Core-shell insulin-loaded nanofibrous scaffolds for repairing diabetic wounds

Nanomedicine: Nanotechnology, Biology, and Medicine xx (xxxx) xxx

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Graphical Abstract

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Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2019) xxx – xxx

Core-shell insulin-loaded nanofibrous scaffolds for repairing diabetic wounds

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Chen-Hung Lee, MD, PhDa, Kuo-Chun Hung, MDa, Ming-Jer Hsieh, MD, PhDa, Shang-Hung Chang, MD, PhDa, Jyuhn-Huarng Juang, MD b, I-Chang Hsieh, MDa, Ming-Shien Wen, MD a, Shih-Jung Liu, PhDc,d,⁎

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Accelerate the healing wound following treatment using functionally active insulin released from insulin-loaded nanofibrous scaffolds.

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Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital-Linkou, Chang Gung University College of Medicine, Tao-Yuan, Taiwan Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung University and Chang Gung Memorial Hospital, Tao-Yuan, Taiwan Department of Mechanical Engineering, Chang Gung University, Tao-Yuan, Taiwan d Department of Orthopedic Surgery, Chang Gung Memorial Hospital-Linkou, Tao-Yuan, Taiwan

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NANO-0000102123; No of Pages 10

Nanomedicine: Nanotechnology, Biology, and Medicine xx (xxxx) xxx

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Core-shell insulin-loaded nanofibrous scaffolds for repairing diabetic wounds

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Chen-Hung Lee, MD, PhD a , Kuo-Chun Hung, MD a , Ming-Jer Hsieh, MD, PhD a , Shang-Hung Chang, MD, PhD a , Jyuhn-Huarng Juang, MD b , I-Chang Hsieh, MD a , Ming-Shien Wen, MD a , Shih-Jung Liu, PhD c, d,⁎

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Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital-Linkou, Chang Gung University College of Medicine, Tao-Yuan, Taiwan b Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung University and Chang Gung Memorial Hospital, Tao-Yuan, Taiwan c Department of Mechanical Engineering, Chang Gung University, Tao-Yuan, Taiwan d Department of Orthopedic Surgery, Chang Gung Memorial Hospital-Linkou, Tao-Yuan, Taiwan Revised 15 October 2019

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Abstract

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Patients with diabetes mellitus have up to a 15% lifetime risk of non-healing and poorly healing wounds. This work develops core-shell nanofibrous bioactive insulin-loaded poly-D-L-lactide-glycolide (PLGA) scaffolds that release insulin in a sustained manner for repairing wounds in diabetic rats. To prepare the biodegradable core-shell nanofibers, PLGA and insulin solutions were fed into two capillary tubes of different sizes that were coaxially electrospun using two independent pumps. The scaffolds sustainably released insulin for four weeks. The hydrophilicity and water-containing capacity of core-shell nanofibrous insulin/PLGA scaffolds significantly exceeded those of blended nanofibrous scaffolds. The nanofibrous core-shell insulin-loaded scaffold reduced the amount of type I collagen in vitro, increased the transforming growth factor-beta content in vivo, and promoted diabetic would repair. The core-shell insulin-loaded nanofibrous scaffolds prolong the release of insulin and promote diabetic wound healing. © 2019 Published by Elsevier Inc.

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Key words: Core-shell nanofiber; Coaxial electrospinning; Diabetic wound; Insulin

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Systemic insulin or topical insulin solution treatment promotes the formation of a wound matrix and accelerates wound healing in patients with diabetes. 11 , 12 However, the application of insulin to wounds can normally only be performed using only a few methods, involving gauzes soaked in insulin 13, insulin cream 14, and subcutaneous injections of insulin. 15 These methods require regular reapplication, exhibiting short-term bioactive properties for only several hours. Therefore, Hrynyk et al reported that bioactive crystalline insulin can be successfully encapsulated within poly (lactic-co-glycolic acid) (PLGA) microspheres, which provide sustained and controlled release

