Nanofiber Dressings Topically Delivering Molecularly Engineered

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Nanofiber Dressings Topically Delivering Molecularly Engineered Human Cathelicidin Peptides for Treatment of Biofilms in Chronic Wounds Yajuan Su, Hongjun Wang, Biswajit Mishra, Jayaram Lakshmaiah Narayana, Jiang Jiang, Debra A Reilly, Ronald R Hollins, Mark A Carlson, Guangshun Wang, and Jingwei Xie Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Molecular Pharmaceutics

Nanofiber Dressings Topically Delivering Molecularly Engineered Human Cathelicidin Peptides for Treatment of Biofilms in Chronic Wounds

Yajuan Su¶, Hongjun Wang¶, Biswajit Mishra§, Jayaram Lakshmaiah Narayana§, Jiang Jiang¶, Debra A. Reilly ♯ , Ronald R. Hollins ♯ , Mark A. Carlson♮, Guangshun Wang§*, and Jingwei Xie¶*

Department of Surgery-Transplant and Mary & Dick Holland Regenerative Medicine

¶

Program, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States §Department

of Pathology and Microbiology, College of Medicine, University of

Nebraska Medical Center, Omaha, Nebraska 68198, United States ♯

Department of Surgery-Plastic Surgery, College of Medicine, University of

Nebraska Medical Center, Omaha, Nebraska 68198, United States ♮

Department of Surgery-General Surgery, College of Medicine, University of

Nebraska Medical Center, Omaha, Nebraska 68198, United States *Corresponding

address to [email protected] (J. Xie) and [email protected] (G.

Wang)

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

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ABSTRACT Biofilms of multidrug-resistant bacteria in chronic wounds post a great challenge in wound care. Herein, we report the topical delivery of molecularly engineered antimicrobial peptides using electrospun nanofiber dressings as a carrier for treatment of biofilms of multidrug-resistant bacteria in diabetic wounds. Molecularly engineered human cathelicidin peptide 17BIPHE2 was successfully encapsulated in the core of pluronic F127/17BIPHE2-PCL core-shell nanofibers. The in vitro release profiles of 17BIPHE2 showed an in initial burst followed by a sustained release over four weeks. The

peptide

nanofiber

formulations

effectively

killed

methicillin-resistant

Staphylococcus aureus (MRSA) USA300. Similarly, the 17BIPHE2 peptide containing nanofibers could also effectively kill other bacteria including Klebsiella pneumoniae (104 - 106 CFU) and Acinetobacter baumannii (104 - 107 CFU) clinical strains in vitro without showing evident cytotoxicity to skin cells and monocytes. Importantly, 17BIPHE2-containing nanofiber dressings without debridement caused five-magnitude decreases of the MRSA USA300 CFU in a biofilm-containing chronic wound model based on type II diabetic mice. In combination with debridement, 17BIPHE2-containing nanofiber dressings could completely eliminate the biofilms, providing one possible solution to chronic wound treatment. Taken together, the biodegradable nanofiber-based wound dressings developed in this study can be utilized

to

effectively

deliver

molecularly

engineered

peptides

to

treat

biofilm-containing chronic wounds.

KEYWORDS: electrospinning, nanofibers, topical delivery, antimicrobial peptides, biofilms, chronic wounds

2


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1. INTRODUCTION Burn, trauma, surgery and chronic diseases induced wound infections caused by multi-drug resistant pathogens pose a major challenge to wound management1, 2. For example, diabetes mellitus affects 23.6 million people in the United States and approximately 20–25% of diabetic patients will develop foot ulceration during the course of the disease3. Among them, 63.4% of diabetic patients develop multidrug-resistant organism infections4. Failure to prevent or manage such infections has resulted in amputation, sepsis, and even death5. There is an urgent need for novel approaches for treating wound infections, particularly those caused by multi-drug resistant pathogens. Current approaches to the treatment of wound infections mainly include the use of antibiotics, silver and surgical management6-8. In short, the use of antibiotics is a primary and foundational tool in the care of the injured personnel9. Current evidence strongly suggests that this routine use of antibiotics may be an important factor in selecting resistant bacterial strains10. Meanwhile, recent findings indicated some antibiotic actually delays the wound-healing process and that silver may have serious cytotoxic activity on various host cells11, 12, as well as potential side effects such as argyria, a medically benign but permanent bluish-gray discoloration of the skin13. Furthermore, while antibiotic development continues to stagnate, antibiotic resistance continues to spread, particularly Gram-negative bacilli14. Additionally, current antibiotic

biomaterial

formulations

suffer

from

limitations

including

non-degradability (e.g., poly(methyl methacrylate) (PMMA)) or too short duration of release (e.g., collagen sponge)15, 16. Peptides have been widely used in biomedical application17. It has been shown that the antimicrobial peptide LL-37, the only cathelicidin-derived host defense peptide found in humans, effectively kills multi-drug resistant pathogens18. LL-37 is a key component of the innate immune system and represents the first line of defense against various invading pathogens19. Although direct application of LL-37 or over-expression by vectors increases local concentrations, significant problems with 3



delivery, host-cell toxicity, tissue destruction and inflammation exist20. Furthermore, the antimicrobial efficacy of LL-37 is heavily dependent on the environment including pH, salt/ions concentration, and proteases21. In fact, LL-37 peptides present low antimicrobial activities under serum and tissue conditions22. LL-37 was found to inhibit biofilm growth of Staphylococcus aureus23. LL-37 was unable to inhibit bacterial attachment or disrupt preformed biofilms24. Towards this end, our recent study demonstrated the successful engineering of human cathelicidin LL-37 into selective, stable and potent antimicrobial peptides, which displayed superior antibiofilm capability24,25. Electrospinning is a versatile technique for generating long fibers with nanoscale diameters26. Electrospun nanofiber wound dressings offer significant advantages over hydrogels or sponges for local drug delivery27. Collagen28, fibrin29, poly(ethylene glycol)(PEG)30, and alginate hydrogels31 are capable of soft tissue-like compliance, but are difficult to suture and are often too weak to support physiologic loads. In addition, it is difficult to encapsulate hydrophobic molecules inside hydrogels32. In sponges, hydrophobic drug molecules are usually crystalized after encapsulation, which slows down the dissolution rate and is unfavorable. On the other hand, electrospun nanofibers serve as ideal materials for topical drug delivery with following unique characteristics: i) ease of incorporation of drugs, especially the hydrophobic molecules inside nanofibers, ii) ease of control of release profiles by controlling the porosity of nanofibers and the degradation profiles, iii) exhibiting an amorphous state for encapsulated hydrophobic drug molecules and thus enhanced solubility of drugs33. The architecture of electrospun nanofibers mimics the collagen structure of the extracellular matrix (ECM)-a 3D network of collagen fibers 50 - 500 nm in diameter; therefore, compared to traditional wound dressings, nanofiber-based wound dressings provide several functional and structural advantages including hemostasis, high filtration, semi-permeability, conformability and scar-free healing34. Even though electrospun nanofibers offer numerous advantages, their full potential has not been realized. Their antimicrobial use is limited to the surface modifications with chitosan, Ag and ZnO nanoparticles, and encapsulation of antibiotics and Ag 4


