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Small Molecular TGF-#1 Inhibitor Loaded Electrospun Fibrous Scaffolds for Preventing Hypertrophic Scars Le Wang, Junchuan Yang, Bei Ran, Xinglong Yang, Wenfu Zheng, Yun-Ze Long, and Xingyu Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09796 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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ACS Applied Materials & Interfaces

Small Molecular TGF-β1 Inhibitor Loaded Electrospun Fibrous Scaffolds for Preventing Hypertrophic Scars Le Wang †,§,‡, Junchuan Yang §,‡, Bei Ran §, Xinglong Yang §,# , Wenfu Zheng §,*, Yunze Long †,*, Xingyu Jiang §,#,*

†

Collaborative Innovation Center for Nanomaterials & Devices, College of Physics,

Qingdao University, Qingdao 266071, China. §

CAS Center of Excellence for Nanoscience, Beijing Engineering Research Center for

BioNanotechnology, CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, Beijing, 100190, China. #

University of Chinese Academy of Sciences, Beijing, 100049, China.

ABSTRACT: Hypertrophic scarring (HS) is a disorder that occurs during wound healing and seriously depresses the quality of human life. The scar-inhibiting scaffolds, though bring promise to HS prevention, face problems such as the incompatibility of the scaffold materials and instability of bioactive molecules. Herein, we present a TGF-β1 inhibitor-doped poly (ε-caprolactone) (PCL)/gelatin (PG) co-electrospun nanofibrous scaffold (PGT) for HS prevention during wound healing. The appropriate ratio of PCL to gelatin can avoid individual defects of the two

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materials and achieve optimized mechanical property and biocompatibility. The TGF-β1 inhibitor (SB-525334) is a small molecule and is highly stable during electrospinning and drug release processes. The PGT effectively inhibits fibroblast (major cell type contributing to scar formation) proliferation in vitro and successfully prevents HS formation during the healing of full-thickness model wounds on rabbit ear. Our strategy offers an excellent solution for potential large-scale production of scaffolds for clinical HS prevention.

KEYWORDS: co-electrospun; scaffolds; TGF-β1 inhibitor; anti-scar; wound healing

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1. INTRODUCTION Hypertrophic scarring (HS) is a dermal disorder that often occurs following deep burns or operations, and the patients always suffer from a reduced quality of life.1,2 In the developed world, 4 million patients acquire scars each year, and the incidence is even greater in developing countries.3-5 The excessive deposition of collagen secreted by fibroblasts is the major cause for HS formation. However, the collagen deposition is a necessary process for wound healing, thus, keeping the balance of collagen secretion and degradation is an important issue in scar research. Current approaches for treating and preventing scars generally include topical intralesional corticosteroids, silicone gel sheeting, pressure therapy, laser therapy, cryotherapy, radiation, and surgical treatment.6 To facilitate HS study, in vivo HS model was built by continuous gradient elastic tension.7 Several cell signaling molecules/pathways have been investigated for their potential scar reductive properties on animal models, such as the matrix metalloproteinase (MMPs),8 basic fibroblast growth factor (bFGF),9 Mitsugumin 53 (MG53) protein,10 transforming growth factor-β (TGF-β),11 and 20(R)-ginsenoside Rg3 (Rg3).12 TGF-β1 receptor, also named activin-like kinase 5 (ALK5), is a serine/threonine kinase that phosphorylates intracellular secondary messengers and downstream transcriptional regulators for genetic expression of procollagen alpha I. The latter will transform into secreted collagen type I and contribute to scar formation during wound healing.13-14 Hence, we reason that the inhibition of the signaling pathway of TGF-β1 receptor will be useful for preventing scar formation. A small molecule, SB-525334

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(6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline), has been characterized as a potent and selective inhibitor of the TGF-β1 receptor, thus, it is a potential candidate for suppressing scar formation. Electrospinning (ES) technology has attracted considerable attention as a kind of effective method to fabricate micro/nano-fibers due to its merits such as large specific surface area, flexibility, wide selectivity of polymer materials and composites.15-19 ES is widely used in biomedicine,20,21 wound dressing,22-24 and drug delivery,