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Approximately 15% of all patients with diabetic wounds undergo surgical amputation, despite intensive medical treatment and a carefully calculated diet. 1–3 A decrease in growth factor activation 4, angiogenic action 5, macrophage phagocytosis 6, stratum corneum barrier hydration, and fibroblast and keratinocyte proliferation and migration 7 all inhibit the wound healing in diabetic patients. Insulin is important in all cases of diabetes because it can induce the development and growth of various cells, and signal the migration, proliferation, and secretion of growth factors through fibroblasts, endothelial cells, and keratinocytes. 8 , 9 10

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Abbreviations: ECM, Extracellular matrix; ELISA, Enzyme-linked immunosorbent assay; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; PBS, Phosphate buffered saline; PLGA, Poly (lactic-co-glycolic acid); SEM, Scanning electron microscopy; TEM, transmission electron microscope; TGF-beta, Transforming growth factor beta; WB, Western blotting Acknowledgments and funding sources: The authors would like to thank the National Science Council of Taiwan (Contract No. NSC-107-2320-B-182A029-) and Chang Gung Memorial Hospital (Contract No. CMRPG3F1551) for supporting this research. Disclosure: The authors report no conflicts of interest with respect to this work. The listed authors wrote this article, and no ghostwriters were used for article writing. ⁎Corresponding author at: Biomaterials Lab, Mechanical Engineering, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan. E-mail addresses: [email protected], (C.-H. Lee), [email protected], (K.-C. Hung), [email protected], (M.-J. Hsieh), [email protected], (S.-H. Chang), [email protected], (J.-H. Juang), [email protected], (I.-C. Hsieh), [email protected], (M.-S. Wen), [email protected]. (S.-J. Liu).

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https://doi.org/10.1016/j.nano.2019.102123 1549-9634/© 2019 Published by Elsevier Inc. Please cite this article as: Lee C.-H., et al., Core-shell insulin-loaded nanofibrous scaffolds for repairing diabetic wounds. Nanomedicine: NBM 2019;xx:09, https://doi.org/10.1016/j.nano.2019.102123

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The commercially available PLGA (mole ratio 50:50, molecular weight 33,000 g/mol, Resomer RG 503) material was used herein. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was used as solvent, which was obtained from Sigma-Aldrich (Saint Louis, MO, USA). Insulin glargine was purchased from SanofiAventis Inc. (Frankfurt, Germany). Two core-shell structured nanofibrous scaffolds and one regular nanofibrous scaffold were prepared by coaxial electrospinning and blending electrospinning techniques, respectively. To fabricate the core-shell structured nanofibers, a special coaxial device that simultaneously delivers two solutions to an aluminum sheet was designed and built. 23 A predetermined mass of PLGA (700 mg) was dissolved in 3 mL of HFIP. The mixture thus obtained was used as the shell solution. The core solution was 1 mL insulin glargine (equivalent to 3.64 mg) (Group A) or phosphate buffered saline (PBS) (1 mL) (Group B). For subsequent coaxial electrospinning, the PLGA and core liquids from needles were put into two separate feeding tubes. During spinning, the liquids were delivered into the aluminum sheet using 0.9 mL/h (volumetric flow rate) for the shell PLGA solution and 0.3 mL/h for the core insulin (or PBS) solution by

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Porosity

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The porosity of the each scaffold was obtained using the following equation.

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Poreð%Þ ¼

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where ρpolymer and ρscaffold are the densities of the polymer and the nanofiber, respectively.

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SEM observation

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Using an SEM, the electrospun nanofibers were coated with gold and then their structure was elucidated. The diameters were analyzed from 100 randomly selected fibers (N = 4) using Image J (National Institutes of Health, Bethesda, MD, USA).

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TEM observation

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The fiber structure of the core-shell and blended nanofibrous scaffolds was observed using a TEM (JEOL JEM-2000EXII, Japan). The samples for TEM observations were prepared by directly depositing the spun fibers onto copper grids.