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nanoparticles/ions35-37. Herein, we aim to increase the potential of these materials for fighting infections by developing nanofiber wound dressings for local delivery of LL-37 engineered antimicrobial peptides. Our hypothesis is that topical delivery of molecularly engineered antimicrobial peptides from nanofiber wound dressings could effectively treat multidrug resistant bacteria caused infections in chronic wounds.

2. MATERIALS AND METHODS 2.1. Materials. Poly(ε-caprolactone) (PCL) (Mw= 70 ~ 90kDa) and pluronic F127 were bought from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Media (DMEM) and RPMI 1640 media were bought from Thermo Fisher Scientific Gibco (Waltham, MA, USA). Dichloromethane (DCM) and N, N-dimethylformamide (DMF) were acquired from Thermo Fisher Scientific (Waltham, MA, USA). The 17BIPHE2 antimicrobial peptide was prepared according to our previous work38. Methicillin-resistant

Staphylococcus

aureus (MRSA)

USA300

LAC

and

Acinetobacter baumannii B2367-12 were obtained from University of Nebraska Medical Center (UNMC), while Klebsiella pneumoniae Klebsiella pneumoniae ATCC13883 and Pseudomonas aeruginosa PAO1 were obtained from American Type Culture Collection (ATCC). Columbia CAN w/5% sheep blood agar medium was purchased from Remel (Lenexa, KS, USA) and Tryptic Soy Broth (TSB) bacterial medium was purchased from Thermo Fisher Scientific Oxoid (Waltham, MA, USA). LIVE/DEAD BacLight bacterial viability kit and Alamar Blue cell viability assay kit was purchased from Thermo Fisher Scientific Invitrogen (Waltham, MA, USA). 2.2. Fabrication of Molecularly Engineered Peptides-loaded Nanofibers. A co-axial electrospinning setup was used to encapsulate peptides in the core of pluronic F127/17BIPHE2-PCL core-shell nanofibers following our previous studies39. Briefly, PCL was dissolved in a solvent mixture consisting of DCM and DMF with a ratio of 4:1(v/v) at a concentration of 10% (PCL) (w/v). To prepare F127-PCL core-shell fibers, 1 g pluronic F127 was dissolved in ddH2O to form the aqueous phase. To 5



prepare F127/17BIPHE2-PCL core-shell fibers, 1 g pluronic F127 and 25 mg 17BIPHE2 were dissolved in ddH2O to form the aqueous phase. The polymer phase was pumped at a flow rate of 0.5 ml/h and the aqueous phase was pumped at a flow rate of 0.02 ml/h while a potential of 20 kV was applied between the spinneret (a 22-gage needle) and a grounded collector located 12 cm apart from the spinneret. A rotating drum was used to collect membranes composed of random fibers with a rotating speed less than 100 rpm. Then, the obtained fiber samples were divided into two parts. One part was stored in 4 oC named as F127/17BIPHE2-PCL (Figure 1A). The other part was coated with 10 mg 17BIPHE2 named as F127/17BIPHE2-PCL-S (Figure 1B). Briefly, the 17BIPHE2 aqueous solution was deposited onto the fibers through electrospraying. All the fiber samples were sterilized by ethylene oxide gas prior to cell culture and in vivo animal study. 2.3. Morphology Characterization of Nanofibers. The morphology of fiber samples was characterized by scanning electron microscopy (SEM) (FEI, Quanta 200, Oregon, USA). To avoid charging, polymeric fiber samples were fixed on a metallic stud with a double-sided conductive tape and coated with platinum for 4 min in vacuum at a current intensity of 10 mA using a sputter coater. SEM images were acquired at an accelerating voltage of 30 kV. To confirm the core-shell structure, the FITC labeled bovine serum albumin (BSA) was used to form F127/FITC-BSA - PCL core-shell nanofibers using the same fabrication procedure described in section 2.2. The obtained fibers were collected on a coverslip and then imaged by a laser scanning confocal microscope (LSCM) (Zeiss, LSM 710). 2.4. Encapsulation Efficiency and In Vitro Drug Release Study. In vitro release of 17BIPHE2 from the fibers was evaluated by immersing 10 mg fiber samples in the 10 mL PBS solution at 37 oC. The supernatants were collected at each time point and replaced by fresh PBS solutions.17BIPHE2 concentrations in samples were determined using a high performance liquid chromatography (HPLC, Agilent Technologies, 1260 Infinity) with a UV detector at 220 nm. The drug encapsulation efficiency was determined as follows. A known mass of nanofiber membrane was dissolved in 1 mL of DCM, and the solution was added dropwise to 20 mL of 6


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methanol in which the polymer was precipitated and peptide was dissolved. After centrifugation of the methanol solution, the liquid supernatant was detected by HPLC at λmax=220 nm. The encapsulation efficiency was calculated by the following equation (1): Encapsulation efficiency %=

𝑡ℎ𝑒 𝑎𝑐𝑡𝑢𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑔) 𝑡ℎ𝑒 𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑔)