25,26

due

to its operational simplicity and extensive applicability.27 Poly (lactic-co-glycolic) acid (PLGA) and Poly (ε-caprolactone) (PCL) are US Food and Drug Administration (FDA)-approved materials for various biomedical applications such as tissue regeneration, wound dressing, and drug delivery.28,29 ES PLGA30,31 or poly(l-lactide) (PLA) 32,33 fibrous scaffold has been modified by Rg3 for synergistic HS inhibition. However, PLGA tends to shrink and become stiffer whereas PLA is difficult to maintain its shape in aqueous conditions.34 Furthermore, Rg3 is sensitive to lights and need to be protected under light shielding to preserve its bioactivity.35 siRNA targeting TGF-β1 receptor has been used for reducing wound scarring, however, siRNA is not stable and needs repeated administration.36 Hence, the selection of appropriate scaffolds and bioactive molecules is significant for the practicable and effective scar inhibiting applications. Herein, we demonstrate a facile approach for preparing TGF-β1 inhibitor (SB-525334)-doped PCL/gelatin (PGT) fibrous scaffold for treating HS. PCL is a widely used biodegradable material for biomedical applications. Gelatin is a natural

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biopolymer derived from collagen37 and has been widely used in the pharmaceutical and medical fields. PCL has more reliable mechanical property compared to gelatin, whereas the latter is more biocompatible than PCL. Thus, the co-ES of PCL/gelatin may balance the mechanic property and biocompatibility of them both.38 SB-525334 is a small molecule and is stable during ES processes and diffusible in aqueous conditions. Hence, the PGT fibrous scaffold is supposed to be a good candidate for inhibiting HS with minimal unfavorable effects. In vitro experiments show that PGT scaffold can effectively inhibit the growth of fibroblast cells. In vivo wound healing assays on rabbit ear model show excellent scar inhibiting effects of the PGT scaffold, implying its potential applications in clinics (Figure 1).

2. MATERIALS AND METHODS 2.1 Materials. Poly (ε-caprolactone) (PCL, Mw = 80,000), gelatin (Type A, 300 Bloom, from porcine skin in powder form) and 1,1,1,2,2,2-Hexafluoro-2-propanol (HFIP)

are

from

Sigma

(USA).

6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H

-imidazol-4-yl]-quinoxaline (SB-525334, Mw =343.42) is from Selleck Chemicals. All other chemicals and solvents are of reagent grade.

2.2 Electrospinning (ES) Process. We dissolve PCL in 10 mL HFIP, and mix gelatin with PCL translucent solution in different proportions (PCL: gelatin = 3:1, PGI; PCL: gelatin = 5:1, PGII; PCL: gelatin = 8:1, PGIII) to yield a 10 wt % blend solution in a 25 mL conical flask. We add TGF-β1 inhibitor (SB-525334) in the PCL/gelatin

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solution with the concentration ranging from 1 to 10 µM after vigorous stirring for 2-4 h. A set of ES device which consists of a high-voltage power supply (HVPS DW-P303-1ACFO, Tianjin Dongwen), a spinneret, and a metal collector is used in the experiment. For the ES process, we draw 5 mL precursor solution into the spinneret with a tip diameter of 0.4 mm, which connects to a precision pump (LSP02-1B, Baoding Longer Precision Pump Co., Ltd) to maintain a steady flow rate of 1.2 mL h-1. ES voltage is applied between the spinneret and the collector at 17 kV with a distance of 8 cm. After 2 h, we obtain consecutive ultrathin fibrous membranes. We dry the ES fibrous membranes overnight under vacuum at room temperature.

