide-based nanofibrous structure shows good biocompatibility and inhibition capacity of MDR bacteria. Secondly, the PCL-30%PCE nanofibrous matrix could efficiently enhanced the formation of skin appendages

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

such as epidermis, dermis, and hair follicle tissues, which are few reported in previous studies.45,46 The biomimetic structure, mechanical properties, anti-MDR bacteria activity, complete skin regeneration capacity make PCL-30%PCE highly promising as a multifunctional biomaterial for potential wound healing application. However, the further in vivo study should be carried out to clarify the mechanism of wound healing though PCL-PCE hybrid nanofibrous matrix. CONCLUSIONS In summary, we fabricated a biomimetic elastomeric, photoluminescent, highly antibacterial, biocompatible polypeptide-based PCL-PCE hybrid nanofibrous matrix for overcoming MDR bacterial infection and enhancing wound healing. PCL-PCE nanofibrous matrix possessed a similar tensile elastomeric modulus with human skin tissue, as well as optimized hydrophilicity. The photoluminescent property of PCL-PCE hybrids enabled the real-time noninvasive fluorescent imaging in vivo during the wound healing. By taking advantage of EPL polypeptide, PCL-30%PCE nanofibrous matrix showed highly efficient antibacterial activity against normal E.coli, P. aeruginosa (Gram-negative) and S.aureus, E. faecalis (Gram-positive), as well as MRSA (MDR Gram-positive), while remaining excellent cytocompatibility and hemocompatibility. Based on the optimized structure and properties, PCL-30%PCE hybrid nanofibrous matrix effectively prevented the MDR bacteria-derived wound infection, enhanced the cutaneous wound healing and skin regeneration through stimulating the formation of epidermis, dermis, and hair follicle tissues. As a multifunctional dressing, PCL-30%PCE hybrid nanofibrous matrix has great potential in enhancing chronic wound healing and cutaneous tissue regeneration. MATERIALS AND METHODS Synthesis of PCE Polymer. The PC prepolymer was obtained by a melt-derived polymerization method,

ACS Paragon Plus Environment

Page 12 of 34

Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

according to our previous report.47 The PCE prepolymer was formed through the reaction of PC and EPL under the excitation of EDC/NHS in DMSO with stirring for 72 h,48 the resulted PCE was also purified via dialysis (MWCO 10000), followed by freeze-drying. The chemical structure was identified through the nuclear magnetic resonance spectrum (1H NMR) and Fourier transformation infrared spectroscopy (FT-IR NICOLET 6700, Thermo). Fabrication and Structure Characterizations of Multifunctional PCL-PCE Nanofibrous Matrix. The PCL-PCE hybrid nanofibrous matrix was fabricated by electrospinning. Briefly, PCE and PCL was dissolved in the mixed solvent (DCM: HAc: DMSO =7:2:1 in volume ratio) and the total concentration was 6 wt% (w/v). PCL-PCE hybrids with 10 wt%, 30 wt% and 50 wt% PCE were prepared. Electrospinning was performed by loading the PCL-PCE solution in a 20 mL Becton Dickinson syringe and the voltage was 18 kV. The final PCL-PCE nanofibers were received through the collector (15 cm away). As a control, pure PCL nanofiber was prepared by the similar method. The surface of the nanofiber mats were observed through scanning electron microscopy (SEM) (XL30, FEI). The nanofiber diameters were calculated by the Image J software (n =50). The chemical structure of PCL-PCE nanofibers were indicated by FT-IR via a KBr method. Hydrophilicity Measurement. The hydrophilicity of PCL-PCE nanofibers were assessed by contact angle (water) and water absorption analysis. Briefly, a drop of water was put on the PCL-PCE nanofibers (2 cm ×2 cm) film. After 0 second, 15 seconds and 30 seconds, the water drop was captured and the contact angle was measured. The water absorption of the nanofiber mats were evaluated via soaking samples (10 cm×4 cm) in PBS buffer at 37 °C. After 4 h the water absorption of the nanofiber mats was obtained by: Water absorption (%) = [(Mw-Md)/Md]×100% Where Md is the original weight of the PCL-PCE nanofiber mats and Mw is the weight of the nanofiber mats after immersed in PBS.

