Gelatin-Cerium Oxide Nanocomposite for Enhanced Excisional

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Gelatin-Cerium Oxide Nanocomposite for Enhanced Excisional Wound Healing I. Selestin Raja, and Nishter Nishad Fathima ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00208 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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ACS Applied Bio Materials

Gelatin-Cerium Oxide Nanocomposite for Enhanced Excisional Wound Healing I. Selestin Raja, Nishter Nishad Fathima∗

Inorganic and Physical Cheimstry Laboratory, Central Leather Research Institute, Council of Scientific and Industrial Research, Adyar, Chennai-600020, India

∗

Author to whom correspondence should be made. Tel: +91 44 24437188

E-mail: [email protected]; [email protected] 1 ACS Paragon Plus Environment

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ABSTRACT Researchers are keen on formulating composites blending biomacromolecules with functional nanoparticles to achieve greater efficacy in order to expedite wound healing process. In the present work, we have engineered a genipin cross linked gelatin hydrogel composite containing optimized concentration of cerium oxide nanoparticles (G-ONp) for the purpose of wound healing. The concentration of cerium oxide nanoparticles in G-ONp has been optimized to be 250 µg/mL, which shows more than 80% cell viability in cytotoxicity study. X-ray diffractogram of ONp displays characteristic lattice planes of cubic fluorite structure and Transmission Electron Micrograph reveals that the particles are sized between 2.5 - 6.5 nm. The genipin dimeric cross linkage in G-ONp has been confirmed by UV-Vis peak at 603 nm. Swelling ratio of G-ONp (25.3 ± 1.2) has been found to be three fold to that of native gelatin (9.2 ± 1.4). As far as pore size distribution is concerned, lyophilized sponges of gelatin and G-ONp had microsized pores in the range of 1-140 µm and 1-19 µm, respectively and hydrogels of the same determined by thermoporometry had nanosized pores in the range of 7-48 nm and 7-24 nm, respectively. The in vivo wound healing and histological examination have revealed that G-ONp treated rat group has shown more infiltration of leukocytes and larger deposition of collagen, when compared to gelatin and control groups and has healed the wound in 12 days. These findings suggest that the composite of G-ONp is superior to gelatin in increasing wound healing and can be envisaged as a wound dressing material in future. KEYWORDS: gelatin, cerium oxide nanoparticles, hydrogel, genipin, wound healing, thermoporometry

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INTRODUCTION Skin, the largest organ in human body, acts as a physical barrier to protect internal organs from foreign invaders. When the skin is damaged under the circumstances of injury, disease and strenuous exercise, it repairs itself to restore original state through a complex cascade of biochemical and cellular events including hemostasis, inflammation, proliferation and maturation.1,2 However, there is a need to design a wound dressing material to accelerate wound healing and restore normal skin integrity in the case of serious wound. Initially, a number of synthetic and naturally available macromolecules have been investigated as wound dressing material in different forms such as fiber, film and gel.3-5 Later on, researchers were inclined to prepare hybrid composites in order to enhance physico-chemical properties such as air permeability, hydrophilicity, tensile strength, elasticity and biodegradation of wound dressing material to create a benign environment around the wound site.6-8 Smaller sized organic molecules (ex. polyphenols) and inorganic nanoparticles have also proved their capacity to treat wounds with ease of permeability into the wound tissue and long term activity.9,10 However, they encounter the drawbacks of excessive accumulation of particles into the tissue cells triggering toxicity and oozing out of the wound site while applying causing ineffectiveness. To overcome the problems, smaller drug molecules and nanoparticles are dispersed physically or conjugated chemically within the matrix of macromolecules in recent times so as to increase efficacy and sustainability. For instance, curcumin loaded oleic acid based polymeric bandage composed of chitosan and sodium alginate has allowed the release of curcumin in a sustained manner proving a better clinical therapeutic activity.11 Micro porous and flexible composite of chitosan/nano ZnO has enhanced keratinocyte migration toward the wound site and promoted healing.12 In this context, we have attempted to formulate a nanocomposite of 3 ACS Paragon Plus Environment