2.3 Characterization of ES Fibrous Membranes. 2.3.1 Morphology Characterization. We characterize the morphology of the ES nanofibers composed of PCL and gelatin in different proportions by scanning electron microscopy (SEM, S4800, Japan) at an accelerating voltage of 10 kV. At least five images are taken for each membrane sample and the fiber diameter of the membranes is measured from the SEM images at 5000× magnification using Image J software (National Institutes of Health, USA). 2.3.2 Hydrophilicity Analysis. We evaluate surface wettability of the ES PCL/gelatin membranes by static water contact angles (WCA, SA100, Germany). Water droplets with a volume of 3 µL are separately dripped onto the surface of nanofibrous membranes at room temperature for measurements. 2.3.3 Water Absorbing Capacity and Degradation Characterization. We investigate

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the water absorbing capacity of the ES membranes by testing the weight increase of the wet membranes. The degradation property is evaluated by comparing the mass loss of the dry membranes after degradation. The water absorption (WA) and the weight loss (WL) are calculated according to the following equations: WA = (W1-W0)/W0×100%

(1)

WL = (W2-W0)/W0×100%

(2)

In equation (1, 2), W0 represents the initial weights of the dry ES samples, W1 represents the weights of the wet ES samples and W2 represents the weights of the wet samples vacuum dried at 37 oC. The mass loss is calculated by comparing the remaining dry weights of the samples with their initial weights at time points of 1, 3, 5, 7, 10, and 14 days. 2.3.4

Fourier

Transform

Infrared

Spectroscopy

(FTIR)

Analysis

and

Thermogravimetric (TG) Analysis. We determine the chemical compositions of the fibers by FTIR (FT-IR Spectrum One, Perkin Elmer Instruments Co., Ltd.) and process the data by Omnic32 software. Each group consists of three samples in the experiments, the average value is reported with standard deviation. We record the thermal properties of ES fibrous membranes by a TG analyzer (TG, Diamond TG/DTA, Perkin Elmer Instruments Co., Ltd.). 2.3.5 Tensile Strength Test. We evaluate the mechanical properties of the ES PGT scaffolds by using a tensile mechanical analyzer (Instron5567, USA) at a fixed speed of 20 mm min−1 at 25 oC, 45% relative humidity. We cut the fibrous membranes into a strip (1 cm × 10 cm) and fix the membranes on the two clamps with a distance of 50

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mm.

2.4 Degradation of PG and Release of TGF-β1 Inhibitor. We carry out in vitro degradation study by measuring the weight of the ES fibrous membranes before and after treatment in phosphate buffered saline (PBS, Solarbio, pH 7.4). We cut the membranes into square pieces (2 × 2 cm2), and measure their thickness (around 150 µm) and weight (around 50 mg) by a digital caliper (Guanglu). We soak all of the fibrous membranes in PBS in glass tubes. At selected time intervals (1, 3, 5, 7, 10, 14 days), we take the samples out and wash them with distilled water three times to remove the residue of PBS and dry them in vacuum at 37 oC for 24 h. We measure the weight loss of the samples to evaluate their degradation behavior. Meanwhile, we test the PGT fibrous membranes with different ratios of TGF-β1 inhibitor (1 µM, PGT1; 5

µM, PGT5; 10 µM, PGT10) as mentioned above. We take an aliquot of (1 mL) solution from the tube for subsequent drug release measurement, and the same amount of fresh PBS mixed with the same portion of TGF-β1 inhibitor is served as sample for drawing standard curves. We measure the concentration of TGF-β1 inhibitor to show the release behavior of the membranes by UV-vis spectrophotometer (UV2450, Japan). The degradation experiments and TGF-β1 inhibitor release assays are repeated in triplicate for each sample.