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Mechanical Properties Assessments. The uniaxial tensile tests including tensile modulus and strength and elongation of PCL-PCE nanofiber mats were executed through a materials mechanical analysis machine (Criterion Model 43, MTS).49 The detailed description on mechanical properties was shown in Supporting Information. In Vitro Antibacterial Assays. For the antibacterial activity assays, non-MDR Gram-negative P. aeruginosa and E. coli, non-MDR Gram-positive S. aureus and E. faecalis, MDR Gram-positive MRSA were selected as bacterium models. The detailed procedure was shown in Supporting Information. In Vivo Assay against MDR Bacteria. The infection model was slightly modified based on previous study.50 Female Kunming mice (8 weeks, 30-32 g) were anesthetized via intraperitoneal injection of chloral hydrate (10%) and a round wound (7 mm in diameter) was made. MRSA suspension (10 µL) with 107 CFU in PBS was put on the wound, and various materials were used to cover the wound. After 3 days the samples were taken and cultured on LB agar plates to check the antibacterial activity. Macroscopic photographs of each group were assessed by a digital camera in order to observe the infection. Cytotoxicity and Hemocompatibility Assay. A mouse-derived fibroblast (L929) was employed to analyze the cytotoxicity for PCL-PCE nanofiber mats. The cell activity on nanofibrous matrix was determined via Live/Dead and Alamar Blue kit (Invitrogen). The process of cell culture and test was according to previous report.

51

The hemocompatibility of nanofibrous matrix was evaluated through testing their hemolysis

property.52 The detailed methods were presented in Supporting Information. Cutaneous Wound Healing Examination. The wound healing investigation in vivo for samples was conducted on female Kunming mice (30-35 g). After anesthetization, the round section of skin injury (7 mm) was made with a skin biopsy apparatus from the back of the mouse skin under anesthetization. Kunming mice

ACS Paragon Plus Environment

Page 14 of 34

Page 15 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

were randomly divided into five groups (A-E). Group A was the untreated group with nothing dressed after skin injury, group B mice received 3M TegadermTM film (3M Health Care, USA), group C mice were dressed with the pure PCL nanofiber mats, mice in group D were treated with PCL-10%PCE nanofiber mats and group E mice received PCL-30%PCE nanofiber mats. After 3, 7, 10 and 14 days, the wounds imaged, the wound area were measured by Image J software according to the equation: wound size (%) = [W(3,7,10,14)/W0)] × 100% where W0 and W(3,7,10,14) represent the wounds areas on 0 day and 3, 7, 10, 14 days, respectively. The healed skin tissues were further evaluated using the histological analysis. Briefly, the wound site with the surrounding skin was isolated and fixed in 4% neutral formalin solution. The samples were embedded by paraffin, sectioned, stained via hematoxylin & eosin (H&E) and Masson’s trichrome. All samples were analyzed and photo-captured by microscope (IX53, Olympus, Japan). At 3, 7, 10 and 14 days, the thickness of the epidermis was measured by H&E stained micrographs and Image J software. In addition, at 14 days, the statistics of the number of follicles, cell density of epidermis and dermis thickness were also obtained by the H&E stained micrographs. Real-time quantitative polymerase chain reaction (RT-PCR) was performed to investigate the transforming growth factor- β (TGF-β1) expression in different regenerated tissue on day 3 and day 7, respectively. Briefly, total RNA of newborn skin was isolated and the cDNA synthesis was performed using All-In-One RT MasterMix (G490, ABM). The RT-PCR using UltraSYBR Mixture (Low ROX, CWBIO) was carried out by a 7500 Fast System (Applied Biosystems). The primer sequence of TGF-β1 was 5’-ACTGGAGTTGTACGGCAGTG-3’ and 5’-GGCTGATCCCGTTGATTTCC -3’. Gapdh was employed as the housekeeping gene. The formed collagen was evaluated through analyzing the amount of Hydroxyproline. The newborn skin was hydrolyzed and tested following the procedure of a Hydroxyproline Assay Kits

