Cerium Oxide Nanoparticle Incorporated Electrospun Poly(3

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Cerium oxide Nanoparticle Incorporated Electrospun Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Membranes for Diabetic Wound Healing Applications Robin Augustine, Anwarul Hasan, Noorunnisa Khanam Patan, Yogesh B Dalvi, Ruby Varghese, Aloy Antony, Raghunath Narayanan Unni, Sandhyarani N, and Ala-Eddin Moustafa ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01352 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Biomaterials Science & Engineering

Cerium Oxide Nanoparticle Incorporated Electrospun Poly(3-hydroxybutyrate-co-3hydroxyvalerate) Membranes for Diabetic Wound Healing Applications Robin Augustine1,2, Anwarul Hasan1,2*, Noorunnisa Khanam Patan1, Yogesh B Dalvi3, Ruby Varghese3, Aloy Antony3, Raghunath Narayanan Unni4, Neelakandapillai Sandhyarani5, AlaEddin Al Moustafa2,6 1Department

of Mechanical and Industrial Engineering, College of Engineering, Qatar

University, Doha-2713, Qatar. 2Biomedical 3Pushpagiri

Research Centre, Qatar University, Doha-2713, Qatar.

Research Centre, Pushpagiri Institute of Medical Science & Research, Tiruvalla,

Kerala-689101, India. 4RP

MedHelix Diagnostics Private limited, Cochin, Kerala-682025, India.

5Nanoscience

Research Laboratory, School of Materials Science & Engineering, National

Institute of Technology Calicut, Kozhikode, Kerala-673601, India. 6College

of Medicine, Qatar University, Doha-2713, Qatar.

Corresponding author, Email: [email protected] Abstract Insufficient cell proliferation, cell migration and angiogenesis are among the major causes for non-healing of chronic diabetic wounds. Incorporation of cerium oxide nanoparticles (nCeO2) in wound dressings can be a promising approach to promote angiogenesis and healing of diabetic wounds. In this paper, we report the development of a novel nCeO2 containing electrospun poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) membrane for diabetic wound healing applications. In vitro cell adhesion studies, chicken embryo angiogenesis assay and in vivo diabetic wound healing studies were performed to assess the cell proliferation, angiogenesis and wound healing potential of the developed membranes. The experimental results showed that nCeO2 containing PHBV membrane can promote cell proliferation and cell adhesion when used in wound dressings. For less than 1% w/w of nCeO2 content, human mammary epithelial cells (HMEC) were adhered parallel to the individual fibers of PHBV. For higher than 1% w/w of nCeO2 content, cells started to flatten and spread over the fibers. In ovo angiogenic assay showed the ability of nCeO2 incorporated PHBV membranes to enhance blood vessel formation. In vivo wound healing study in diabetic rats confirmed the wound healing potential of nCeO2 incorporated PHBV membranes. The study suggests that nCeO2 incorporated PHBV membranes have strong potential to be used as wound dressings to enhance cell proliferation, vascularization and promote the healing of diabetic wounds. 1 ACS Paragon Plus Environment

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Key words: PHBV, electrospinning, cerium oxide, angiogenesis, diabetic wound healing Introduction Diabetes wounds are among the major causes of morbidity and mortality in diabetic patients involving complications such as prolonged inflammations, severe infections and non-closure of the wounds, resulting in amputation of limbs and often losses of lives1. Prolonged inflammatory phase and elevated oxidative stress are the major causes of complications associated with diabetic wounds2. Application of suitable antioxidant agents is a useful strategy to promote the healing of diabetic wounds3,4. Several earlier studies have established that the use of antioxidant and anti-inflammatory agents can accelerate diabetic wound healing5,6,7. Insufficient blood vessel formation is another major issue in diabetic wounds8. Despite the ability of angiogenic growth factors to improve the vascularization in diabetic wounds9, the poor stability of the growth factors under processing and application conditions, in addition to their excessively high costs, limit their clinical application10. Recent advances in nanotechnology-based approaches are highly promising to overcome the difficulties associated with the conventional medical interventions11. For instance, catalytic properties of several metal oxide nanoparticles have been exploited to develop novel therapeutic modalities for oxidative stress induced health issues12. However, the potential for application of such metal oxide nanoparticles in diabetic wound healing is yet to be thoroughly explored. Cerium oxide nanoparticles (nCeO2) have been proven to exhibit antioxidant and enzyme-mimetic activity in biological systems13. Due to the antioxidant14 and angiogenic15,16 properties of nCeO2, they are effectively used in several biomedical applications17. In a recent report, a significantly faster wound healing was observed when ceria nanocrystals decorated mesoporous silica nanoparticles were used as tissue adhesive18. Earlier research also demonstrated the potential of nCeO2 to improve the cell proliferation and vascularization in tissue engineering scaffolds19,20. However, the applicability of nCeO2 in biodegradable wound coverage matrices to improve angiogenesis and wound healing in diabetic wounds has not been explored. For the past few decades, biodegradable polymers have extensively been used as potential biomaterials for augmenting the repair and regeneration of damaged tissues21,22. Various biodegradable polymers have been effectively utilized for the development of biomaterial matrices for drug delivery23,24, tissue engineering25,26 and wound healing27 applications. One class among them is poly (hydroxyalkonate)s (PHA) which are biocompatible, biodegradable polyesters and are obtained from various microorganisms28. Poly (3-hydroxybutyrate) (PHB)29 2 ACS Paragon Plus Environment