2.5 In vitro Cell Culture and Material Effect Evaluation. We culture NIH/3T3 fibroblast cells on the ES membranes to evaluate their biocompatibility. The NIH/3T3

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cells and Madin-Daby canine kidney cells (MDCK) are cultured in Dulbecco's Modified Eagle Media (DMEM) containing 10% fetal bovine serum (FBS, Invitrogen, USA) and 1% penicillin-streptomycin (PS, MP Biomedicals, USA). We count the number of cells with a hemocytometer. We seed 1 mL of the cell suspension (105 cells mL-1) in 6 well culture plates (Costar, Corning, NY, USA) containing culture medium supplemented with different concentrations of TGF-β1 inhibitor and culture the cells for 3 days in a humidified incubator at 37 oC with 5% CO2. We remove the culture medium and rinse the cells three times with PBS. The cells are fixed with 4% formaldehyde solution (PFA, Leagene) for 10 min and rinsed with PBS three times with an interval of 5 min. We stain the cells with TM 488 phalloidin (Invitrogen, 5 µg mL-1) and Hoechst 33342 (Sigma, 10 µg m L-1) to visualize cytoskeleton and cell nucleus respectively. The morphologies of the cells and the number of nucleus are analyzed by confocal microscopy (Zeiss LSM 710，Germany). We assess the cell density by counting cell number in three stochastic areas. We cut the sterilized ES fibrous scaffolds into small squares (2 × 2 cm2) and soak them in cell culture medium for 4 h before use. We seed the trypsinized cells onto the PGI and PGT scaffolds and incubate them for 3 days. The adhesion of NIH/3T3 cells on the membrane are counted after staining with Calcein-AM/PI (Dojindo, Japan, Calcein-AM (2 µM), PI (4.5 µM)). All the experiments are performed in triplicate.

2.6 Rabbit Ear Model of HS. We study the anti-scarring property of the PGT fibrous membranes on full thickness wounds in a rabbit ear HS model. Twelve healthy New

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Zealand white female rabbits weighting 2.5-3 kg are purchased from HFK (HFK Bioscience Co., LTD, Beijing). The rabbit model of HS is created as previously described.39 We perform all animal experiments according to the guideline of Chinese People's Liberation Army general hospital. We anesthetize the animals by pelltobarbitalum natricum (Sigma, 30 mg kg-1) and operate under sterile conditions. We remove the epidermis, dermis and perichondrium in each wound thoroughly. Four round-shaped wounds of 10 mm diameter, kept away from the central ear artery and marginal ear veins, are created on the ventral surface of each ear. The minimum distance between the wounds is larger than 12 mm. We divide the wound treatments into four groups (n = 3 for each group) randomly: group 1 (blank control); group 2 (gauze); group 3 (blank PGI scaffolds); group 4 (PGT5 scaffolds). In order to prevent the shedding of the scaffolds, we peel 2 mm normal epidermis skin around the wound. We sterilize the ES fibrous membranes by ultraviolet irradiation (30 min per side). The wounds in group 1 are not treated after operation, while the wounds in group 2-4 are covered with corresponding scaffolds respectively.

2.7 Macroscopic Evaluation and Histological Analysis. We observe and record the healing of each wound every day until the rabbits are sacrificed at the 1st, 2nd, 4th, and 8th week after surgery. We excise scars with a 2 mm margin of surrounding unwounded tissue. The excised rabbit ear scar tissue samples in each group are divided into three parts. Samples are fixed in 4% PFA for 24 h, embedded in paraffin, cross-sectioned, and stained by hematoxylineosin (HE) for histological analysis and

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determination of scar elevation index (SEI), which is measured by histomorphometric analysis (200× magnification) of the HE stained tissue sections.40,41 For Masson’s trichrome stained tissue, five randomly chosen fields of dermis are photographed at 200× magnification. We calculate the collagen in each group by normalizing to the collagen density of the unwounded skin.