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Confocal microscopy

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To assess the distribution of biomolecules in the coaxially electrospun nanofibers, core-shell scaffolds with PLGA as the shell and reGFP (6H1-38, Shanghai PrimeGene Bio-Tech, China) as the core were prepared. After spinning, a thin layer of spun fibers was collected on a coverslip and then imaged using a laser scanning confocal microscope (Leica TS SP8X, Japan). Nanofibers of blended PLGA (with and without reGFP) were also formed as a control. The excitation wavelength for reGFP was 487 nm.

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Mechanical properties of materials

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Lloyd tensiometer (AMETEK, USA) with a 0.1 kN load cell was used to analyze the core-shell insulin/PLGA and the PBS/ PLGA nanofibrous scaffolds, and the blended insulin/PLGA scaffold by the ASTM D638 standard. A strip with an area of 5.0 cm by 1.0 cm was prepared from each of the scaffolds and kept between two clamps (3 cm distance). Before returning to the starting point of clamp, the material was moved by the top clamp at 1.0 mm/second through a distance of 10 cm. The force on scaffolds and elongation of the samples were stored after the scaffold broke. Tests on each scaffold were performed four times. The elongation and tensile strength at breakage were

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two independently controlled pumps. Using the blending electrospinning technique, PLGA and insulin (PLGA, 700 mg; insulin 1 mL) (Group C) were mixed directly and electrospun to form nanofibrous scaffolds. All spinning experiments were conducted at room temperature. The thickness of the nanofibrous scaffolds thus obtained was approximately 0.3 mm. All electrospun scaffolds were taken into a vacuum oven at 40 °C for 72 h in order to evaporate off the HFIP, and thereafter stored at 4 °C until they were used.

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for ten days. The difficult scaling-up process and expense of the large-scale production of microspheres limit their availability. 18 Ideal drug delivery systems, including a controllable drug delivery rate, the precise targeting of specific sites, prolonged therapeutic effects in vivo, and prolongation of the drug's activity, will help treat wounds that are related to diabetes. Recently, nanofiber-based scaffolds that closely resemble the natural architecture of the extracellular matrix (ECM) for use in tissue engineering have been extensively investigated. 19 Coreshell structure of biodegradable nanofibers that are prepared using the electrospinning technique with coaxial nozzles has been developed and used as special implants, in which biologically active molecules are incorporated into them. 20 , 21 The coaxial electrospinning method has numerous advantages over the traditional electrospinning process, especially in that it can be used to encapsulate drugs that are only water-soluble and will likely lose their bioactivity when they dissolve in organic solvents. 22 In this work, we hypothesize that the topical route of a coreshell nanofibrous scaffold that is loaded with insulin promotes wound healing in an animal model susceptible to diabetes. Insulin-loaded core-shell nanofibers are fabricated using the coaxial electrospinning process. Following the spinning procedure, the structure of the electrospun scaffolds was determined using scanning electron microscopy (SEM) and transmission electron microscope (TEM). The release data of pharmaceutical from the scaffolds were performed using enzyme-linked immunosorbent assay (ELISA). The effect of core-shell nanofibrous insulin-loaded scaffolds on the diabetic wound repairing was also examined using immunofluorescence, histology, and western blotting (WB).

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Tensile strength ðMPaÞ
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Contact angle with water

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Using a water contact angle analyzer, the core-shell nanofibrous scaffolds were carried out for water contact angles (First Ten Angstroms, USA). Scaffolds with an area of 10 mm by 10 mm were prepared. Dropping gently on to the different scaffolds surfaces of the testing plate was analyzed using a video monitor (n = 4).

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Water absorption capacity

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The water absorption capacities of the core-shell insulin/ PLGA or PBS/PLGA, and the blended insulin/PLGA scaffolds were performed. The immersion of electrospun nanofibers in PBS after 0.5, 1, 2, 3, 8, and 24 h were weighted following the surface PBS was taken using filter paper. The composition (%) of water is evaluated by the following formula.

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WC ð%Þ ¼ ðW –W 0 Þ=W 0  100

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In vitro release of insulin

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By employing an elution method, the in vitro release patterns of insulin of the scaffolds were obtained. Before the eluent was collected for analysis, scaffolds with an area of 10 mm by 10 mm were prepared (N = 5) in 1.0 mL of PBS and were incubated at 37 °C. Solution was changed using fresh PBS (1 mL) daily for 28 days. The bioactive insulin concentrations were obtained using an insulin enzyme-linked immunosorbent assay (ELISA) kit (Thermo Scientific, USA).