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and its copolymers with varying ratios of hydroxyvalerate (PHBV) are the most widely exploited PHA in biomedical applications. The clinical success of bare PHBV based biomaterials in wound healing is limited due to the lack of bioactivity of these polymers30. To tackle this issue, novel functional agents that can promote the proliferation of host cells and angiogenesis at the site of implantation can be incorporated in PHBV based wound coverage matrices31,32. Electrospinning is an excellent technique to fabricate highly porous submicrometer-diameter fibers from natural and synthetic polymers33. Electrospun fibers have several applications in bioengineering such as tissue engineering scaffolds, wound dressings, biomedical implants and drug delivery systems34. One of the major advantages of electrospun membranes in wound dressing application is their microbial barrier property which can help to protect wounds from pathogenic colonization35. Integrating active components such as metallic36 or metal oxide37 nanoparticles in electrospun fibres is a highly promising approach to enhance their functional properties. In this study, we designed electrospun PHBV membrane based novel wound coverage matrices to enhance cell proliferation, cell migration and angiogenesis by exploiting the therapeutic potential of nCeO2 for promoting diabetic wound healing. The nCeO2 incorporated PHBV membranes were fabricated using electrospinning technique, characterized by various physicomechanical techniques and investigated for their biocompatibility, cell migration property, angiogenic potential and wound healing ability using in vitro, in ovo and in vivo models. The experimental results showed that nCeO2 incorporated PHBV membranes have strong potential for application in wound dressings to enhance cell proliferation, blood vessel formation and accelerate the healing of diabetic chronic wounds. Experimental Synthesis and characterization of nCeO2 Cerium nitrate (Ce(NO3)3·6H2O, Alfa Aeser, USA) was used as the precursor of nCeO2. Gelatin (CDH, India) was used as the stabilizing agent for the formed nanoparticles. Briefly, 500 mL of 0.5 M cerium nitrate solution containing gelatin (2%) was prepared and stirred vigorously for 30 min. Aqueous ammonia (HIMEDIA, India) was added slowly to the cerium nitrate/gelatin solution until light yellow precipitate was formed. This was stirred in a magnetic stirrer for 10 h (26 °C). The solution, containing formed nanoparticles, was centrifuged at 8000 rpm to separate the nanoparticles. The centrifugate was washed several times alternatively with 3 ACS Paragon Plus Environment


deionized water and ethanol. The washed precipitate was dried in oven at 80 C for 12 h and then calcined at 600 C for 10 h (at a heating rate of 15 °C min-1). Fourier transform infrared (FT-IR) analysis was performed with a Perkin Elmer Frontier MIR spectrophotometer with a PIKE Gladi ATR (USA) attachment at 4 cm−1 resolution in the range 4000–400 cm−1. The purity and crystallinity of the samples were characterized by X-ray diffraction (XRD) analysis using a MiniFlex X-ray diffractometer in the 2θ range of 10°–90. Crystalline particle size was calculated using Debye’s Scherrer equation (1) D = K λ/ βcos(θ)

(1)

where, K is the shape factor, λ is the wave length of X-ray, β is the full width at half the maximum (FWHM) and θ is the Bragg angle. Transmission electron microscope (TEM) was used to determine the morphology and size of synthesized nanoparticles. nCeO2 sample was dispersed in isopropyl alcohol (IPA) and drop casted on a carbon film coated TEM grid. JEOL JEM 2100 HR TEM was used to analyse the sample. Selected area electron diffraction (SAED) pattern was collected to identify the crystal structure of synthesized nanoparticles. Fabrication of PHBV/nCeO2 membranes PHBV and PHBV/nCeO2 nanocomposite membranes were fabricated by electrospinning technique. Electrospinning instrument was consisted of a DC power supply, a syringe controller and a metallic plate collector. PHBV solutions with various amounts of nCeO2 were prepared in chloroform-dimethyl formamide (9:1) mixture. Prior to the addition of PHBV pellets (Sigma-Aldrich, USA) into chloroform-dimethyl formamide mixture, various amounts of nCeO2 were dispersed in the solvent by ultrasonication (15 min). Then, the PHBV pellets were dissolved in the above dispersions to get 20% w/v PHBV solution. Finally, the prepared solutions (10 mL) were electrospun at a tip to collector distance of 10 cm. The applied flow rate and voltage were 2.5 mL·h–1 and 18 kV, respectively. Fibrous membranes deposited on the collector were carefully removed and stored in a desiccator at room temperature until used. Bare electrospun PHBV (no nanoparticle loading) membranes were named as PHBV. Membranes with 0.5%, 1%, 2% and 4% (w/w) of nanoparticle loading were designated as PHBV/nCeO2-0.5, PHBV/nCeO2-1, PHBV/nCeO2-2 and PHBV/nCeO2 -4, respectively. 4 ACS Paragon Plus Environment