×100%

2.5. In Vitro Antibacterial Efficacy Test. The antibacterial activity of F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S core-shell nanofibers were investigated. Single bacterial colonies of MRSA, K. pneumoniae, A. baumannii and P. aeruginosa were picked up by inoculating loops and cultured at 37 oC and 200 rpm in liquid TSB overnight. Ten μL bacterial culture was added into 2 mL fresh TSB and incubated for additional ~2 h. Then, the cultures were centrifuged and washed with PBS twice. Bacteria were re-suspended and then diluted into 1.0×107 CFU/mL in PBS. One mg of F127-PCL, F127/17BIPHE2-PCL, and F127/17BIPHE2-PCL-S nanofiber membranes was co-incubated with the bacteria solution for 2 h at 37 oC, respectively. Total living bacteria were determined by culturing on agar plates, and the log reduction of bacteria was calculated by the following equation (2): Log reduction = log (cell count of control) - log (survivor count in peptide treatment group) 2.6. In Vitro Cytotoxicity Test. The in vitro cytotoxicity of nanofiber membranes to skin cells and monocytes was investigated by determining the cell viability of co-incubated HaCaT cells (human keratinocyte cell line) and U937 cells as described in the previous publication40. Nanofiber membranes were firstly sterilized by ethylene oxide. HaCaT cells were cultured in DMEM with 10% FBS, and U937 cells were cultured in RMPI1640 with 10% FBS. HaCat and U937 cells were seeded in 24-well plates. Each well contains 2.5×104 cells and 1 mL culture media. The cells were treated by the following the procedure described in the section of cell culture and treatments. The pre-sterilized slides were placed into the wells with the surface coatings contacting with the cells. The plate containing cells and slides was cultured

7



for 5 days and the culture medium was refreshed every 2 day. On days 1, 3, and 5, the cell viability was investigated by Alamar Blue assay. 2.7. In Vivo Antibiofilm Efficacy Test. To evaluate the antibacterial and antibiofilm efficacy in vivo, we established a biofilm-containing chronic wound model following previous studies41. Briefly, MRSA or P. aeruginosa was grown in TSB overnight. Subsequently, 100 μL bacterial strain was pipetted into 4 mL fresh TSB medium and cultivated for 3 h followed by PBS washing for three times. Then, the bacterial concentration was adjusted to 1×108 CFU/mL and stored in the ice box before use. One hundred female 005314-TALLYHO/JngJ diabetic defective mice (male, 10-11 weeks, 30-35 g, GLU ＞ 200 mg/dL) fed with standard pellet diet and water were used. Our study was approved by the Institutional Animal Care and Use Committee of University of Nebraska Medical Center (Protocol # 18-003-03-FC). The biofilm was established in 005314-TALLYHO/JngJ mice excisional wounds. Two 6 mm-diameter full thickness wounds were created on the back of a mouse using a disposable biopsy punch (Integra Miltex®, Kai Medical, USA) and fixed with a wound splint (Grace Bio-labs, Inc, Bend, Oregon, USA). The wounds were all inoculated with 10 μL of 1×108 CFU/mL MRSA instantly after surgery, and Mupirocin 2% was applied to treat the wounds at day 2 (24 h after surgery). Then, the surrounding and wound tissue was collected using an 8 mm-diameter punch and the biofilm was confirmed by CFU count, Live/Dead staining and SEM observation. The biofilm formation was performed as above-mentioned. We then divided the mice into control groups and peptide groups. Control groups’ wounds were treated with 6-mm diameter F127-PCL core-shell nanofiber discs alone. The peptide groups’ wounds were treated with 6-mm diameter F127/17BIPHE2-PCL-S core-shell nanofiber discs. In this case, we divided the mice into two groups. One group was treated with fiber dressings once during the period of 3 days. The other group was treated with fiber dressings with daily replacement for 3 and 7 days. After treatment, the mice were euthanized and the wound and surrounding tissue was collected by an 8-mm diameter punch into sterilized tubes. Then, 1 mL sterilized PBS was added in each tube, which was blended by a homogenizer (Fisherband, USA). Subsequently, 8


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the mixed liquid was diluted and plated on agar dishes. All the dishes were inoculated in a 37 ℃ microbial incubator for 18 h and the CFU numbers were counted. 2.8. Histological and Immunohistochemical Analysis. To evaluate healing and inflammation in wound areas, half of the samples collected after euthanasia were fixed with 4% paraformaldehyde for 24 h and then embedded in paraffin and cross sectioned to 4 μm thickness slices. Partial slides were stained with Hematoxylin-Eosin (Thermofisher, USA). 2.9. Ex Vivo Human Skin Antibacterial Efficacy Test. F127/17BIPHE2-PCL-S core-shell nanofiber membranes and F127/PCL nanofiber membranes were cut into 8-mm diameter discs and sterilized by ethylene oxide before skin culture. The human skin tissues were collected from patients who underwent plastic surgery and the IRB protocol was approved at University of Nebraska Medical Center. After collection, skin tissues were kept on ice. All the samples were incubated with DMEM within 2 h after surgery. Fat tissues were removed and the skin tissue was rinsed in PBS twice in order to remove blood. Then, the skin tissue was cut into 2 cm × 2 cm. PCL was melt in a customized mold and form a sheet with a size of 2 cm × 2 cm × 0.2 cm. The tissue was fixed on PCL sheet by three or four staple clips on the corners and then placed in a 6 cm-diameter culture dish. The liquid-air-culture was applied. We added approximately 7 ml DMEM medium with 10% FBS in each dish and kept dermal layer soaking in the medium and epidermal layer exposed to air. All the cultures were maintained at 37 oC under 95% air and 5% CO2. After 1-day incubation, a wound was generated by an 8-mm punch in the center of each skin fragment. The wound depth was around 1 mm. P. aeruginosa was prepared by the same method introduced above. Then, P. aeruginosa was diluted by sterilized PBS into 1×104 CFU/ml as a bacterial inoculation liquid. Twenty μL inoculation bacterial liquid was added into wound. F127/17BIPHE2-PCL-S core-shell nanofibers membranes or PCL nanofiber discs were put on wound immediately. All the cultures were maintained at 37 oC under 95% air and 5% CO2 for 2 h. The fiber membranes were then removed and the wound area and wound edge of skin sample was punched off by a 10-mm punch. The skin samples were weighted and then put into 15 ml centrifuge tube followed by adding 1 9



ml PBS. Ten W ultrasonication was applied to isolate bacteria from the tissue. Total living bacteria were determined by culturing on agar plates. 2.10. Statistical Analysis. All the quantitative data are represented as mean ± standard deviation. The obtained data were analyzed for statistical significance using one-way ANOVA tests and p < 0.05 was considered statistically significant for all tests.