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(A030-2, Nanjing Jiancheng). The VEGF protein (vascular endothelial growth factor) expression in newborn skin tissue on day 7 was tested using a western blotting method. The detailed procedure for western blotting evaluation was shown in Supporting Information. Statistical Analysis. All experiments data were exhibited as the mean value ± standard deviation (SD). Statistically significant value was assessed on student’s T-test, and the differences were significant statistically when *P < 0.05, **P < 0.01. AUTHOR INFORMATION Corresponding authors

* Email (B. Lei) [email protected] Author Contributions # Y.

Xi and J. Ge contributed equally to this work.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (Grant No. 51502237, 51872224), China Postdoctoral Science Foundation (Grant No. 2017M613148), Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University (Grant No. 2018LHM-KFKT004), the Joint Funds of the National Natural Science Foundation of China (Grant no. U1501245). ASSOCIATED CONTENT Supporting Information Experimental sections of materials, detailed mechanical test, photoluminescence and bioimaging evaluations, in vitro antibacterial assays, cytotoxicity and hemocompatibility evaluation, western blotting analysis; 1H NMR

ACS Paragon Plus Environment

Page 16 of 34

Page 17 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

spectra of PCE; fatigue properties of the electrospun nanofiber mats; photoluminescent properties of the PCL-PCE nanofibrous matrix; antibacterial activity against E. faecalis and P. aeruginosa in vitro; number of blood vessels; H&E stained normal kunming mice skin; H&E stained hair follicles; Micrographs of Masson’s trichome stained tissues; gene and protein expression in wound healing; pictures of CD31 stained tissues. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.

REFERENCES (1) Chen, W. Y.; Chang, H. Y.; Lu, J. K.; Huang, Y. C.; Harroun, S. G.; Tseng, Y. T.; Li, Y. J.; Huang, C. C.; Chang, H. T. Self-Assembly of Antimicrobial Peptides on Gold Nanodots: Against Multidrug-Resistant Bacteria and Wound-Healing Application. Adv. Funct. Mater. 2015, 25, 7189-7199. (2) Levy, S. B.; Marshall, B. Antibacterial Resistance Worldwide: Causes, Challenges and Responses. Nat. Med. 2004, 10, S122-S129. (3) Bing, W.; Chen, Z. W.; Sun, H. J.; Shi, P.; Gao, N.; Ren, J. S.; Qu, X. G. Visible-Light-Driven Enhanced Antibacterial and Biofilm Elimination Activity of Graphitic Carbon Nitride by Embedded Ag Nanoparticles. Nano Res. 2015, 8, 1648-1658. (4) Xu, Q.; Zheng, Z.; Wang, B.; Mao, H.; Yan, F. Zinc Ion Coordinated Poly(Ionic Liquid) Antimicrobial Membranes for Wound Healing. ACS Appl. Mater. Interfaces 2017, 9, 14656-14664. (5) Ruparelia, J. P.; Chatteriee, A. K.; Duttagupta, S. P.; Mukherji, S. Strain Specificity in Antimicrobial Activity of Silver and Copper Nanoparticles. Acta Biomater. 2008, 4, 707-716. (6) Asghari, S.; Johari, S. A.; Lee, J. H.; Kim, Y. S.; Jeon, Y. B.; Choi, H. J.; Moon, M. C.; Yu, I. J. Toxicity of Various Silver Nanoparticles Compared to Silver Ions in Daphnia Magna. J. Nanobiotechnol. 2012, 10, 14-24 (7) Fjell, C. D.; Hiss, J. A.; Hancock, R. E. W.; Schneider, G. Designing Antimicrobial Peptides: form Follows Function. Nat. Rev. Drug Discovery 2012, 11, 37-51. (8) Shih, I. L.; Shen, M. H.; Van, Y. T. Microbial Synthesis of Poly(ε-Lysine) and Its Various Applications.