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Characterization of PHBV/nCeO2 membranes Scanning electron microscopy (SEM) Morphological features of fabricated samples were analysed using field emission scanning electron microscopy (Hitachi SU6600). Gold coated samples were imaged at 15 kV accelerating voltage. ImageJ software was used for the calculation of individual fibre diameter and the average value was calculated from at least 100 fibres. Energy dispersive X-Ray spectroscopy (EDS) analysis was used to detect the presence of nCeO2 in PHBV membranes (Nova NanoSEM 450, FEI, USA). FTIR and XRD analysis Electrospun membranes were analysed by XRD and FTIR to confirm the loading of nCeO2 in PHBV fibers. Instrumentation used for these characterizations were similar to those employed for the analysis of nanoparticles. Differential Scanning Calorimetry (DSC) Thermal behaviour of the samples was studied by DSC analysis (TA Instruments, DSC Q-20). The bare PHBV and nanocomposite membranes (5 mg) were heated from 0°C to +200°C at 10°C min-1 under the flow of nitrogen (20 mL/min). The samples were kept for 1 min at +200°C and then cooled at 10°C min-1 to 0°C. Crystallinity of the polymer at various nanofiller content was calculated using the equation (2). % Crystallinity = [ΔHm/ΔHm°] X 100%

(2)

ΔHm is the enthalpy of fusion of the samples used in this experiment and ΔHm° is the enthalpy of fusion of 100% crystalline PHBV from the literature (ΔHm° = 146 J/g)38. Uniaxial tensile mechanical properties of PHBV and PHBV/nCeO2 membranes The mechanical properties of wound coverage materials are desired to be similar to those of the skin to avoid mechanical mismatch and associated complications. A Universal Testing Machine (UTM, Tinus Olsen H50 KT) in compliance with the ASTM D 882 standard was used to measure the tensile mechanical properties of the PHBV and PHBV/nCeO2 membranes. PHBV and PHBV/nCeO2 membranes with 6×1 cm2 size were used for the experiments. The samples were stretched at a crosshead speed of 1 mm/min using a 100 N load cell. Results of tensile measurements were calculated from the average values of five different tests and given as the mean ± standard deviation (SD). 5 ACS Paragon Plus Environment


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In vitro cell culture studies Cell lines used Human Oral Epithelial cells (HOEC)39 and Human Mammary Epithelial Cells (HMEC)40 used in this study were immortalized by transfection with retroviral vectors containing E6/E7 oncoproteins of High-Risk human papillomavirus (HPV) type 16. HaCat keratinocyte cell line (spontaneously transformed immortal cell line), obtained from adult human skin, was used at passage 48 in this study. Evaluation of cell adhesion and cell viability HOEC and HMEC cells were used to evaluate cytocompatibility of the membranes. Both of the cells were seeded on the developed membranes at 50,000 cells/cm2 and cultured with Keratinocyte-SFM (Gibco, USA) containing Bovine pituitary extract (Gibco, USA) supplement and 1% Penicillin-Streptomycin solution (Gibco, USA) for 7 days. To understand the effect of nCeO2 loading on cell adhesion, cell seeded membranes were stained with DAPI (Thermo Fisher Scientific, USA) and phalloidin; and imaged with a fluorescent microscope (Leica DMi8 S). To further verify the adhesion behavior and morphology of cells on individual fibers, crystal violet (HIMEDIA, India) staining was used. To determine the viability of cells grown over the membranes, MTT cell viability assay was carried out as per the procedure obtained from the manufacturer (Thermo Fisher Scientific, USA). All the experiments were performed in triplicates. In vitro wound healing assay using human keratinocytes The in vitro wound healing assay (scratch assay) was performed using HaCat cells to determine the effect of PHBV/nCeO2 membranes on keratinocyte cell migration. Cells were cultured on 24-well cell culture plates in Keratinocyte-serum free medium containing 1% penicillinstreptomycin-neomycin solution and bovine pituitary extract. Then, a scratch was made in the middle of every well with the help of pipet tip (100-µL). After washing and media replacement, sterilized 1X1 cm sized PHBV and PHBV/nCeO2 membranes were placed over the scratched area. Scratched areas were imaged using a microscope (Leica DMi1 Inverted Microscope) soon after the procedure and after 20 hours. Quantification of wound contraction was performed by measuring the gap between the borders of remaining cell-free space. Wound contraction (%) was calculated using equation (3). Wound contraction (%) = (Wd0 – Wdt)/Wd0

(3)

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Whereas Wd0 and Wdt are the gaps between scratch boundaries before and after time ‘t’ of the application of samples. Evaluation of the angiogenic potential of the membranes The angiogenic potential of the developed membranes were evaluated by chorioallantoic membrane assay based on the reported protocol41,42. Fertilized white leghorn hens’ eggs were purchased from Arab Qatari for Poultry Production, Doha, Qatar. As received eggs were incubated in an egg incubator at 37ºC with 60% humidity and used for the experiments on 4th day of incubation. Wound healing study in diabetic rats Male Sprague Dawley rats (180-260g) were used for the wound healing experiment. The rat breed was purchased from Kerala Veterinary and Animal Sciences University, Mannuthy, India. The surgical experiments were performed with the approval of institutional animal ethics committee (No.602/PO/Re/S/2002/CPCSEA) by firmly following the rules of CPCSEA constituted by the Ministry of Environment, Forests and Climate Change, Animal Welfare Division of Government of India in Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla, Kerala, India. Diabetes was induced in overnight fasting rats by the intraperitoneal injection of streptozotocin (Sigma Aldrich, USA) at 40 mg/kg body weight. Blood glucose level was measured at regular intervals to determine the development of diabetes (Supporting information, Table S2). Prior to the implantation, PHBV and PHB/nCeO2 membranes (1.5 × 1.5 cm) were sterilized as described in earlier works43. Intraperitoneal administration of combination of xylazine (5 mg/kg) and ketamine hydrochloride (50 mg/kg) was used to anaesthetise the animals. Dorsal area of the animals was shaved, surface sterilized with 70% ethanol and two 1.5 x 1.5 cm full thickness excision wounds were made at distance of 3 cm from each other. In one of the wounds PHBV membrane and in the other one PHBV/nCeO2-1 membrane was sutured. No external secondary dressings were used to cover the wounds. For visualizing the general appearance and extend of wound healing, photographs of the wounds were taken with a digital camera on 0, 5, 10 and 15 days. In addition, length and breadth of wounds were measured using a measurement scale with µm precision once in every 2 days. The wound contraction was expressed as percentage of wound contraction which was calculated using Equation (4). Wound contraction (%) = [(WA0-WAt)/WA0] x 100