gelatin/nano cerium oxide (G-ONp) as wound dressing material to exploit the combined effect of gelatin macromolecules and cerium oxide nanoparticles to treat excisional skin wound in rat model. Gelatin has been extensively used as wound dressing material by many researchers due to its porosity, biocompatibility and degradation.13 Gelatin is a denatured form of collagen, one of the extracellular matrix proteins abundantly present in skin matrix.14 Genipin is an aglycon of geniposide extracted from the fruits of Gardenia jasminoides Ellis. The exact mechanism of genipin cross linking reaction is not yet known. However, many researchers have proposed that genipin cross links two primary amine groups of polymers involving a number of pathways and intermediates to yield a dimeric product, which promotes blue color to the solution.15,16 Cerium oxide (CeO2) belongs to lanthanide and rare earth metal groups. In recent times, cerium oxide has been found to have many biological applications, as it exhibits oxygen transporting ability, multi-enzyme mimetic activity and free radical scavenging activity involving in redox reaction between Ce3+ and Ce4+.17,18 It is important to note that the wound site normally maintains hypoxic environment and be a source of generation of free radicals. Depending on the magnitude of wound, cells confront hypoxic challenge supports adaptation and survival in mild and moderate conditions. In contrast, extreme hypoxia leads to tissue loss.19 In severe pathological conditions, inflammation is generally associated with production of free radicals at much quantity, which further complicates wound healing.20 Cerium oxide nanoparticle has the ability to overcome these problems by oxygen buffering and free radical scavenging ability of nanoparticles. Cerium oxide nanoparticle has been used as a sol for wound healing application. It has been reported that cerium oxide nanoparticle with 3–5 nm size enhance the proliferation and migration of keratinocytes and fibroblasts, and accelerate the 4 ACS Paragon Plus Environment

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migration and tube-forming ability of vascular endothelial cells.21 In the present work, the nanoparticles are dispersed into cross linked gelatin matrix in order to achieve sustained activity of functional cerium oxide nanoparticles. In brief, the aim of the present work is to prepare a highly cross linked nanocomposite of GONp with the help of cross linking agent, genipin and study the composite with required physico-chemical and biological characterizations for the application of excisional wound healing using the optimized ratio of functional CeO2 nanoparticles in G-ONp.

EXPERIMENTAL SECTION Materials Gelatin, type B for bacteriology (120 kDa) was purchased from Hi Media. Oleylamine (≥ 98 %), cerium nitrate hexahydrate (Ce(NO3)3. 6H2O), 1-octadecene (90 %), genipin (≥ 98%, (1R, 4aS, 7aS)-methyl 1, 4a, 5, 7a-tetrahydro-1-hydroxy-7-(hydroxymethyl)-4a, 7a-dimethylcyclopenta [c] pyran-4-carboxylate) and dialysis tubing cellulose membrane (MWCO 14 kDa) were availed from Sigma Aldrich. The solvents used throughout the work were of analytical grade. Preparation and Optimization of G-ONp Oleylamine protected cerium oxide nanoparticles were synthesized by thermal decomposition following the procedure given in previous literature.22 Briefly, 1 mM of Ce(NO3)3. 6H2O was dissolved in the mixture of 3 mM of oleylamine and 5 g of 1-octadecene at 80 °C for 30 min. Nanoparticles were grown at 260 °C for 2 h under nitrogen gas atmosphere. The as-prepared CeO2 nanoparticles were purified using the mixture of methanol and acetone (1:1, v/v) to remove unreacted cerium precursor and additives. The purified nanoparticles were re-dispersed in CHCl3 5 ACS Paragon Plus Environment


and phase transferred to stabilize the nanoparticles using the surfactant, oleylamine, by probe sonication method as follows. 2 µM of oleylamine was added to 2.0 mL of CeO2 nanoparticles in CHCl3 and mixed with 8.0 mL of distilled water. The mixture was then probe sonicated following the conditions 3 min, 70 % amplitude and full cycle. The resulting phase transferred nanoparticles were evaporated to remove organic solvent using rotavapor at 40 °C. Purification of water soluble oleylamine protected cerium oxide nanoparticles (ONp) was carried out by ultracentrifugation at 12,000 rpm for 5 min. The pellet containing ONp was kept in hot air oven for 12 h to obtain dried form of nanoparticles for further use. Gelatin stock solution (5 %, w/v) was prepared by dissolving in double distilled water at 60 °C. For the preparation of composite of gelatin impregnated with oleylamine protected nanoparticles (G-ONp), a 4.0 mL of 5% of gelatin was blended with 1.0 mL of ONp (0.1%, w/v) and magnetically stirred. To the above mixture, 1.0 mL of genipin (5 mM) was added drop wise at 40 °C. At 30 min of the reaction, the solution turns to blue indicating the formation of cross linkage among the polymeric chains of gelatin and ONp, as summarily given in Figure 1. After 6 h, the stirring was discontinued and the resulting dark bluish gel was dialyzed against double distilled water to remove un-reacted genipin. Both gelatin and G-ONp were lyophilized to obtain respective sponges for further characterizations. Literature reports reveal that gelatin can be classified into type A and type B depending on preparation procedure either acid or alkali treated processes, respectively. Type B gelatin differs from type A in terms of isoelectric point and presence of amine groups available for cross linkage. Type B gelatin has isoelectric point at 4.85.4 and is negatively charged at neutral pH, whereas type A has isoelectric point at 8-9 and is positively charged at neutral pH.23 Generally, negatively charged particles have long shelf life in blood circulation when compared to positively charged particles as the latter significantly