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Cell migration assay and insulin/PLGA scaffold test

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A Corning Costar Transwell filter chamber with an 8.0 μm pore size was used in the migration assay. Commercially available atrial fibroblasts (Food Industry Research and Development Institute, Hsinchu, Taiwan) 24 were seeded at a density of 5 × 10 5 cells per filter. To initiate the chemotaxis assay, cells in 200 μL DMEM were added to the upper chamber, and the bottom chamber was filled with 540 μL DMEM plus 60 μL various test fluid (eluent of insulin/PLGA scaffold (on day 7, 14, 21, and 28), insulin (10 mU/mL), and a control of only contains PBS) as the chemotaxis factor for cell motion. Atrial fibroblasts were allowed to migrate at 37 °C in a highconcentration glucose solution (50 mM) for 24 h. Liu's stain was used for cells on the lower surface of the filter membrane

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where W0 and W are the weights of the samples before and after being immersed in PBS for the specified periods, respectively.

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Thirty Sprague–Dawley rats (about 300 g) were treated and cared for under the supervision of a licensed veterinarian, in a manner consistent with the regulations of the National Institute of Health of Taiwan. All animal related procedures were approved by the Institutional Animal Care and Use Committee of Chang Gung University (IACUC Approval No.: CGU16060). The single intraperitoneal injection with sterile streptozotocin (STZ) (70 mg/kg) (Sigma, St Louis, MO, USA) in sodium citrate (0.1 mol/L, pH 4.5) was performed to induce experimental diabetic rat. The diabetes was verified 3 days after the injection by measuring glucose levels (N300 mg/dL). The diabetic rats were divided into three groups — each group consists of 10 rats; group A with core insulin and shell PLGA, group B with core PBS and shell PLGA, and group C with blend insulin and PLGA. Following anesthetization with isoflurane inhalation (4%) and intraperitoneal injection of pentobarbital (75 mg/kg), a sterile biopsy punch with a diameter of 10 mm was used to excise the skin on the middle of the back of each rat with a full-thickness wound to the deep fascia. Wound closure was expressed as an area (mm 2) using Image J software. Blood from tail veins was measured daily in all groups using OneTouch strips and OneTouch UltraEasy meter (LifeScan, Milpitas, CA, USA). On days 3, 7, and 14, the wound with a 5 mm area of unwounded skin was cut down to the fascia. Before undergoing frozen sectioning on a microtome-cryostat, the obtained tissue samples were stored and maintained at the optimal cutting temperature.

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Immunofluorescence

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Frozen sections were washed in PBS and blocked with 2% bovine serum albumin for a half hour at room temperature. The sections were then incubated overnight at 4 °C with primary antibodies against transforming growth factor beta (TGF-beta) (Abcam, Cambridge, MA), followed by Cy3-conjugated secondary antibody (Chemicon, Temecula, CA). Nuclei were visualized by DAPI-staining. All experiments were carried out in triplicate.

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Western blot analysis

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The protein (20 μg) was loaded and performed for blotting and then they were incubated overnight at 4 °C with anticollagen I (Abcam, MA, USA) as primary antibodies. The amount of protein of interest was expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Image J to quantify of the protein bands for their densitometry.

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Elongation at breakage ð%Þ ð2Þ ¼ Increase in length at breaking point ðmmÞ=Original length ðmmÞ

staining. The six random fields of each well were photographed and counted. Then, the type I collagen in each group was determined for different weeks in eluent from group A, positive control group with insulin and negative control without insulin.

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Figure 1. Scanning electron microscopy photographs of three groups of electrospun nanofibers; (A) insulin/PLGA nanofibers (B) PBS/PLGA nanofibers, and (C) blended insulin/PLGA (Scale bar: 5 μm). Diameters of group A and group B were 432 ± 106 nm (D) and 467 ± 144 nm (E), respectively. Blended PLGA nanofibrous scaffolds had larger diameters (1056 ± 376 nm) (F) (P b 0.001).