(4)

Where ‘WA0’ is the area of wound at 0th day (on the day of creation of wound) and ‘WAt’ is the area of wound at ‘t’ days of healing.



Skin was excised from the healed area after 30 days of healing. Fixed and processed samples were embedded in paraffin wax and 3 micrometer-thick sections were cut with a rotary microtome (Thermoscientific HM325 microtome). The slides were stained with hematoxylin and eosin. The stained sections were independently examined by two researchers for angiogenesis, cellular infiltration, granulation tissue formation, and re-epithelialization with a Leica light microscope. The sections of healed wounds were also compared with the normal skin sections based on histology. Statistical analysis Un-paired Student’s t-test and “One-way ANOVA” were performed using Graph- Pad Prism Version 6.04, San Diego, California, USA to find out the statistical significance of the obtained results. P < 0.05 considered as statistically significantly different than the control groups.

Results and Discussion Morphology and physical properties of nCeO2 The XRD pattern which was recorded from 2 degrees 10 to 90 of synthesized and calcined nCeO2 is shown in Fig. 1A. As expected, well defined characteristic diffraction patterns of cerium oxide were observed at 2θ = 28.49°, 33.04°, 47.45°, 56.38°, 59.11°, 69.42°, 76.71°,79.17° and 88.51° which were corresponding to the miller indices (111), (200), (220), (311), (222), 400), (331), (420) and (422), respectively. Obtained patterns indicated the presence of cubic lattice of pure CeO2 and were matching with the JCPDS Data (898436)44. Based on Debye’s Scherrer equation, average crystalline size was 12.5 ± 4.4 nm. FTIR spectrum of synthesized nCeO2 is shown in Fig. 1B. The bands at 3387 cm-1 and 1641 cm-1 were due to O–H groups of bound moisture45 as observed by others.46 The bands at 1537 cm-1 and 1328 cm-1 might be from the precursors. The peak below 700 cm-1 is ascribed to the O-Ce-O stretching mode vibration of nCeO247. TEM micrograph of prepared nCeO2 powder is given in Fig. 1C. As expected, spherical-ovoid shaped nanoparticles with average diameter of 8.6 ± 3.8 nm were observed from the TEM image. HR-TEM image showed the lattice fringes of individual nCeO2 (Fig. 1D). nCeO2 particles showed relatively uniform crystal orientation. They possessed lattice fringes with d spacing of approximately 0.32 nm which correspond to the (111) plane of nCeO248,49. The nanocrystalline features of the nCeO2 was determined by SAED analysis (Inset of Fig. 1D). The high crystallinity of nCeO2 produced clear Debye–Scherrer diffraction rings that


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correspond to (111), (200), (220), (311), (222), (400), (331) and (420) of cubic CeO2. Both the structures which were determined by XRD and SAED were comparable.

Fig. 1. XRD pattern (A), FTIR spectrum (B) and TEM image (C) of nCeO2 powder. HR-TEM image of nCeO2 (D). SAED pattern of nCeO2 is given in inset of (D). Morphological and physical properties of PHBV/nCeO2 membranes SEM micrographs of electrospun PHBV and PHBV/nCeO2 membranes are given in Fig. 2A. Highly porous interconnected network of polymeric fibres with varying diameters was observed. To quantify the fiber diameter, we measured the fiber diameter and given in Table 1. Average fiber diameter of neat PHBV membrane was 2.85 ± 2.42 µm. In the case of PHBV/nCeO2 membranes, we observed a marginal variation in fibre diameter with respect to the increase in nCeO2 concentration. Fibre diameter of PHBV/nCeO2-0.5 membrane was comparable to the neat PHBV. For PHBV/nCeO2 -1, the fibre diameter was slightly reduced (1.87 ± 2.27 µm) but without any statistically significant difference. PHBV/nCeO2-2 and 9 ACS Paragon Plus Environment


PHBV/nCeO2-4 showed 2.14 ± 1.67 µm and 2.36 ± 1.68 µm average fiber diameter, respectively. The minor difference in fibre diameter while incorporating nCeO2 nanoparticles can be described in terms of probable variation in solution properties, especially the viscosity50. Representative higher magnification images are shown in the supporting information (Fig. S1). In order to allow cell penetration, migration, subsequent cell proliferation and the effective elimination of exudates produced in the wound, the membrane should be highly porous51. The obtained porosities of the PHBV and nanocomposite membranes were in the range of 88.15 ± 6.46% to 91.23 ± 3.12% (Supporting information Table S1). Based on the morphological features, all the membranes showed sufficient porosity which can provide enough space for cell migration during wound healing. Table 1. Fibre diameter of PHBV and PHBV/nCeO2 membranes. Sample