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioresour. Technol. 2006, 97, 1148-1159. (9) Li, Y. Q.; Han, Q.; Feng, J. L.; Tian, W. L.; Mo, H. Z. Antibacterial Characteristics and Mechanisms of ε-Poly-Lysine against Escherichia coli and Staphylococcus aureus. Food Control 2014, 43, 22-27. (10) Ye, R.; Xu, H.; Wan, C.; Peng, S.; Wang, L.; Xu, H.; Aguilar, Z. P.; Xiong, Y.; Zeng, Z.; Wei, H. Antibacterial Activity and Mechanism of Action of ε-Poly-L-Lysine. Biochem. Biophys. Res. Commun. 2013, 439, 148-153. (11) Zhou, L.; Xi, Y.; Yu, M.; Wang, M.; Guo, Y.; Li, P.; Ma, P. X.; Lei, B. Highly Antibacterial Polypeptide-Based Amphiphilic Copolymers as Multifunctional Non-viral Vectors for Enhanced Intracellular siRNA Delivery and Anti-infection. Acta Biomater. 2017, 58, 90-101. (12) Sittinger, M.; Bujia, J.; Minuth, W. W.; Hammer, C.; Burmester, G. R. Engineering of Cartilage Tissue Using Bioresorbable Polymer Carriers in Perfusion Culture. Biomaterials 1994, 15, 451-456. (13) Martin-Lopez, E.; Nieto-Diaz, M.; Nieto-Sampedro, M. Differential Adhesiveness and Neurite-Promoting Activity for Neural Cells of Chitosan, Gelatin, and Poly-L-Lysine Films. J. Biomater. Appl. 2012, 26, 791-809. (14) Rieger, K. A.; Birch, N. P.; Schiffman, J. D. Designing Electrospun Nanofiber Mats to Promote Wound Healing - a Review. J. Mater. Chem. B 2013, 1, 4531-4541. (15) Ge, J.; Liu, K.; Niu, W.; Chen, M.; Wang, M.; Xue, Y.; Gao, C.; Ma, P. X.; Lei, B. Gold and Gold-Silver Alloy Nanoparticles Enhance the Myogenic Differentiation of Myoblasts through p38 MAPK Signaling Pathway and Promote in vivo Skeletal Muscle Regeneration. Biomaterials 2018, 175, 19-29. (16) Dong, R. H.; Jia, Y. X.; Qin, C. C.; Zhan, L.; Yan, X.; Cui, L.; Zhou, Y.; Jiang, X. Y.; Long, Y. Z. In situ Deposition of a Personalized Nanofibrous Dressing via a Handy Electrospinning Device for Skin Wound Care. Nanoscale 2016, 8, 3482-3488. (17) Zhao, F.; Lei, B.; Li, X.; Mo, Y.; Wang, R.; Chen, D.; Chen, X. Promoting in vivo Early Angiogenesis with Sub-Micrometer Strontium-Contained Bioactive Microspheres through Modulating Macrophage Phenotypes. Biomaterials 2018, 178, 36-47. (18) Kandhasamy, S.; Perumal, S.; Madhan, B.; Umamaheswari, N.; Banday, J. A.; Perumal, P. T.; Santhanakrishnan, V. P. Synthesis and Fabrication of Collagen-Coated Ostholamide Electrospun Nanofiber Scaffold for Wound Healing. ACS Appl. Mater. Interfaces 2017, 9, 8556-8568. (19) Yu, M.; Lei, B.; Gao, C.; Yan, J.; Ma, P. X. Optimizing Surface-Engineered Ultra-Small Gold Nanoparticles for