Statistics analysis

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All data are presented as mean ± standard deviation. Oneway ANOVA was performed to analyze the data and calculate statistical differences. Between pairs of groups, the post hoc Bonferroni procedure was used to identify their significant differences. Differences were considered statistically significant at P b 0.05. Data were analyzed using SPSS software (version 17.0 for Windows; SPSS Inc., Chicago, IL, USA).

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Results

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Core-shell and blended scaffolds

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Three nanofibrous scaffolds using suitable process parameters were fabricated. Figure 1 displays the structures of the electrospun nanofibers (SEM, 3000× magnification). The distribution of the diameters of the coaxially electrospun insulin/ PLGA nanofibers (432 ± 106 nm, dispersion index = 26.0) (Figure 1, A) was comparable to that of the PBS/PLGA nanofibers (467 ± 144 nm, dispersion index = 44.4) (Figure 1, B) (P = 0.304), and the average diameter of blended insulin/ PLGA was greater than the other two (1056 ± 376 nm) (P b 0.001). The porosity of the core-shell insulin/PLGA nanofiber (75.6 ± 2.0%) exceeded that of blended insulin/ PLGA (64.7 ± 1.5%) (P b 0.001).

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Figure 2 displays TEM (A-B) and laser scanning confocal microscopic (C-E) images. A liquid solution is observed (between the two red dashed lines) in the core-shell nanofibers (Figure 2, A) whereas the blended PLGA nanofibers did not exhibit a core-shell structure (Figure 2, B). The laser scanning confocal microscopic image in Figure 2, C revealed string-like reGFP within the core-shell reGFP/PLGA nanofibers, only a few reGFP string within the blended nanofibers (Figure 2, D), and none within the nanofibers without reGFP (Figure 2, E).The mechanical properties of the spun nanofibrous scaffolds were analyzed. The experimental data in Figure 2, F shows that blended PLGA nanofibers type (Group C) had a lower tensile strength (2.00 ± 0.22 MPa) than the core-shell nanofibers (Group A 2.87 ± 0.07 and Group B 3.31 ± 0.19 MPa) (P = 0.001 and P b 0.001, respectively). The nanofibers in Group A exhibited similar elongation at breakage (214.8 ± 16.9 vs. 251.7 ± 14.5%, P = 0.068) to those in group C, while those in group B (164.3 ± 27.2%) exhibited significantly less than those in group C (P = 0.002). Figure 3 presents the measured contact angle with water of core-shell and blended PLGA scaffolds. The measured contact angles for nanofibers in groups A and B were 124.7 ± 1.3 o and 128.6 ± 0.1 o, respectively, which did not differ significantly (P = 0.110). The nanofibers in group C were more hydrophobic (135.1 ± 2.5 o) (vs. group A, P = 0.004; vs. group B, P = 0.020). Differences between the water contents of the core-shell nanofibers and blended PLGA nanofibers were also examined.

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Figure 2. Transmission electron microscopic (A-B) and laser scanning confocal microscopic (C-E) observations. (A) Core-sheath nanofibers; liquid solution between red dashed lines. (B) Blended PLGA nanofibers (Scale bar: 1 μm). (C) Sheath-core structured nanofibers with PLGA as sheath and reGFP as core were prepared. (D) Blended PLGA nanofibers with reGFP and (E) without reGFP (Scale bar: 50 μm). (F) Stress–strain curves of core-sheath (group A and B) and blended PLGA (group C) nanofibrous scaffolds. Top tracing is for PBS/PLGA scaffold with tensile strength of 3.9 MPa and elongation at breakage of 164.6%. Middle tracing is for insulin/PLGA scaffold with tensile strength of 2.9 MPa and elongation at breakage of 214.8%. Bottom tracing is for blended PLGA nanoscaffolds with tensile strength of 2.0 MPa and elongation at breakage of 251.7%.