3 RESULTS AND DISCUSSION 3.1. Fabrication and Characterization of Peptides-loaded Nanofibers. In this work, we chose PCL as model materials because it is biocompatible and biodegradable polymers and has been approved by FDA for certain clinical applications42. 17BIPHE2 was produced by following our previous studies38. Due to the water solubility of the peptide, co-axial electrospinning (Figure 1) was used to encapsulate peptides to the core following our previous studies. Figure 2A shows the photography of F127/17BIPHE2-PCL nanofiber membranes. Figure 2(B-D) shows SEM images of F127-PCL, F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S nanofiber samples, indicating a fibrous and porous structure. Figure 2E shows a LSCM image of F127/FITC-BSA-PCL core-shell nanofiber, exhibiting a fluorescent core and a lightless shell, confirming the core-shell structure of the nanofibers. In general, the fibrous and porous structure could mimic the extracellular matrix (ECM) and nanofiber scaffolds could serve as an artificial ECM suitable for wound healing43. Moreover, the core-shell structure could protect the encapsulated biological agents from a hostile microenvironment. Hence, in this study, the antimicrobial peptide could retain its biological activity after encapsulation in the core-shell nanofibers44. 3.2. In Vitro Release of Antimicrobial Peptides 17BIPHE2. The encapsulation efficiencies of 17BIPHE2 for F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S nanofiber samples were 93.5 ± 2.8% and 89.7 ± 3.9%, respectively. The amount of 17BIPHE2 incorporated into F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S nanofiber samples were 23.38±0.70 and 44.85±1.95 mg/g. 10


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After deposition of peptides, the encapsulation efficiency decreased, probably because of the loss of some amount of the aggregated drug during the electrospray deposition. As shown in Figure 3, the antimicrobial peptide 17BIPHE2 could be gradually released from the fibers. Such a release was the most important factor affecting the antimicrobial and antibiofilm activities during an extended period. To evaluate the gradual release effect of 17BIPHE2, a 28-day release profile was determined. Figure 3 shows the in vitro release kinetics of 17BIPHE2 peptides from F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S nanofiber samples, suggesting F127/17BIPHE2-PCL-S showed a higher initial burst release when compared with F127/17BIPHE2-PCL. Therefore, the initial burst from F127/17BIPHE2-PCL-S nanofiber samples could allow the peptides to reach an effective concentration rapidly and the subsequent sustained release may help maintain an effective concentration for 4 weeks, thereby treating infections by a sustained release over 28 days. It is noteworthy that the 17BIPHE2 deposited nanofiber membranes could elute peptides more rapidly from nanofiber formulations. It reached the peak release of non-coated fibers on day 8. The increased level of 17BIPHE2 burst release would result in an increase in the antibacterial and antibiofilm efficacy. The larger burst release could provide a greater efficacy in inhibiting the bacteria growth and disrupting biofilms. Thus, the F127/17BIPHE2-PCL-S nanofibers were selected as the wound dressing for in vivo efficacy test. 3.3. In Vitro Antibacterial Efficacy Test. Before challenging the biofilm, it was necessary to evaluate the in vitro antibacterial efficacy of peptide nanofiber formulations. Four pathogens including MRSA USA300, K. pneumoniae, A. baumannii and P. aeruginosa related to clinic infections were applied to determine the in vitro antibacterial activities of the antimicrobial peptides-loaded nanofiber membranes. The pathogens were cultured overnight and then re-inoculated till the bacterial growth to OD600 0.5. Then, the pathogen suspension was diluted to 1.0×107 CFU/mL in PBS and co-incubated with 1 mg of F127-PCL, F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S nanofibers for 2 h at 37 oC, respectively. The bacterial Log reduction was then determined by culturing on LB agar plates. As shown in 11



Figure 4, the F127/17BIPHE2-PCL core-shell nanofibers effectively killed four typical infection-related bacterial strains, with 3.2, 3.6, 3.8, and 3.6-log reduction of MRSA USA300, K. pneumoniae, A. baumannii, and P. aeruginosa, respectively. Moreover, the F127/17BIPHE2-PCL-S nanofibers also effectively killed the bacteria clinical strains in vitro, with 3.4, 3.6, 3.9, and 3.7-log reduction of MRSA, K. pneumoniae, A. baumannii and P. aeruginosa, respectively (Figure 4). 3.4. In Vitro Cytotoxicity Test. It is critical for the antimicrobial wound dressing to effectively kill bacteria45. However, the cytotoxicity of the dressing should be evaluated46. In order to evaluate the in vitro cytotoxicity on the skin cells and immune cells, we tested the effect of peptide nanofiber membranes on the proliferation of HaCaT and U937 cells. As shown in Figure 5(A, B), F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S core-shell nanofibers membranes had no significant influences on the proliferation of HaCaT and U937 cells comparing with control group TCPS and F127-PCL. Importantly, at this concentration (1 mg/mL), the 17BIPHE2 peptide-loaded nanofibers were able to effectively kill bacteria (Figure 4). Overall, the cell viability results displayed no significant cytotoxicity of F127/17BIPHE2-PCL and F127/17BIPHE2-PCL-S in direct contact tests. Therefore, the antimicrobial peptides-loaded PCL nanofibers had excellent cytocompatibility allowing their application as potential trauma wound dressings. 3.5. In Vivo Antibiofilm Efficacy Test. It is widely known that 99% of clinic infections are caused by bacteria in biofilms instead of planktonic ones47. As shown in the antibiotic agar inhibition test (Figure S1), the antibiotic could kill the planktonic bacteria very effectively. To further test the antibiofilm efficacy, we established a biofilm-containing, full-thickness excisional wound model in type II diabetic mice following previous studies41 (Figure 6). Figure 6A shows the typical wound created and fixed with splint and inoculated with MRSA for 24 h. LIVE/DEAD staining indicated that a huge number of bacteria were densely assembled in the tissue (Figure 6B). SEM image also shows numerous bacteria gathered around the tissue, forming dense matrix structure, which further confirms the formation of biofilms (Figure 6C). We further quantified the CFU on the wound. It is found that there were 1010 CFU/g 12