Average fibre diameter ± S.D. (µm)

PHBV PHBV/nCeO2-0.5

2.85 ± 2.42 2.62 ± 1.38

PHBV/nCeO2-1

1.87 ± 2.27

PHBV/nCeO2-2 PHBV/nCeO2-4

2.14 ± 1.67 2.36 ± 1.68

EDS of the membranes showed the successful incorporation of nCeO2 in PHBV membranes. The corresponding EDS spectra are shown in Fig. 2A (bottom part), which reveals the presence of elemental Ce and oxygen signals from nCeO2 along with a signal of carbon from PHBV. As a preliminary test, XRD analysis was used to identify the presence of nanoparticles in the PHBV matrix and given in Fig. 2B. This also provided insight on the influence of nCeO2 on the crystalline behaviour of PHBV membranes. Crystalline nature of the polymers play a major role in their physical and mechanical properties. The peaks at 13.5° and 16.9° correspond to α phase of PHBV polymer52. As expected, in the case of PHBV/nCeO2 membranes, the matching diffraction patterns of nCeO2 were detected at 2 angles 28.49°, 33.04°, 47.45° and 56.38°. Presence of nanoparticles in the PHBV fibres was apparent from the diffraction peaks of nCeO2 along with the peaks of PHBV in the XRD patterns of the nanocomposite membranes. The FT-IR spectra of pure PHBV membrane and PHBV/nCeO2 nanocomposite membranes are shown in Fig. 2C. The intense band at 1747 cm-1 corresponds to the stretching vibration of ester carbonyl groups (C=O) in the amorphous region of semicrystalline PHBV53. The band around 1452 cm−1 corresponds to asymmetric deformation of methylene groups and peak at 1380 cm−1 was due to the symmetrical wagging of CH3 groups. Other major peak at 1177 cm-1 10 ACS Paragon Plus Environment

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was due to the amorphous regions of PHBV. Characteristic peaks of symmetric –C–O–C– stretching vibration were present from 650 cm−1 to 1200 cm−1 and the antisymmetric –C–O– C–stretching between 1050 and 1160 cm−1.54 PHBV/nCeO2 membranes also presented similar characteristic peaks as obtained for PHBV polymer. Absence of the specific IR signatures of nCeO2 in the spectra of nanocomposite membranes might be due to the low concentration of nanoparticles which were entrapped in the polymer matrix. There was no noticeable shift in the peak representing the carbonyl groups of PHBV which indicate the absence of direct interaction between nCeO2 and PHBV polymer55.



Fig. 2. SEM micrograph and EDS spectra of PHBV and PHBV/nCeO2 membranes (A). XRD patterns of PHBV/nCeO2 membranes (B). FTIR spectra of PHBV/nCeO2 membranes (C). DSC curves of the PHBV and PHBV/nCeO2 membranes shows melting (D) and crystallization (E) peaks during the heating and cooling cycles, respectively. To understand the effect of nCeO2 in the thermodynamic properties during the physicochemical transformations of the PHBV membranes, DSC analysis was performed (Fig. 2D, E and Table 2). The endothermic peaks which were present between 180 and 190 °C during the heating 12 ACS Paragon Plus Environment

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ramp, correspond to the melting whereas the exothermic peaks observed between 99 and 101°C during subsequent cooling was due to the crystallization of PHBV. Minor exothermic peaks were also observed between 60 and 70 °C. There was no considerable shift in the melting point (Tm) or crystallization temperature (Tc) of PHBV after the loading of various amounts of nCeO2. However, melting (ΔHm) and crystallization (ΔHc) enthalpies of the nanocomposite membranes were varied to some extent. A comparable tendency was apparent for the crystallinity (Xc%) of the nanocomposites at lower nCeO2 content whereas at higher loading there was a reduction in overall crystallinity. It is plausible that the nanofillers could induce or alter the crystallization of polymers by acting as nucleating agents56. DSC analysis shows the appearance of dual melting peaks during the second heating process. Such multiple melting peaks can be due to the heterogeneity in composition, presence of several crystalline arrangements, or from melting–recrystallization– remelting during the heating ramp 57,58. In this case, during first heating step, all the crystalline phases and the thermal history of the samples were removed. The first melting peak corresponds to the melting of secondary crystallites formed during cooling. Melting of secondary crystalline phase happens at a lower Tm59. Melted polymer may crystallize and melt again during the second heating. Regardless of nanofiller content PHBV membranes showed comparable Tm and Tc. Moreover, crystallinity of PHBV was not significantly affected by the addition of relatively low amount of nCeO2 (1% w/w with respect to PHBV). On contrary, those membranes which were loaded with higher amount of nCeO2 (≥2% w/w) showed a considerable reduction in ΔHm and thus Xc%. This variation in crystalline fraction might be due to the formation of nanofiller agglomerates in PHBV matrix which could disturb the nucleation and formation of crystallites60. However, such minor reduction in crystallinity may not have a considerable effect on the biological performance of the fabricated membranes. Table 2. Temperatures and enthalpies of various endothermic and exothermic transitions obtained from DSC analysis of PHBV and PHBV/nCeO2 membranes Tm (°C) PHBV 188.6 PHBV/nCeO2-0.5 188.9 PHBV/nCeO2-1 188.5 PHBV/nCeO2-2 188.8 PHBV/nCeO2-4 189.4