2.8 Western Blot Analysis. We homogenate every specimen (100 mg skin) in 1 mL tissue lysis solutions (20 mM Tris-CI, pH 7.4, 1 mM EDTA) on ice for 30 min. We directly mix 1 mL of each sample with 1 mL SDS-PAGE (sodium dodecylsulfate polyacrylamide gel) loading buffer, and the mixture is centrifuged at 12,000 rpm for 15 min to remove insoluble precipitates. We use SDS gel electrophoresis to separate all the freshly prepared collagen and electrotransfer onto PVDF membranes in the buffer (Tris 3.0 g, Gly 14.4 g, M-OH 200 mL, add deionized water to 1L) at 400 mA for 4 h. The membranes are blocked with blocking buffer containing TBST (20 mM Tris-HCI, pH 7.40,150 mM NaCl, 1% Tween-20, 5% nonfat dry milk) 1 h at 4 oC. We add 1:300 dilution of Collagen Type I primary antibody (Abcam, USA) overnight at 4 o

C, followed by treatment with 1:1000 dilution of HRP-conjugated goat antimouse

IgG secondary antibody (Jackson, USA) for 1 h at 37 oC.

3. RESULTS AND DISCUSSION 3.1 Characterization of the ES Fibrous Membranes. ES technology allows us to fabricate the PCL, gelatin, and their blended fibers in various blending ratios with 11



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average diameters ranging from sub-micrometer to nanometer. The scale of the ES fibrous membrane is determined by the size of the collector and the time of the ES procedure. We can fabricate fibrous membranes with 5 mL precursor in a dimension of 300 mm×180 mm×80 µm (Figure S1A) or 260 mm×80 mm×200 µm (Figure S1B). One piece of the ES fibrous membrane can be used to prepare at least 200 pieces of wound dressings (the wound size is 1 cm in diameter) in the animal model. By using an industrial ES apparatus, we can produce ES products with the size up to 1 m2 or larger. SEM images show the morphology of the ES nanofibers with different PCL/gelatin ratios (Figure 2A-E). All the nanofibers are evenly distributed and have uniform sizes. The diameters of the ES fibers of gelatin, PGI (PCL/gelatin, 3:1), PGII (PCL/gelatin, 5:1), PGIII (PCL/gelatin, 8:1), and PCL are 0.45 ± 0.11 µm, 0.54 ± 0.21

µm, 0.65 ± 0.29 µm, 0.6 ± 0.17, and 1.42 ± 0.06 µm, respectively (Figure 2F). The incorporation of gelatin does not significantly influence the morphology of the fibers even when its content reaches up to 30 wt %. We characterize the wettability of the ES membranes by water contact angle analysis because the surface property has great influence on cell adhesion and proliferation. Pure ES PCL membrane has a contact angle of 120 o, demonstrating its high hydrophobicity (Figure 3). As for the ES membranes of gelatin, PGI, PGII, and PGIII, the water droplets infiltrate into the membranes immediately when contacting the membrane surfaces (Figure 3). The time for the complete water absorption is 4.86 ± 0.45 s, 6.52 ± 0.33 s, 7.44 ± 0.38 s and 9.64 ± 0.35 s on the surfaces of the ES membranes of gelatin, PGI, PGII and PGIII, respectively, demonstrating that the

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absorption time of the drops decreases with the increasing ratio of gelatin in the ES fiber (Figure S2). The water droplets completely disappear within 10 s for all the ES membranes of gelatin, PGI, PGII, and PGIII. These results indicate that all the membranes except PCL are hydrophilic. The substantial increase of the surface hydrophilicity of the ES fibers demonstrates the successful blending of gelatin with PCL. We characterize the water uptake capability of the ES membranes. The PCL fibrous membrane, although being hydrophobic, can absorb certain amount of water due to the porous property of the membranes (Figure 4A). With the increase of gelatin content, the PG fibrous membranes become hydrophilic with the water uptake capacity reaching up to 600% for the PGI, PGII, and PGIII membranes (Figure 4A). This means that the incorporation of gelatin significantly increases the water uptake capability of the membranes. We evaluate the degradation property of the ES membranes. A desirable feature of implantable scaffolds is the synchronized degradation of the scaffolds and the replacement by natural tissues. PGI, PGII, and PGIII membranes are immersed in PBS for different periods of time and their mass are weighed. All these membranes degrade with the increase of incubation time. PGI shows the highest degradation ratio among the three PG membranes (Figure 4B). Considering the appropriate degradation ratio of PGI for drug release, we select PGI (PCL/gelatin, 3:1) as the representative scaffold for the following experiments. The degradation of the PG membrane could be attributed to the hydrolysis of the