ACS Paragon Plus Environment

Page 18 of 34

Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Highly Efficient miRNA Delivery to Enhance Osteogenic Differentiation of Bone Mesenchymal Stromal Cells. Nano Res. 2017, 10, 49-63. (20) Song, D. W.; Kim, S. H.; Kim, H. H.; Lee, K. H.; Ki, C. S.; Park, Y. H. Multi-Biofunction of Antimicrobial Peptide-Immobilized Silk Fibroin Nanofiber Membrane: Implications for Wound Healing. Acta Biomater. 2016, 39, 146-155. (21) Lorden, E. R.; Miller, K. J.; Ibrahim, M. M.; Bashirov, L.; Hammett, E.; Chakraborty, S.; Quiles-Torres, C.; Selim, M. A.; Leong, K. W.; Levinson, H. Biostable Electrospun Microfibrous Scaffolds Mitigate Hypertrophic Scar Contraction in an Immune-Competent Murine Model. Acta Biomater. 2016, 32, 100-109. (22) Del Bakhshayesh, A. R.; Annabi, N.; Khalilov, R.; Akbarzadeh, A.; Samiei, M.; Alizadeh, E.; Alizadeh-Ghodsi, M.; Davaran, S.; Montaseri, A. Recent Advances on Biomedical Applications of Scaffolds in Wound Healing and Dermal Tissue Engineering. Artif. Cells, Nanomed., Biotechnol. 2018, 46, 691-705. (23) Lei, B.; Shin, K. H.; Noh, D. Y.; Jo, I. H.; Koh, Y. H.; Choi, W. Y.; Kim, H. E. Nanofibrous Gelatin-Silica Hybrid Scaffolds Mimicking the Native Extracellular Matrix (Ecm) Using Thermally Induced Phase Separation. J. Mater. Chem. 2012, 22, 14133-14140. (24) Ma, C.; Gerhard, E.; Lu, D.;Yang, J. Citrate Chemistry and Biology for Biomaterials Design. Biomaterials 2018, 178, 383-400. (25) Li, Y.; Guo, Y.; Ge, J.; Ma, P. X.; Lei, B. In Situ Silica Nanoparticles-Reinforced Biodegradable Poly (citrate-siloxane) Hybrid Elastomers with Multifunctional Properties for Simultaneous Bioimaging and Bone Tissue Regeneration. Appl. Mater. Today 2018, 10, 153-163 (26) Jiang, B.; Suen, R.; Wang, J. J.; Zhang, Z. J.; Wertheim, J. A.; Ameer, G. A. Vascular Scaffolds with Enhanced Antioxidant Activity Inhibit Graft Calcification. Biomaterials 2017, 144, 166-175. (27) Du, Y.; Ge, J.; Li, Y.; Ma, P. X.; Lei, B. Biomimetic Elastomeric, Conductive and Biodegradable Polycitrate-Based Nanocomposites for Guiding Myogenic Differentiation and Skeletal Muscle Regeneration. Biomaterials 2018, 157, 40-50. (28) Prabhakaran, M. P.; Nair, A. S.; Kai, D.; Ramakrishna, S. Electrospun Composite Scaffolds Containing Poly(octanediol-co-citrate) for Cardiac Tissue Engineering. Biopolymers 2012, 97, 529-538. (29) Zhu, L.; Zhang, Y.; Ji, Y. Fabricating Poly(1,8-Octanediol Citrate) Elastomer Based Fibrous Mats via