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bacteria resided on each surgical site and surrounding tissues (Figure 6D). Our results showed that after inoculation of 1×108 CFU/mL MRSA for 24 h and subsequent 24 h treatment of Mupirocin 2%, the biofilm was successfully formed on the wound. After 24 h of MRSA and P. aeruginosa inoculation and subsequent 24 h of Murpirocin 2% treatment, we divided the mice into unclean and clean groups. Unclean groups represented treatment with fiber dressings without debridement, and clean groups performed debridement (a clinical viable approach for wound infection management) before administration of fiber dressings. The F127-PCL and F127/17BIPHE2-PCL-S nanofiber dressings were applied to treat the wound against MRSA and P. aeruginosa biofilms. And two different dressing change frequencies were applied during the treatment. After 3 and 7 days, all the tissue was collected and homogenized, and tissue suspension was diluted to a proper concentration and plated on agar. Without debridement (the unclean group), around 1.57×108 CFU/g MRSA were detected in wounds treated once with F127/17BIPHE2-PCL-S dressing for 3 days, with a 1.68-log reduction compared to the F127-PCL control group (Figure 7A). With debridement (the clean group), approximately 7.41 × 105 CFU/g MRSA were detected in the wounds treated once with F127/17BIPHE2-PCL-S dressing for 3 days, with a 2.99-log reduction compared to the F127-PCL control group (Figure 7A). To further improve the treatment efficacy, we also attempted to change the dressing daily (i.e. During the treatment, F127/17BIPHE2-PCL-S dressings were replaced daily). Without debridement, around 6.17 × 106 CFU/g MRSA were detected in wounds treated with F127/17BIPHE2-PCL-S dressings daily for 3 days, with a 3.08-log reduction comparing to the F127-PCL control group (Figure 7B). Intriguingly, with debridement, no colonies were detected in the wounds treated with F127/17BIPHE2-PCL-S dressing daily for 3 days, with a 9.86-log reduction comparing to the F127-PCL control group (Figure 7B). In addition, after 7-day treatment, without debridement, around 1.72 × 105 CFU/g MRSA were detected in wounds treated with F127/17BIPHE2-PCL-S dressings daily, with a 5.01-log reduction comparing to the F127-PCL control group (Figure 7C). Similarly, with debridement,

no

colonies

were

observed

in

the

13


wounds

treated

with


Page 14 of 29

F127/17BIPHE2-PCL-S dressing daily for 7 days, with a 10.24-log reduction comparing to the F127-PCL control group (Figure 7C). Besides, to explore the antibiofilm activities for Gram-negative bacteria, we also established a P. aeruginosa biofilm wound model and the wound was treated with the F127/17BIPHE2-PCL-S dressing daily for 3 days. Without debridement, around 1.74 ×

107

CFU/g

P.

aeruginosa

were

detected

in

wounds

treated

with

F127/17BIPHE2-PCL-S dressings daily for 3 days, with a 3.61-log reduction comparing to the F127-PCL control group (Figure 7D). Moreover, similar to the results of Gram-positive bacteria, with debridement, no colonies were detected in the wounds treated with F127/17BIPHE2-PCL-S dressing daily for 3 days, with a 10.75-log reduction comparing to the F127-PCL control group (Figure 7D). Thus, we conclude that the biofilms consisting of either Gram-negative or Gram-positive bacteria on the diabetic wounds could be eliminated entirely after the combinatorial treatment of debridement and the antimicrobial peptide-loaded nanofiber dressings with daily changes for 3 and 7 days. 3.6. Histology. To examine the wound healing and inflammation, we carried out histology and cytokine assay. As shown in Figure 8A, the inflammatory exudates, necrosis, and granulation tissue, accompanied by acute and chronic inflammatory cell infiltration, were found in the wounds with treatment of F127-PCL nanofiber dressings. In contrast, the inflammatory exudation and inflammatory cell infiltration were reduced in the wounds with debridement and treatment of F127-PCL nanofiber dressings (Figure 8B). The inflammatory exudation and inflammatory cell infiltration were further reduced in the wounds with treatment of F127/17BIPHE2-PCL-S nanofiber dressings (Figure 8C). The inflammatory level was the lowest in the wounds with debridement and treatment of F127/17BIPHE2-PCL-S nanofiber dressings compared to the other three groups (Figure 8D). In addition, new blood vessels, granulation tissues, and re-epithelialization were formed (Figure 8D). 3.7. Ex Vivo Antibacterial Efficacy Test. In order to further test the antibacterial efficacy of peptide-loaded nanofiber dressings, we developed an artificial wound infection model by culturing human skin tissues ex vivo. Pathogens including MRSA, 14


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K. pneumoniae, and A. baumannii were not successfully grown in the ex vivo culture after inoculation to the artificial wounds. Herein, we chose P. aeruginosa as a model bacterial for inoculation to human skin artificial wounds. A 2 cm diameter partial thickness wound was created using a surgical scalpel and inoculated with 200 CFU/mL P. aeruginosa. After administration of nanofiber dressings to the wounds, the tissues were cultured for 2 and 8 h and the bacterial burden was quantified as shown in Figure 10. It is seen that P. aeruginosa grew rapidly in the inoculated wounds with PCL nanofiber dressing covering. In contrast, the bacterial growth was inhibited in the inoculated wounds with 17BIPHE2-loaded PCL nanofiber dressing covering. At day 8, the CFU was reduced by 5 logs.

4. CONCLUSIONS In summary, we have demonstrated the successful incorporation of human cathelicidin

engineered

peptides

to

electrospun

nanofibers

using

co-axial

electrospinning and electrospray deposition. Such peptide-loaded nanofiber membranes showed an initial burst release followed by a sustained release of antimicrobial peptides for at least 28 days. We further demonstrated the antibacterial efficacy of peptide-loaded nanofiber membranes against multiple antibiotic resistant bacteria in vitro, ex vivo and in vivo. These results indicate that the LL-37 engineered 17BIPHE2 retained activity against both S. aureus and P. aeruginosa under different scenarios, confirming the usefulness of nanofibers as a peptide carrier. Our results also lay a foundation for our testing co-incorporation of this peptide with conventional antibiotics for an improved antibiofilm capability48,49. Thus, our study offers a possible treatment for chronic wounds by combining the engineered LL-37 peptide with nanofibers. Together, nanofiber membranes topically delivering engineered peptides hold great promise in the prevention of wound infections. Aside from this, electrospun nanofibers developed from this study could serve as sutures, coating materials of biomedical devices, or hemostasis materials for preventing infections in different scenarios.