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gelatin in the fibers, since the degradation of PCL is very slow in the absence of a biocatalyst.42 PCL and PLGA are all US Food and Drug Administration (FDA)-approved biomaterials. In our previous study, the PLGA scaffolds tend to shrink and become stiffer during long-term culture.35 Before the design of the PCL/gelatin membrane, we compare the degradation properties of PCL and PLGA. After the incubation of PCL and PLGA ES membranes in PBS for 2 weeks, significant changes take place in PLGA (Figure S3A, B), while PCL keeps homogenous fibrous structure, demonstrating that PCL has the potential for keeping morphological cues to guarantee cell growth in vivo for long time. Thus, we select PCL to composite with gelatin for better performance as a wound dressing. We characterize morphological variations of the PGI membranes by SEM after soaking them in PBS at 37 oC for 1, 3, 7, 10, and 14 days (Figure 4D-H). The diameter of the fibers, which is 0.58 ± 0.06 µm, 0.76 ± 0.18 µm, 0.81 ± 0.25 µm, 0.95 ± 0.28 µm, and 0.99 ± 0.21 µm on day 1, 3, 7, 10, and 14, respectively, increases with the extension of the incubation time (Figure 4C). The SEM images also show that the fibers of PGI gradually melt together with the extended incubation time (Figure 4D-H). We co-ES TGF-β1 inhibitor and PGI to form fibrous membranes to construct TGF-β1 inhibitor releasing scaffolds (PGT). With the elevation of the proportion of TGF-β1 inhibitor from 1 µM to 10 µM, the shape and homogeneity of the fibers show no significant alteration. With the high doping concentration of TGF-β1 inhibitor, more dendritic structure and irregularly shaped fibers are collected (Figure 5A-D).

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Although the morphology of the bi-component fiber changes, the integral fibrous structure of the membrane could maintain well enough to support cell adhesion and growth. The average diameter of the PGT ES fibers is around 650 nm, which is similar to that of the pure PGI (0 µM) (ANOVA, P>0.05) (Figure 5E). Thus, the co-ES of TGF-β1 inhibitor does not significantly affect the morphology of the PGI fibrous scaffolds. We characterize PCL, gelatin and their blended ES fibers in different proportions by FT-IR (Figure 6A). The characteristic peaks of PCL appear at 2953 cm-1 (asymmetric CH2 stretching), 2840 cm-1 (symmetric CH2 stretching), 1728 cm-1 (C=O stretching), 1340cm-1 (C-O and C-C stretching), and 1240 cm-1 (asymmetric C-O-C stretching). The absorption peak at 1650 cm-1 is assigned to the light active amide I of gelatin, implying that gelatin is incorporated within PCL during the ES process. After the addition of TGF-β1 inhibitor, there is no obvious change in the characteristic peaks (Figure 6B), indicating that the incorporation of TGF-β1 inhibitor has no significant influence on the chemical composition of the polymer matrix. We analyze the thermostability of the ES fibers by thermogravimetric analysis (TGA). The decomposition temperature (Td) of the gelatin fibrous membranes with 5% and 10% weight losses are 162.42 oC and 195.41 oC, respectively, while the Td of the PGI fibers with 5% and 10% weight losses rise to 277.17 oC and 310.93 oC, respectively (Figure 6C). The addition of TGF-β1 inhibitor does not significantly change the Td of the PGI (Figure 6D). Thus, the ES fibrous membranes of PGI and PGT are stable enough for sterilization at high temperature (usually

Small Molecular TGF-Î²1-Inhibitor-Loaded ... - ACS Publications

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