Figure 3. Measured contact angles of (A) core sheath insulin/PLGA, (B) PBS/PLGA, and (C) blended PLGA nanofibrous scaffolds. Contact angles with water were 124°, 129°, and 132°, respectively. (D) Variation of water content of nanofibers over time. Core-sheath with insulin scaffold reached peak water content (317 ± 43%) in 2 h and PLGA reached peak content (50 ± 16%) in 30 min.

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As depicted in Figure 3, D, whereas the water content of the blended nanofibers (group C) peaked at 50 ± 16% after half an hour, that of the core-shell insulin/PLGA (group A) nanofibers peaked at 317 ± 43% at 2 h, and was still 211 ± 60% at 24 h.

Figure 4. In vitro release of insulin from core-shell nanofibrous scaffolds. Release pattern for 4 weeks: stable release from day 1 (12.8 ± 4.8 mU/ml) to day 21 (15.7 ± 1.2 mU/ml), followed by gradual decrease in concentration to 8.5 ± 1.9 mU/ml on day 28.

Clearly, the capacity of the core-shell structured insulin/PLGA scaffolds to hold water for the first 24 h greatly exceeded that of the blended scaffolds (P b 0.001).

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Figure 4 displays the daily in-vitro release curves of insulin. The core-shell insulin/PLGA scaffolds (group A) continuously released insulin for four weeks steadily from day 1 (12.8 ±

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Figure 5. In vitro study of eluent of core-sheath insulin/PLGA scaffolds at high glucose levels for bioactivity of insulin. Fibroblast migration test. (A) Significantly less migration of cells without insulin was noticed than (B) day 28 eluent from core-sheath insulin/PLGA scaffolds and (C) insulin solution (10 mU/ml), (D) **, P b 0.01; ****, P b 0.001. Collagen I/ GAPDH using Western blotting showed treatment with (E) PBS, (F) day 28 eluent, and (G) insulin. More collagen was noted upon treatment with PBS P = 0.005 (# vs. day 28 eluent), P b 0.001 ($, vs. insulin).

were significantly lower at 5.6 ± 0.3 mm 2. The core-shell insulin/PLGA scaffolds (group A) promoted wound repair over that achieved using core-shell PBS/PLGA scaffolds (group B) or the blended insulin/PLGA (group C) (Post hoc P all b0.004). Figure 7 shows the histological and immunofluorescence microscopic examinations of TGF-β. In all treatment animals, there was no remarkable inflammation around the treated areas of nanofibrous scaffolds. Moreover, core-shell insulin/PLGA scaffolds improved wound healing over that in the other two groups. There was significantly more infiltration of keratinocytes in group A than in other two groups after one week. Fourteen days following surgery, the wounds in the three groups had almost healed but the amount of TGF-β in group A (3.2 ± 0.3) exceeded that in the other groups (group B, 1.1 ± 0.2, vs. group A; group C, 0.8 ± 0.3 vs. group A, all P b 0.001). Figure 8 presents the results of western blotting for type I collagen content in the wound area on day 14. The type I collagen/GAPDH ratio in group A (1.22 ± 0.07) was higher than that in group B (0.65 ± 0.06) (P b 0.001) and group C (0.56 ± 0.01) (P b 0.001).

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A cell migration study was performed at a high glucose concentration (50 mM). The fibroblast migration test revealed significantly fewer migrating cells in the control (PBS only) group (13.7 ± 3.2 per field) (Figure 5, A) than in the other groups (vs. core-she core-shell insulin/PLGA eluent on day 7 (20.0 ± 2.0 per field), 14 (23.3 ± 2.5 per field), 21 (21.6 ± 1.5 per field), and 28 (22.0 ± 2.6 per field, all P b 0.01); vs. pure insulin solution (10 mU/ml) (22.7 ± 2.1 per field, P = 0.006) (Figure 5, B-D). The protein analysis (Figure 5, E-G) revealed a higher ratio of the amount of type I collagen without insulin and day 28 sample eluent treatment to GAPDH in the control group (0.68 ± 0.10) (Figure 5, F) than in the other groups (vs. core-she core-shell insulin/PLGA eluent on day 7 (0 7 ± 0.04), P = 0.005 (Figure 5, G); vs. pure insulin (0.29 ± 0.01), P b 0.001 (Figure 5, H)).