15



Page 16 of 29

ACKNOWLEDGMENTS This work was partially supported from startup funds from University of Nebraska Medical Center, National Institute of General Medical Science (NIGMS) Grant 2P20 GM103480 and NIGMS Grant R01GM123081 of the National Institutes of Health, and Otis Glebe Medical Research Foundation to JX and the NIAID grant R01 AI105147 to GW. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

SUPPORTING INFORMATION Figure S1 showing MRSA inhibition zone test with commercial antibiotic 2% Mupirocin, indicating the antibiotic could kill the planktonic bacteria very effectively

REFERENCES 1.

Branski, L. K.; Al-Mousawi, A.; Rivero, H.; Jeschke, M. G.; Sanford, A. P.; Herndon, D. N.

Emerging infections in burns. Surg. Infect. 2009, 10, 389-397. 2.

Wang, Y.; Beekman, J.; Hew, J.; Jackson, S.; Issler-Fisher, A. C.; Parungao, R.; Lajevardi, S. S.;

Li, Z.; Maitz, P. K.

Burn injury: challenges and advances in burn wound healing, infection, pain and

scarring. Adv. Drug Deliver. Rev. 2018, 123, 3-17. 3.

Wukich, D. K.; Lowery, N. J.; McMillen, R. L.; Frykberg, R. G.

Postoperative infection rates in

foot and ankle surgery: a comparison of patients with and without diabetes mellitus. J. Bone Joint Surg. Am. 2010, 92, 287-295. 4.

Lavery, L. A.; Peters, E. J.; Armstrong, D. G.; Wendel, C. S.; Murdoch, D. P.; Lipsky, B. A.

Risk factors for developing osteomyelitis in patients with diabetic foot wounds. Diabetes Res. Clin. Pr. 2009, 83, 347-352. 5.

Sen, C. K.; Gordillo, G. M.; Roy, S.; Kirsner, R.; Lambert, L.; Hunt, T. K.; Gottrup, F.; Gurtner,

G. C.; Longaker, M. T.

Human skin wounds: a major and snowballing threat to public health and the

economy. Wound Repair Regen. 2009, 17, 763-771. 6.

Kalan, L. R.; Pepin, D. M.; Ul-Haq, I.; Miller, S. B.; Hay, M. E.; Precht, R. J.

Targeting

biofilms of multidrug-resistant bacteria with silver oxynitrate. Int. J. Antimicrob. Ag. 2017, 49, 719-726. 7.

Hingorani, A.; LaMuraglia, G. M.; Henke, P.; Meissner, M. H.; Loretz, L.; Zinszer, K. M.; Driver,

V. R.; Frykberg, R.; Carman, T. L.; Marston, W. The management of diabetic foot: a clinical practice guideline by the Society for Vascular Surgery in collaboration with the American Podiatric Medical Association and the Society for Vascular Medicine. J. Vasc. Surg. 2016, 63, 3S-21S. 8.

Abbas, M.; Uckay, I.; Lipsky, B. A.

In diabetic foot infections antibiotics are to treat infection,

not to heal wounds. Expert Opin. Pharmaco. 2015, 16, 821-832. 16


Page 17 of 29 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


9.

Das, D.; Chen, J.; Srinivasan, S.; Kelly, A. M.; Lee, B.; Son, H.-N.; Radella, F.; West, T. E.;

Ratner, D. M.; Convertine, A. J.

Synthetic Macromolecular Antibiotic Platform for Inhalable Therapy

against Aerosolized Intracellular Alveolar Infections. Mol. Pharm. 2017, 14, 1988-1997. 10. Du, J.; Bandara, H.; Du, P.; Huang, H.; Hoang, K.; Nguyen, D.; Mogarala, S. V.; Smyth, H. D. Improved biofilm antimicrobial activity of polyethylene glycol conjugated tobramycin compared to tobramycin in Pseudomonas aeruginosa biofilms. Mol. Pharm. 2015, 12, 1544-1553. 11. Bourdillon, K. A.; Delury, C. P.; Cullen, B. M.

Biofilms and delayed healing-an in vitro

evaluation of silver-and iodine-containing dressings and their effect on bacterial and human cells. Int. Wound J. 2017, 14, 1066-1075. 12. Atiyeh, B. S.; Costagliola, M.; Hayek, S. N.; Dibo, S. A.

Effect of silver on burn wound

infection control and healing: review of the literature. Burns 2007, 33, 139-148. 13. Kwon, H. B.; Lee, J. H.; Lee, S. H.; Lee, A. Y.; Choi, J. S.; Ahn, Y. S.

A case of argyria

following colloidal silver ingestion. Ann. Dermatol. 2009, 21, 308-310. 14. Dik, D. A.; Fisher, J. F.; Mobashery, S.

Cell-wall recycling of the Gram-negative bacteria and

the nexus to antibiotic resistance. Chem. Rev. 2018, 118, 5952-5984. 15. Tong, C.; Hao, H.; Xia, L.; Liu, J.; Ti, D.; Dong, L.; Hou, Q.; Song, H.; Liu, H.; Zhao, Y. Hypoxia pretreatment of bone marrow-derived mesenchymal stem cells seeded in a collagen-chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia. Wound Repair Regen. 2016, 24, 45-56. 16. Shi, M.; Kretlow, J. D.; Nguyen, A.; Young, S.; Baggett, L. S.; Wong, M. E.; Kasper, F. K.; Mikos, A. G.

Antibiotic-releasing porous polymethylmethacrylate constructs for osseous space

maintenance and infection control. Biomaterials 2010, 31, 4146-4156. 17. Shi, W.; Ogbomo, S. M.; Wagh, N. K.; Zhou, Z.; Jia, Y.; Brusnahan, S. K.; Garrison, J. C. The influence of linker length on the properties of cathepsin S cleavable 177Lu-labeled HPMA copolymers for pancreatic cancer imaging. Biomaterials 2014, 35, 5760-5770. 18. Steinstraesser, L.; Kraneburg, U.; Jacobsen, F.; Al-Benna, S. Host defense peptides and their antimicrobial-immunomodulatory duality. Immunobiology 2011, 216, 322-333. 19. Chieosilapatham, P.; Ikeda, S.; Ogawa, H.; Niyonsaba, F.