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Electrospinning for Soft Tissue Engineering Scaffold. J. Mater. Sci.: Mater. Med. 2017, 28, 93-102. (30) Lu, T. J.; Xu, F. Mechanical Properties of Skin: a Review. Adv. Mech. 2008, 38, 393-426. (31) Chong, E. J.; Phan, T. T.; Lim, I. J.; Zhang, Y. Z.; Bay, B. H.; Ramakrishna, S.; Lim, C. T. Evaluation of Electrospun PCL/Gelatin Nanofibrous Scaffold for Wound Healing and Layered Dermal Reconstitution. Acta Biomater. 2007, 3, 321-330. (32) Zhong, S. P.; Zhang, Y. Z.; Lim, C. T. Tissue Scaffolds for Skin Wound Healing and Dermal Reconstruction. Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2010, 2, 510-525. (33) Yang, J.; Zhang, Y.; Gautam, S.; Liu, L.; Dey, J.; Chen, W.; Mason, R. P.; Serrano, C. A.; Schug, K. A.; Tang, L. Development of Aliphatic Biodegradable Photoluminescent Polymers. Proc. Natl. Acad. Sci. U. S. A 2009, 106, 10086-10091. (34) Fischbach, M. A.; Walsh, C. T. Antibiotics for Emerging Pathogens. Science 2009, 325, 1089-1093. (35) Ong, S. Y.; Wu, J.; Moochhala, S. M.; Tan, M. H.; Lu, J. Development of a Chitosan-Based Wound Dressing with Improved Hemostatic and Antimicrobial Properties. Biomaterials 2008, 29, 4323-4332. (36) Barrientos, S.; Stojadinovic, O.; Golinko, M. S.; Brem, H.; Tomic-Canic, M. Growth Factors and Cytokines in Wound Healing. Wound Repair Regen. 2008, 16, 585-601. (37) Mao, C.; Xiang, Y.; Liu, X.; Cui, Z.; Yang, X.; Yeung, K. W. K.; Pan, H.; Wang, X.; Chu, P. K.; Wu, S. Photo-Inspired Antibacterial Activity and Wound Healing Acceleration by Hydrogel Embedded with Ag/Ag@Agcl/ZnO Nanostructures. ACS Nano 2017, 11, 9010-9021. (38) Bhang, S. H.; Jang, W. S.; Han, J.; Yoon, J. K.; La, W. G.; Lee, E.; Kim, Y. S.; Shin, J. Y.; Lee, T. J.; Baik, H. K.; Kim, B. S. Zinc Oxide Nanorod-Based Piezoelectric Dermal Patch for Wound Healing. Adv. Funct. Mater. 2017, 27, 1603497 (39) Yang, X.; Yang, J.; Wang, L.; Ran, B.; Jia, Y.; Zhang, L.; Yang, G.; Shao, H.; Jiang, X. Pharmaceutical Intermediate-Modified Gold Nanoparticles: Against Multidrug-Resistant Bacteria and Wound-Healing Application via an Electrospun Scaffold. ACS Nano 2017, 11, 5737-5745. (40) Xiao, J.; Zhu, Y.; Huddleston, S.; Li, P.; Xiao, B.; Farha, O. K.; Ameer, G. A. Copper Metal-Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes. ACS Nano 2018, 12, 1023-1032.