Tc (°C) 99.7 100.6 99.5 98.9 99.6

ΔHm (J/g) 78.9 80.5 79.6 68.6 70.7

ΔHc (J/g) 65.6 74.9 68.4 69.4 73.9

Xc% 54.0 55.1 54.5 46.9 48.4

Uniaxial mechanical properties of PHBV and PHBV/nCeO2 membranes



Appropriate mechanical strength, elasticity and modulus are necessary for the acceptable clinical performance of the wound healing matrices. PHBV membranes loaded with upto 2% w/w nCeO2 showed improvement in tensile strength with respect to bare PHBV membrane (Table 3). However, PHBV/nCeO2-4 membranes showed a significantly lower tensile strength than the other samples studied. Bare PHBV and PHBV/nCeO2-0.5 membranes showed a relatively similar elongation at break (71 ± 11% and 68 ± 14%, respectively). Elongation at break of the membranes were decreased continuously with higher concentration of nCeO2 and this was very significant in the case of PHBV/nCeO2-4 (43 ± 12%). Results also demonstrated that PHBV/nCeO2 membranes incorporated with 1% w/w nCeO2 had the highest tensile strength and modulus (4.38 ± 0.36 MPa and 11.18 ± 3.14 MPa, respectively). A modulus value close to the skin is considered as ideal for wound healing applications since it can reduce stressshielding effect61. It is plausible that nanofillers at optimum concentrations can play vital role in improving the tensile strength of polymeric materials by acting as reinforcing agents62. This improvement might be due to the availability of more interfacial area for the transfer of stress from the polymer chains to the filler materials63. In contrast, a statistically significant reduction of elongation at break, tensile strength and modulus was observed where more than 2% w/w nCeO2 were loaded (P≤0.05). As expected, nCeO2 may tend to agglomerate each other at higher concentrations which results in the poor and uneven distribution of them in the polymer matrix. During mechanical stretching, agglomerates of nCeO2 might have acted as stress concentration centers and subsequent breaking points in the PHBV matrix64. This decreased the overall mechanical performance of PHBV/nCeO2 nanocomposite membranes with higher filler loading. Our results were in agreement with those reported in the case of PHBV/clay nanocomposites65. Table 3. Uniaxial tensile properties of PHBV and PHBV/nCeO2 membranes. Sample

Tensile strength (MPa) PHBV 3.68 ± 0.58 PHBV/nCeO2 -0.5 3.84 ± 0.41 PHBV/nCeO2 -1 4.38 ±0.36 PHBV/nCeO2 -2 4.43 ±0.18 PHBV/nCeO2 -4 2.53 ±0.84

Elongation at break (%) 71 ± 11 68 ± 14 65 ± 8 62± 15 43 ± 12

Modulus (MPa) 7.8 1± 2.34 8.56 ± 3.47 11.18 ± 3.14 9.24 ± 3.18 4.54 ± 2.32

PHBV/nCeO2 membranes enhance cell viability and adhesion of HOEC and HMEC cells


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To understand the effect of nCeO2 loaded in the membranes on human cell adhesion, we cultured HOEC and HMEC on the membranes and determined the cell adhesion behaviour. Fig 3A shows the representative images of the cell adhesion characteristics on PHBV and PHBV/nCeO2 membranes after 3 days of cell culture. Interestingly, higher cell adhesion was observed on PHBV membranes loaded with nCeO2. Considerably a smaller number of cells were only adhered on bare PHBV and PHBV/nCeO2-0.5 membranes. However, higher number of cells were observed on PHBV/nCeO2-1 and PHBV/nCeO2-2 membranes compared to the bare PHBV membranes. Adhesion of both the cells were almost same on PHBV/nCeO2-4 membranes also. Fig. 3B shows the morphological features of the cells adhered on PHBV and PHBV/nCeO2 fibers. From the crystal violet stained images, the difference in the cell density and the morphology of adhered cells on different membranes was highly evident. Neither of the studied cells showed good affinity with bare PHBV membranes. A considerable improvement in cell adhesion was observed on PHBV/nCeO2-0.5 membranes. HOEC showed a spreading kind of morphology on all nanocomposites whereas HMEC adhered on individual fibers and grown on the orientation of fiber length at lower nCeO2 content. At higher nCeO2 content (PHBV/nCeO2-2 and PHBV/nCeO2-4), HMEC also showed spreading cell morphology. Despite the relative increase in cell number, both kind of cells adhered on PHBV/nCeO2-1 showed a similar morphology as observed on PHBV/nCeO2-0.5. To understand the proliferation of HOEC and HMEC cultured on PHBV and PHBV/nCeO2 membranes, MTT assay was carried out upto 7 days and the result is given in Fig. 3C and Fig. 3D. A wound healing biomaterial should be biocompatible and able to maintain the original viability and physiology of the cells cultured on their surface. We observed that incorporation of nCeO2 did not affect the cell viability of either HOEC or HMEC cells when compared to PHBV membranes at 1, 3- and 7-days period. Both kind of cells seeded on nanocomposite membranes possessed more than 90% of cell viability as compared to the bare PHBV membranes. Interestingly, HOEC seeded on PHBV/nCeO2-1 membranes presented higher set of viability than neat PHBV membranes and other nanocomposite membranes. In the case of HMEC seeded membranes, PHBV/nCeO2-1, PHBV/nCeO2-2 and PHBV/nCeO2-4 showed relatively higher cell viability than other membranes studied.