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Discussion

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Two full-thickness circular wounds were prepared on the back of each rat, parallel to the vertebral column in studies on the diabetic wound repair. Figure 6 shows representative gross images of diabetic wounds on the animals in each group (coreshell insulin/PLGA, core-shell PBS/PLGA, and blended insulin/ PLGA groups) on different days following nanofibrous scaffolds treatment. On days 3, 7 and 14, the wound area in group A was apparently less than that in groups B and C; the wound areas in groups B and C were comparable. Whereas the areas of wounds in groups B and C had been slowly reduced to 16.9 ± 2.4 mm 2 and 17.7 ± 4.7 mm 2, respectively, on day 14, the areas of the wounds that were treated using the core-shell insulin/PLGA mats

This work developed core-sheath nanofibrous bioactive insulin-loaded PLGA scaffolds that sustainably release insulin for four weeks and for treating wound in diabetic rats. The hydrophilicity and water-containing capacity of core-shell nanofibrous insulin/PLGA scaffolds exceeded those of blended nanofibrous scaffolds. The nanofibrous core-shell insulin-loaded scaffold reduced the amounts of type collagen I in vitro, increased TGF-β content in vivo and promoted wound repair in diabetic wounds. The experimental results suggest that nanofibrous core-sheath insulin-loaded scaffolds maintain insulin biologic activity for the treatment of diabetic wounds and may effectively enhance wound healing in the early stage.

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4.8 mU/mL), day 3 (10.7 ± 1.1 mU/mL), day 21 (15.7 ± 1.2 mU/mL) and then with a gradually decreasing concentration to 8.5 ± 1.9 mU/mL on day 28. In group C, insulin was lower than the detection limit.

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Local and topical delivery of insulin to the target tissue provides high and sustained local concentrations without having to provide large systemic doses, minimizing the possibility of hypoglycemia risk. Adding insulin or PBS at the core of PLGA scaffold reduces the percentage of polymeric materials and thereby reduces the resistance to elongation externally applied force. Electrospun core-shell nanofibers (groups A and B) thus had smaller ranges of diameters than blended nanofibers (group C). Scaffolds for skin tissue engineering should be permeable to carbon dioxide and oxygen for nutrition and waste for promoting healing with a three-dimensional biodegradable structure and favorable mechanical strength. 25,26 They should also be easily

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Figure 6. Wound healing on different days following treatment (Scale bar = 5 mm). Group A showed its wound closure was quicker than group B and group C. (**, post hoc P b 0.01; ****, post hoc P b 0.001.)

processed and manufactured. Under moist conditions, wound healing usually recovers more rapidly than under dry conditions. 27,28 Dehydrated environments around wounds might lead to rapid cell death which induces the formation of scabs and delays wound healing. 29 Whereas the blended PLGA nanofibers herein were hydrophobic, the insulin-loaded nanofibers were somewhat hydrophilic. Increasing the surface hydrophilicity of hydrophobic materials reportedly improves the adherence of cells to them and the growth of cells on them. Moderate wettability on surfaces can also improve cell attachment and promoting wound healing. 30 In tissue engineering, electrospun PLGA fibers have been widely studied for clinical applications because of their specific

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Figure 7. Histological and immunofluorescence microscopic images of wounds in three groups on days 3 (A, D, and G), 7 (B, E, and H), and 14 with TGF-beta immune stain (C, F, and I) (Scale bar = 100 μm). Group A had highest TGF-beta index (J) (***, post hoc P b 0.001).