ACS Paragon Plus Environment

Page 20 of 34

Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

(41) Wang, X.; Lv, F.; Li, T.; Han, Y.; Yi, Z.; Liu, M.; Chang, J.; Wu, C. Electrospun Micropatterned Nanocomposites Incorporated with Cu2s Nanoflowers for Skin Tumor Therapy and Wound Healing. ACS Nano 2017, 11, 11337-11349. (42) Gao, W.; Jin, W.; Li, Y.; Wan, L.; Wang, C.; Lin, C.; Chen, X.; Lei, B.; Mao, C. A Highly Bioactive Bone Extracellular Matrix-Biomimetic Nanofibrous System with Rapid Angiogenesis Promotes Diabetic Wound Healing. J. Mater. Chem. B 2017, 5, 7285-7296. (43) Dhand, C.; Venkatesh, M.; Barathi, V. A.; Harini, S.; Bairagi, S.; Leng, E. G. T.; Muruganandham, N.; Low, K. Z. W.; Fazil, M. H. U. T.; Loh, X. J. Bio-Inspired Crosslinking and Matrix-Drug Interactions for Advanced Wound Dressings with Long-Term Antimicrobial Activity. Biomaterials 2017, 138, 153-168. (44) Wang, Y.; Beekman, J.; Hew, J.; Jackson, S.; Issler-Fisher, A. C.; Parungao, R.; Lajevardi, S. S.; Li, Z.; Maitz, P. K. M. Burn Injury: Challenges and Advances in Burn Wound Healing, Infection, Pain and Scarring. Adv. Drug Delivery Rev. 2018, 123, 3-17. (45) Liu, J. Q.; Zhao, K. B.; Feng, Z. H.; Qi, F. Z. Hair Follicle Units Promote Re-Epithelialization in Chronic Cutaneous Wounds: A Clinical Case Series Study. Exp. Ther. Med. 2015, 10, 25-30. (46) Lucas, T.; Schafer, F.; Muller, P.; Eming, S. A.; Heckel, A.; Dimmeler, S. Light-Inducible antimiR-92a as a Therapeutic Strategy to Promote Skin Repair in Healing-Impaired Diabetic Mice. Nat. Commun. 2017, 8, 15162. (47) Du, Y.; Yu, M.; Ge, J.; Ma, P. X.; Chen, X.; Lei, B. Development of a Multifunctional Platform Based on Strong, Intrinsically Photoluminescent and Antimicrobial Silica-Poly(citrates)-Based Hybrid Biodegradable Elastomers for Bone Regeneration. Adv. Funct. Mater. 2015, 25, 5016-5029. (48) Wang, M.; Guo, Y.; Yu, M.; Ma, P. X.; Mao, C.; Lei, B. Photoluminescent and Biodegradable Polycitrate-Polyethylene Glycol-Polyethyleneimine Polymers as Highly Biocompatible and Efficient Vectors for Bioimaging-Guided siRNA and miRNA Delivery. Acta Biomater 2017, 54, 69-80. (49) Du, Y.; Yu, M.; Chen, X.; Ma, P. X.; Lei, B. Development of Biodegradable Poly(citrate)-Polyhedral Oligomeric Silsesquioxanes Hybrid Elastomers with High Mechanical Properties and Osteogenic Differentiation Activity. ACS Appl. Mater. Interfaces 2016, 8, 3079-3091. (50) Zhou, L.; Xi, Y.; Chen, M.; Niu, W.; Wang, M.; Ma, P. X.; Lei, B. A Highly Antibacterial Polymeric Hybrid Micelle with Efficiently Targeted Anticancer siRNA Delivery and Anti-infection in vitro/in vivo. Nanoscale. 2018,

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10, 17304-17317. (51) Xue, Y.; Guo, Y.; Yu, M.; Wang, M.; Ma, P. X.; Lei, B. Monodispersed Bioactive Glass Nanoclusters with Ultralarge Pores and Intrinsic Exceptionally High miRNA Loading for Efficiently Enhancing Bone Regeneration. Adv. Healthc. Mater. 2017, 6, 1700630 (52) Zhou, L.; Qu, X.; Guo, Y.; Wang, M.; Lei, B.; Ma, P. X. Branched Glycerol-based Copolymer with Ultrahigh p65 siRNA Delivery Efficiency for Enhanced Cancer Therapy. ACS Appl. Mater. Interfaces 2018, 10, 4471-4480.

ACS Paragon Plus Environment

Page 22 of 34

Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 1. Fabrication and characterizations of PCL-PCE hybrid nanofibrous matrix. (A) Synthesis of PCE; (B) SEM images of the PCL and PCL-PCE nanofibrous matrix; (C) Diameter distribution of PCL and PCL-PCE nanofibers; (D) FT-IR spectra of the PCL and PCL-PCE nanofibrous matrix.

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Hydrophilicity analysis of the PCL-PCE nanofibrous matrix. (A) Water drop change on various matrix as the contact times; (B) Water contact angle test at 0, 15, 30 seconds; (C) Water absorption ability of the PCL-PCE nanofibers after immersion in PBS for 4h. (*p < 0.05 and **p < 0.01)

ACS Paragon Plus Environment

Page 24 of 34

Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 3. Biomimetic elastomeric mechanical properties of PCL and PCL-PCE nanofibrous matrix. (A) Stress-strain curves; (B) Tensile modulus; (C) Elongation at break; (D) Tensile strength. (*p < 0.05 and **p < 0.01).