Fig. 3. Effect of developed PHBV/nCeO2 membranes on the adhesion and proliferation of HOEC and HMEC cells. Adhesion of HOEC and HMEC (A) to the membranes after 3 days of cell culture by DAPI-phalloidin staining (A) and crystal violet staining (B). Viability of HOEC (C) and HMEC (D) on PHBV and PHBV/nCeO2 membranes, as determined by MTT assay. Data are expressed as the mean ± S.D of three different set of experiments. 16 ACS Paragon Plus Environment

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Ability of native cells to adhere to the wound dressing and proliferate over and inside them determines the pace of wound healing and the overall success of the wound coverage matrix66. Results of the present study were highly promising since the developed PHBV membranes containing nCeO2 promoted both the cell adhesion and proliferation. A slight reduction in cell viability at higher concentration of nCeO2 might be due to the over expression of proinflammatory factors which might have affected cellular functions67. However, this effect was marginal since there was no detectable variation in cell density on PHBV/nCeO2-4 membranes compared to the nearest lower concentration studied (PHBV/nCeO2-2). This clearly indicated that nCeO2 at an optimum concentration could promote HOEC and HMEC adhesion and subsequent proliferation on the PHBV membranes. Previous studies also support this observation68. In vitro wound healing assay To determine the wound healing potential of the developed membranes, in vitro wound healing test69 was carried out and the results are presented in Fig. 4. After 20 h of treatment, 40-60% of wounded area were healed in the case of control, PHBV and PHBV/nCeO2-0.5 membrane. Interestingly, higher wound healing was observed for PHBV/nCeO2-1, PHBV/nCeO2-2 and PHBV/nCeO2-4 membranes compared to the control (P≤0.05). Although, difference in scratch contraction percentage between PHBV/nCeO2-1, PHBV/nCeO2-2 and PHBV/nCeO2-4 groups was negligible, PHBV/nCeO2-2 and PHBV/nCeO2-4 membrane groups showed the presence of higher number of dead cells as compared to the other groups in the culture plates. Results of wound healing assay suggest that incorporation of an appropriate amount of nCeO2 in PHBV membranes can promote cell migration and repair of the wound. The observed improvement in in vitro wound contraction might be due to the ability of nanoceria to induce the expression of biomolecules that can stimulate cell migration70. However, in agreement with the results of cell viability studies, higher number of dead cells which were observed in the case of membranes containing higher concentration of nCeO2 gives a caution for their application above 1% w/w in therapeutic settings.



Fig. 4. Evaluation of wound healing activity using HaCat keratinocyte cells showing the improvement in in vitro cell migration upon treatment with nCeO2 containing PHBV membranes. (A) Microscopic images of wounded area prior to the treatment with samples (0 hour) and after 20 hours of treatment, (B) Percentage of wound healing in terms of scratch contraction. Evaluation of the angiogenic potential of the membranes by chicken chorioallantoic membrane assay CAM assay was used to assess the ability of PHBV/nCeO2 membranes to enhance the angiogenesis. Overall performance of PHBV/nCeO2-1 membranes were considerably better than other compositions in terms of tensile strength, cell adhesion and cell viability. Thus, we used PHBV/nCeO2-1 membranes for CAM angiogenesis assay. Photographs of CAM before 18 ACS Paragon Plus Environment

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and after the treatment with the membranes are provided in Fig. 5A. Quantitative data of angiogenesis is provided in Fig. 5B and 5C. After 24 h of study, higher number of capillary branches were observed in PHBV/nCeO2 membranes compared to that of bare PHBV membranes. Higher number of branch points indicates the presence of higher number of newly formed blood vessels. The number of capillary junctions on the CAM treated with PHBV/nCeO2-1 membrane group was significantly higher compared to the controls and neat PHBV membrane group (P< 0.005). However, we did not observe a significant difference in fold of increase in blood vessel diameter between different experimental groups. Overall, an improvement in angiogenesis was observed on CAM treated with PHBV/nCeO2-1 membranes compared to the bare PHBV membranes.

Obtained results were in agreement with earlier

report which suggest the ability of an optimum concentration of nCeO2 to enhance angiogenesis15. They suggest that this enhancement of angiogenesis can happen due to the modulation of oxygen in intracellular environments by antioxidant nanoparticles.