Tissue-specific Regulation of Innate

Immune Responses by Human Cathelicidin LL-37. Curr. Pharm. Design 2018, 24, 1079-1091. 20. Li, D.; Wang, X.; Wu, J.-L.; Quan, W.-Q.; Ma, L.; Yang, F.; Wu, K.-Y.; Wan, H.-Y. Tumor-produced versican V1 enhances hCAP18/LL-37 expression in macrophages through activation of TLR2 and vitamin D3 signaling to promote ovarian cancer progression in vitro. Plos One 2013, 8, (2), e56616. 21. Xhindoli, D.; Pacor, S.; Benincasa, M.; Scocchi, M.; Gennaro, R.; Tossi, A.

The human

cathelicidin LL-37-A pore-forming antibacterial peptide and host-cell modulator. BBA-Biomembranes 2016, 1858, 546-566. 22. Hancock, R. E.; Haney, E. F.; Gill, E. E.

The immunology of host defence peptides: beyond

antimicrobial activity. Nat. Rev. Immunol. 2016, 16, 321. 23. Haisma, E. M.; de Breij, A.; Chan, H.; van Dissel, J. T.; Drijfhout, J. W.; Hiemstra, P. S.; El Ghalbzouri, A.; Nibbering, P. H. LL-37-derived peptides eradicate multidrug-resistant Staphylococcus aureus from thermally wounded human skin equivalents. Antimicrob. Agents Ch. 2014, 58, 4411-4419. 24. Mishra, B.; Golla, R.; Lau, K.; Lushnikova, T.; Wang, G. Anti-Staphylococcal biofilm effects of human cathelicidin peptides. ACS Med. Chem. Lett. 2016, 7, 117-121.

17



Page 18 of 29

25. Mishra, B.; Wang, G. Individual and combined effects of engineered peptides and antibiotics on the Pseudomonas aeruginosa biofilms. Pharmaceuticals 2017, 10(3), 58. 26. Romano, L.; Camposeo, A.; Manco, R.; Moffa, M.; Pisignano, D.

Core-shell electrospun fibers

encapsulating chromophores or luminescent proteins for microscopically controlled molecular release. Mol. Pharm. 2016, 13, 729-736. 27. Chen,

C.-K.;

Huang,

S.-C.

Preparation

of

reductant-responsive

N-maleoyl-functional

chitosan/poly (vinyl alcohol) nanofibers for drug delivery. Mol. Pharm. 2016, 13, 4152-4167. 28. Wang, G.; Babadağli, M. E.; Uludağ, H.

Bisphosphonate-derivatized liposomes to control drug

release from collagen/hydroxyapatite scaffolds. Mol. Pharm. 2011, 8, 1025-1034. 29. Spicer, P. P.; Mikos, A. G.

Fibrin glue as a drug delivery system. J. Control Release 2010, 148,

49-55. 30. Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S.

Poly (ethylene glycol) in drug

delivery: pros and cons as well as potential alternatives. Angew. Chem. Int. Edit. 2010, 49, 6288-6308. 31. Augst, A. D.; Kong, H. J.; Mooney, D. J.

Alginate hydrogels as biomaterials. Macromol. Biosci.

2006, 6, 623-633. 32. Hoare, T. R.; Kohane, D. S.

Hydrogels in drug delivery: Progress and challenges. Polymer

2008, 49, 1993-2007. 33. Khansari, S.; Duzyer, S.; Sinha-Ray, S.; Hockenberger, A.; Yarin, A.; Pourdeyhimi, B. Two-stage desorption-controlled release of fluorescent dye and vitamin from solution-blown and electrospun nanofiber mats containing porogens. Mol. Pharm. 2013, 10, 4509-4526. 34. Sundaran, P. S.; Bhaskaran, A.; Alex, S. T.; Prasad, T.; Haritha, V.; Anie, Y.; Kumary, T.; Kumar, P. A.

Drug loaded microbeads entrapped electrospun mat for wound dressing application. J.

Mater. Sci.-Mater. M. 2017, 28, 88. 35. Wang, B.-L.; Liu, X.-S.; Ji, Y.; Ren, K.-F.; Ji, J.

Fast and long-acting antibacterial properties of

chitosan-Ag/polyvinylpyrrolidone nanocomposite films. Carbohyd. Polym. 2012, 90, 8-15. 36. Shafei, A. E.; Abou-Okeil, A.

ZnO/carboxymethyl chitosan bionano-composite to impart

antibacterial and UV protection for cotton fabric. Carbohyd. Polym. 2011, 83, 920-925. 37. Li, P.; Li, J.; Wu, C.; Wu, Q.; Li, J.

Synergistic antibacterial effects of β-lactam antibiotic

combined with silver nanoparticles. Nanotechnology 2005, 16, 1912-1917. 38. Wang, G.; Hanke, M. L.; Mishra, B.; Lushnikova, T.; Heim, C. E.; Chittezham Thomas, V.; Bayles, K. W.; Kielian, T.

Transformation of human cathelicidin LL-37 into selective, stable, and

potent antimicrobial compounds. ACS Chem. Biol. 2014, 9, 1997-2002. 39. Jiang, J.; Chen, G.; Shuler, F. D.; Wang, C.-H.; Xie, J.

Local sustained delivery of

25-hydroxyvitamin D3 for production of antimicrobial peptides. Pharm. Res. 2015, 32, 2851-2862. 40. Su, Y.; Zhi, Z.; Gao, Q.; Xie, M.; Yu, M.; Lei, B.; Li, P.; Ma, P. X.

Autoclaving-derived surface

coating with in vitro and in vivo antimicrobial and antibiofilm efficacies. Adv. Healthcare Mater. 2017, 6, 1601173. 41. Brackman, G.; De Meyer, L.; Nelis, H.; Coenye, T.

Biofilm inhibitory and eradicating activity

of wound care products against Staphylococcus aureus and Staphylococcus epidermidis biofilms in an in vitro chronic wound model. J. Appl. Microbiol. 2013, 114, 1833-1842. 42. Dash, T. K.; Konkimalla, V. B.

Polymeric modification and its implication in drug delivery:

poly-ε-caprolactone (PCL) as a model polymer. Mol. Pharm. 2012, 9, 2365-2379. 43. Smith, L.; Ma, P.