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Broad-spectrum antibacterial activity against normal and MRSA in vitro. (A) Pictures of agar plates and (B) corresponding statistical data of colonies of S. aureus, E. coli and MRSA treated with different PCL-PCE nanofiber mats. (*p < 0.05 and **p < 0.01).

ACS Paragon Plus Environment

Page 26 of 34

Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 5. SEM analysis showing the morphology changes of S. aureus, E.coli and MRSA after incubated with PCL and PCL-30%PCE nanofibrous matrix, red arrows indicate the morphological changes on the bacterial cell membrane.

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Antibacterial activity against MRSA infection in vivo. (A) Photographs of MRSA-infected mice skin treated with PBS, 3M TegadermTM film, Ampicillin, PCE film, PCL nanofiber mats, PCL-10%PCE nanofiber mats and PCL-30%PCE nanofiber mats after 3 days of bacterial infection (scale bar = 2 mm); (B-C) Pictures of MRSA colonies growing on LB agar plates derived from tissues treated with PBS, 3M TegadermTM film, Ampicillin, PCE film and various nanofibrous matrixes after 3 days of bacterial infection (B), and the statistical data of colonies of MRSA treated with different dressings (C). (*p < 0.05 and **p < 0.01).

ACS Paragon Plus Environment

Page 28 of 34

Page 29 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 7. Cytocompatibility and hemocompatibility analysis of vairous nanofibrous matrixes. (A) Live/Dead staining fluorescent images of fibroblast (L929 cells) after treated with (a) PCL, (b) PCL-10%PCE, (c) PCL-30%PCE, and (d) PCL-50%PCE for 5 d (scale 40 × ); (B) L929 cells viability after cultured for 1 d, 3 d and 5 d; (C) Images of RBCs after treated with (a) PBS, (b) PCL, (c) PCL-10%PCE, (d) PCL-30%PCE, (e) PCL-50%PCE and (f) Triton-X100; (D) Hemolysis ratio anlaysis of RBCs after incubation with various samples. (*p < 0.05 and **p < 0.01).

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. Macroscopic wound healing evaluation. (A) Photographs of wounds treated with 3M TegadermTM film (3M) and variuos nanofibrous matrixes for 0 d, 3 d, 7 d, 10 d and 14 d (scale bar = 2 mm); (B) Changes in wound size after various treatments; (C) Complete wound closure times for various treatments.

ACS Paragon Plus Environment

Page 30 of 34

Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 9. Low magnification H&E stained images showing the wound healing and skin regeneration for untreated group as well as wounds treated with 3M TegadermTM film, pure PCL, PCL-10%PCE and PCL-30%PCE nanofibirous matrixes at 3nd, 7th, 10th and 14th day. The red arrow reperesents the thickness of the dermis (scale bar = 200 µm).

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10. High magnification H&E stained images showing the wound healing and skin regeneration for untreated group as well as wounds treated with 3M TegadermTM film, pure PCL, PCL-10%PCE and PCL-30%PCE nanofibirous matrixes at 3rd, 7th, 10th and 14th day. Green dotted lines indicate the epithelium border, yellow arrows show the adipose cells and black arrows present the hair follicles (scale bar = 50 µm).

ACS Paragon Plus Environment

Page 32 of 34

Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 11. Key parameters statistics showing the wound healing and skin regeneration after variuos treatments, using normal skin tissue as a control. (A) Epidermis thickness at day 3, day 7, day 10, day 14; (B) Cell density of epidermis at day 14; (C) Numbers of hair follicles at day 14; (D) Thickness of dermis at day 14. (*p < 0.05 and **p < 0.01)

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Only

ACS Paragon Plus Environment

Page 34 of 34