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porosity, the core-shell structured nanofibers had greater water retention capacity than blended one. Their porous binding structure provided ideal moisture and controllably released the pharmaceuticals, avoiding the wound area to become dry and thereby enhance repairing. It also eliminated the necessary for continuous wound redressing and cleaning, helping the body to repair better with wounding and decreasing the pain and illness of the patient. 32 Generally, drug release from a pharmaceutical embedding biodegradable device comprises three phases, which are burst liberation, diffusion-dominated elution, and degradationdominated release. 33 In group C, the insulin was blended with PLGA and solvent directly. The insulin might have been deactivated due to its contact with the solvent. It was thus not detectable in the subsequent analysis. Owing to the protective effect of the shell PLGA layer, no initial burst from the core-shell nanofibers occurred. The release of insulin was mainly controlled by the diffusion of the drugs through the PLGA materials. A relatively constant-speed, slow elution of insulin was thus observed. Overall, the electrospun core-shell nanofibrous scaffolds sustainably release insulin for four weeks. Diabetes impairs immune function and inhibits wound repair, both of which are important for survival and recovery when patients suffer from a major wound injury. 34 , 35 A high glucose level and insulin resistance contribute to epithelial downgrowth and fibrosis following skin injury. 30 , 36 The ability of insulin to increase collagen expression has key functions for various physiological settings such as development, growth, and wound healing. 37 , 38 Proper assembly of the subunit molecules of collagen type I is important for its macroscopic fibril structure and is needed for its stability, tensile strength, and biological function. 39 Appropriate insulin treatment for high sugar level can reduce collagen level from fibroblasts in vitro but wounds in vivo for promoting diabetic wounds healing. 40 Among the extracellular factors that govern signaling pathways and cell behavior, TGF-β is a potent regulator of the proliferation and differentiation of cells of many types as it directs the expression of hundreds of target genes. An increase in TGF-β level in response to insulin promotes synergism between TGF-β and insulin signaling. The enhancement in TGF-β

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Figure 8. Western blotting for collagen type I of wound area on day 14. (A) Core-shell insulin/PLGA group, (B) core-shell PBS/PLGA group, and (C) blended PLGA with insulin group.

extra-cellular matrix that mimics a non-woven micro-/nanofibrous structure. 31 Desirable properties of nanofibers include a highly porous structure, a high aspect ratio, and high mechanical strength because they can improve the dressing wounds. 30 The insulin-loaded nanofibrous PLGA scaffolds showed good flexibility and extensibility, supporting the possibility of their application in wound dressings that allow for the skin contraction during recovery. Coaxial electrospinning helpfully separated solvent from biomolecules during the fabrication process. The bioactivity of insulin was preserved. Owing to their higher

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responsiveness arises mainly from the biological effects of insulin. 41 TGF-β signaling is also important for reepithelialization, inflammation, angiogenesis, and the formation of granulation tissue during wound healing. 42 In severe diabetes, insulin receptors are sometimes significantly decreased in impaired wounds. 43 The local administration of insulin in skin wounds can enhance the expression of vascular endothelial growth factor from keratinocytes. 14 , 44 It also changes the patterns of release of wound chemokine and cytokine, having pleiotropic effects and increasing the rate of wound healing. 45 An ideal wound dressing can have faster healing and a better final result, with lowered probability of pain, scarring, and infection. It can also decrease costs by enhancing the healing rate, shortening the duration of therapy, and allowing for simpler and less often requisite actions for medical procedures. This study shows that the biodegradable core-shell insulin/PLGA scaffolds that were fabricated in this work had good biocompatibility and the ability to deliver sustainably insulin to accelerate diabetic wound healing. This finding indicates that insulinloaded PLGA nanofibers can be as an effective scaffold for tissue regeneration of diabetic wounds. Some limitations in this study exist. Diabetic wound dressing should consider insulin activity under drying status at different time points for clinical application. Collagen I and TGF-beta might be not good enough for the assessment of the diabetic wound healing, and other fibroblast growth factors which have shown the potential effects on the repair and regeneration of tissues 46 should also be investigated. These will be the topics of our future studies. This work developed core-sheath nanofibrous bioactive insulin-loaded PLGA scaffolds that provide locally and sustainably release insulin in diabetic wounds. The fabricated nanofibrous scaffolds supported accelerated wound healing and favored epithelial cell proliferation, mainly because the insulin in the scaffolds promoted the epithelialization. Nanofibrous coreshell insulin/PLGA scaffolds were functionally effective and active in the initial stages of the diabetic wound healing and care.

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