Fig. 5. Images showing the angiogenic potential of PHBV/nCeO2-1 membranes by CAM angiogenesis assay (A). Fold change in blood vessel junctions (B) and diameter (C) upon treatment with PHBV and PHBV/nCeO2-1 membrane after 24 h. 19 ACS Paragon Plus Environment


Wound healing potential of PHBV/nCeO2 membranes in diabetic wounds Considerably higher wound healing was observed in PHBV/nCeO2-1 membrane group from the beginning of the study as compared to bare PHBV (P≤0.05) group (Fig. 6A and 6B). The most plausible reason for the higher wound healing might be related to the antioxidant property of cerium oxide nanoparticles71. Unlike normal wound healing, in diabetic wounds, reactive oxygen species (ROS) could enhance the chronicity of wounds and delay wound healing72. Chronicity of diabetic wounds can be reduced by antioxidant therapy73. Antioxidant therapy improves healing of chronic diabetic wounds by inhibiting ROS generation 74. Owing to the ability of PHBV/nCeO2 membranes to promote wound healing, it can be utilized as a suitable biomaterial for diabetic wound healing applications. There are a few studies that demonstrated the potential of cerium oxide nanoparticles in wound healing68, however, there is no investigation that explored the potential of cerium oxide nanoparticles in diabetic wounds. Moreover, PHBV were utilized for the fabrication of wound dressings and demonstrated their potential to enhance wound healing75. However, best of our knowledge, there is no other reported studies regarding the wound healing application of electrospun PHBV membranes containing cerium oxide nanoparticles so far.

Fig. 6. Representative pictures of the wounds treated with bare PHBV membranes and PHBV/nCeO2-1 membranes showing the healing of full thickness excision wounds during 15


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days of study (A). Rate of wound contraction (B) and the histological analysis of healed wound (C). Histological analysis showed a considerable improvement in cell infiltration and granulation tissue formation in PHBV/nCeO2 membranes treated diabetic wounds compared to those treated with PHBV membranes alone at 30th day of healing (Fig. 6C). Those wounds treated with PHBV/nCeO2 membranes developed thick granulation tissue composed of several macrophages, fibroblasts, and new capillaries. Application of PHBV/nCeO2 for 30 days produced more erythema and thicker-appearance of granulation tissue than that was observed in PHBV alone. There was more cellular infiltration and capillary ingrowth in the PHBV/nCeO2 treated wounds. Despite the clear differences in histological appearance in the PHBV/nCeO2 treated wounds and bare PHBV treated wounds, the epidermis formation was very thin when compared with normal skin sections. Previous report demonstrated that topical application of nanoceria can accelerate the healing of full-thickness dermal wounds in mice68. They have observed a higher proliferation and migration of fibroblasts, keratinocytes and vascular endothelial cells on the wounds treated with nanoceria. We did not observe nondegraded PHBV fibers in histological sections. The implanted membranes might have degraded, or some part might have been expelled with the wound debridement76. Unlike in vitro conditions, the rate of degradation of PHBV in vivo is considerably faster at the same temperature and pH77. Under in vivo conditions, along with the role of body’s immune system, the hydrolytic activity of esterase and lysozyme enzymes boost up the degradation process78. However, detailed studies need to be performed to find out the role of nCeO2 in the degradation behaviour of PHBV if any which is out of the scope of this specific work. Based on the results of morphological, physicomechanical and biological characterization, PHBV membranes in combination with nCeO2 can provide appropriate morphology, mechanical support for growing cells, niche for cell adhesion/proliferation and signals for angiogenesis. This significantly enhanced the diabetic wound healing process. It was remarkable that the relatively small amount of nCeO2 in the PHBV membranes could achieve such a significant improvement in the overall performance, suggesting their potential and costeffective way to modulate diabetic wound regeneration. Further detailed studies in large animal models to fully verify biodegradation, toxicity as well as oncogenicity should be performed prior to the clinical testing of the PHBV/nCeO2 membranes. Conclusions 21 ACS Paragon Plus Environment


In this work, nanoceria (nCeO2) incorporated electrospun PHBV membranes were fabricated and evaluated for the physicomechanical and biological performance. An optimum loading of nCeO2 (1% w/w) improved the overall tensile strength of the PHBV membranes. Developed membranes loaded with nCeO2 (especially with 1% w/w) displayed excellent in vitro cytocompatibility and cell adhesion properties. In vitro cell migration was considerably higher for nanocomposite membranes. Results of CAM assay showed nCeO2 loaded membranes enhanced angiogenesis. Finally, results of wound healing experiments using diabetic rats clearly demonstrated the ability of nCeO2 containing membranes to enhance diabetic wound healing. Collectively, our study shows that PHBV membranes loaded with low concentration of nCeO2 can be utilized as a promising biomaterial for diabetic wound healing application where spontaneous tissue regeneration is critical. Notes The authors declare no competing financial interest Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI-------. Detailed experimental procedures for the measurement of porosity of the membranes and measurement of blood glucose in diabetic rats were provided. Table S1 and S2, Porosity of PHBV membranes in percentage; Blood glucose level in streptozotocin treated rats after various days of treatment. Acknowledgment This article was made possible by the NPRP9-144-3-021 grant funded by Qatar National Research Fund (a part of Qatar Foundation). The statements made here are sole responsibility of the authors. References (1)

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For Table of Contents Use Only Cerium Oxide Nanoparticle Incorporated Electrospun Poly(3-hydroxybutyrate-co-3hydroxyvalerate) Membranes for Diabetic Wound Healing Applications Robin Augustine, Anwarul Hasan, Noorunnisa Khanam Patan, Yogesh B Dalvi, Ruby Varghese, Aloy Antony, Raghunath Narayanan Unni, Neelakandapillai Sandhyarani, AlaEddin Al Moustafa


Cerium Oxide Nanoparticle Incorporated Electrospun Poly(3

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