Nano-fibrous scaffolds for tissue engineering. Colloid. Surface. B 2004, 39,

125-131. 18


Page 19 of 29 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


44. Zhang, Y.; Huang, Z.-M.; Xu, X.; Lim, C. T.; Ramakrishna, S.

Preparation of core-shell

structured PCL-r-gelatin bi-component nanofibers by coaxial electrospinning. Chem. Mater. 2004, 16, 3406-3409. 45. Hoque, J.; Adhikary, U.; Yadav, V.; Samaddar, S.; Konai, M. M.; Prakash, R. G.; Paramanandham, K.; Shome, B. R.; Sanyal, K.; Haldar, J.

Chitosan derivatives active against

multidrug-resistant bacteria and pathogenic fungi: in vivo evaluation as topical antimicrobials. Mol. Pharm. 2016, 13, 3578-3589. 46. Archana, D.; Singh, B. K.; Dutta, J.; Dutta, P.

Chitosan-PVP-nano silver oxide wound dressing:

in vitro and in vivo evaluation. Int. J Biol. Macromol. 2015, 73, 49-57. 47. Gupta, P.; Sarkar, S.; Das, B.; Bhattacharjee, S.; Tribedi, P.

Biofilm, pathogenesis and

prevention-a journey to break the wall: a review. Arch. Microbiol. 2016, 198, 1-15. 48. Mishra, B.; Wang, G. Individual and combined effects of engineered peptides and antibiotics on the Pseudomonas aeruginosa biofilms. Pharmaceuticals 2017, 10(3), 58. 49. Mishra, B.; Reiling, S.; Zarena, D.; Wang, G. Host defense antimicrobial peptide as antibiotics: design and application strategies. Curr. Opin. Chem. Biol. 2017, 38, 87-96.

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Page 20 of 29

Figure 1. (A) Schematic illustrating co-axial electrospinning and preparation of F127/17BIPHE2-PCL nanofiber dressings. (B) Schematic illustrating electrospraying deposition

of

the

human

cathelicidin

engineered

peptide

17BIPHE2

to

F127/17BIPHE2-PCL nanofiber membranes to form F127/17BIPHE2-PCL-S nanofiber dressings.

20


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Figure

2.

Morphology

of

nanofiber

dressings.

(A)

Photograph

of

F127/17BIPHE2-PCL core-shell nanofiber membrane. (B-D) SEM images of F127-PCL core-shell nanofibers, F127/17BIPHE2-PCL core-shell nanofibers, and F127/17BIPHE2-PCL-S nanofibers. (E) LSCM image of F127/FITC-BSA-PCL core-shell nanofibers.

21


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

D128G release(ug/mg)


35 30 25 20 15 10 5 0

Page 22 of 29

F127/17B1PHE2-PCL F127/17B1PHE2-PCL-S

0

5

10

15

20

25

30

Time (day) Figure

3.

In

vitro

release

profiles

of

the

17BIPHE2

peptide

from

F127/17BIPHE2-PCL core-shell nanofibers and after electrospraying deposition of 17BIPHE2 (F127/17BIPHE2-PCL-S).

22


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Figure 4. In vitro antibacterial efficacy test of F127/17BIPHE2-PCL core-shell nanofibers. The bacteria solution was diluted into 1.0×107 CFU/mL in PBS. One mg of PCL or F127/17BIPHE2-PCL core-shell nanofiber membranes was co-incubated with the bacteria solution for 2 h at 37 oC. Total remaining bacteria were determined by culturing on agar plates.

23



Figure 5. In vitro cytotoxicity test of 17BIPHE2 peptide-loaded PCL nanofiber membranes. (A) Alamar Blue cell viability test against HaCat cells. (B) Alamar Blue cell viability test against U937 cells. Each data point represents arithmetic mean ± SD values from four samples.

24


Page 24 of 29

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Figure 6. MRSA USA300 biofilm formation in chronic wounds based on type II diabetic mice. (A) Wounds were created and fixed with splint, and MRSA was inoculated for 24 h. (B) LIVE/DEAD staining for the tissue collected from wounds after 24 h of MRSA inoculation and subsequent 24 h Mupirocin 2% treatment. (C) SEM observation of the tissue collected from wounds after 24 h of MRSA inoculation and subsequent 24 h Mupirocin 2% treatment. (D) Quantification of bacterial load in the wound after 24 h of MRSA inoculation and subsequent 24 h and 48 h Mupirocin 2% treatment.

25



Figure 7. In vivo antibiofilm efficacy test of 17BIPHE2/F127-PCL-S nanofiber dressings. The MRSA and P. aeruginosa biofilm-containing chronic wounds created in type II diabetic mice were treated by 17BIPHE2/F127-PCL-S nanofiber dressings without and with debridement. (A): 3 days 1 change 17BIPHE2 against MRSA: the wounds were treated by 17BIPHE2/F127-PCL-S nanofiber dressings once for 3 days. (B): 3 days 3 changes 17BIPHE2 against MRSA: the wounds were treated by 17BIPHE2/F127-PCL-S nanofiber dressings three times for 3 days with daily replacement. (C): 7 days 7 changes 17BIPHE2: the wounds were treated by 17BIPHE2/F127-PCL-S nanofiber dressings seven times for 7 days with daily replacement. (D): 3 days 3 changes 17BIPHE2 against P. aeruginosa: the wounds were treated by 17BIPHE2/F127-PCL-S nanofiber dressings three times for 3 days with daily replacement. (p < 0.05 marked as *).

26


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Figure 8. H&E staining for the tissue collected from biofilm-containing wounds treated by (A) F127-PCL nanofiber dressings with once-daily dressing change for 7 days, (B) F127-PCL nanofiber dressings in combination with debridement with once-daily dressing change for 7 days, (C) F127/17BIPHE2-PCL nanofiber dressings with once-daily dressing change for 7 days and (D) F127/17BIPHE2-PCL nanofiber dressings in combination with debridement with once-daily dressing change for 7 days.

27



Page 28 of 29

Figure 9. Ex vivo antibacterial efficacy of F127/17BIPHE2-PCL core-shell nanofibers. P. aeruginosa (200 CFU/mL) were inoculated to the artificial wounds created on the human

skin

tissues.

The

wounds

were

covered

by

F127-PCL

and

F127/17BIPHE2-PCL-S nanofiber dressings for 2 and 8 h. Bacteria burden was quantified based on the tissue lysis solution.

28


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TOC

29


Nanofiber Dressings Topically Delivering Molecularly Engineered

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