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undergo phagocytosis.24 It is expected that increased shelf life of type B gelatin would ensure sustained activity of gelatin matrix as well as functional nanoparticles around the wound site. Further, type B gelatin has been reported to have more number of reactive amine groups than type A.23 As genipin involves in cross linking amine containing polymers, type B gelatin can have more degree of cross linkage when compared to type A gelatin. Based on these advantages, type B gelatin has been preferred in the present work for the preparation of G-ONp. The concentration of ONp was varied from 50 µg/mL to 500 µg/mL and dispersed into gelatin solution (5%, w/v) and various composite (G-ONp) were prepared by adding constant amount of genipin solution (5 mM) into the reaction mixture. MTT assay was carried out to optimize the concentration of oleylamine coated cerium oxide nanoparticles in G-ONp samples by evaluating cytotoxicity using mouse embryonic ﬁbroblast cells (NIH−3T3). The cell line was procured from National Centre of Cell Science (NCCS), Pune, India. Cells were cultured in Dulbecco's modiﬁed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% CO2, and 100 units per mL of penicillin and streptomycin. NIH−3T3 cells were seeded in 96–well plates (1 X 104 cells per well) at 37 °C and subsequently the samples were added to the cell cultures. The control was the cell line without any additives. After 24 h incubation, MTT solution (0.5 mg/mL) was mixed into each well followed by the addition of 100 µL dimethyl sulfoxide (DMSO) to the mixture. Cell viability was determined by measuring the optical density at 570 nm using a microplate reader.



Figure 1. Preparation of oleylamine protected CeO2 nanoparticles (ONp) and engineering the composite of cross linked gelatin-CeO2 (G-ONp) from the mixture of ONp, gelatin and genipin have been shown schematically. W/O type cerium oxide nanoparticles were produced from precursor, cerium nitrate using thermal decomposition procedure. The as-prepared nanoparticles were transformed into O/W type water dispersed ONp nanoparticles. Blending of nanoparticles into gelatin along with cross linking agent, genipin, has yielded a composite of cerium oxide nanoparticles containing cross linked gelatin. The composite was further subjected to dialysis for purification followed by lyophilization to obtain lyophilized sponge for further characterization. Chemical structure of genipin dimeric cross linkage has also been shown.


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Characterizations UV-Vis spectra were obtained using Shimadzu UV-Visible spectrophotometer UV-1800. The Xray diffractograms of the samples were recorded in the range of 2θ = 10 ‒ 80°, in steps of 2 min−1 using a Shimadzu XRD‒6000 equipped with a Cu-kα radiation source. Dynamic light scattering (DLS) was performed to determine colloidal stability of ONp in terms of hydrodynamic diameter, zeta potential and polydispersity index, using a Malvern Nano ZS system equipped with a HeNe 633 nm laser (Malvern Zetasizer Nanoseries, Malvern, U.K.). The experiment was repeated in triplicate and the average values are reported with standard deviation (SD). Transmission Electron Micrograph (TEM) was carried out by evaporating one drop of ONp onto ultrathin carbon type–A 400 mesh copper grids (Ted Pella Inc.) and the image was captured by a JEOL 2100 field emission TEM gun operating at accelerating voltage of 200 kV with a single tilt holder. Scanning Electron Micrographs (SEM) were performed by the means of SEM Hitachi S–3400. The sponge was fixed over the carbon tape and surface of the samples was monitored with the operating conditions 15 kV and 300× magnifications. Porosity Measurement PMI capillary air flow porometer was employed to study porous nature of the sponges. The samples were cut into pieces of 20 mm diameter. A fluid namely Galwick with a defined surface tension of 15.9 dynes cm−1 was used as a wetting liquid. During the measurement, a non-reacting N2 gas was passed through the dry sample at first. Subsequently, the sample was wetted with Galwick and N2 gas was allowed again. The changes in flow rate of gas were measured as a function of pressure in both dry and wet conditions of the samples. Half-dry curve was computed from the software, which yields half of the gaseous flow through the pore.25 9 ACS Paragon Plus Environment


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Thermoporometry analyses were carried out to study porosity of hydrogel composite using Q200 TA. The measurement was run from −25 °C to +5 °C with a heating rate of 0.5 °C min−1 passing N2 gas. Hermetic aluminum pan was used for placing the samples. The measurements were done in triplicate and the mean value transition temperature (Tm) is reported with S.D. Our previous studies have demonstrated in detail the estimation of pore radii and pore volume with respect to pore radii from the derived equations using the obtained thermodynamic parameters, temperature and heat flow.26,27 Swelling Test The lyophilized sponges of gelatin and G-ONp were placed in deionized water to swell them. At regular time intervals, the samples were removed from the swelling medium and excess water on the surface was removed by the application of dry filter paper. The weight of the samples in swollen state was measured using analytical balance (accuracy ± 0.1 mg). The samples were returned to the swelling medium and the measurements were carried out until the equilibrium reached. The swelling ratio was calculated from the eq 1. Each measurement was repeated thrice and the values are reported with S.D. =

(1)

In vitro Degradation Freshly prepared trypsin solution (0.1 mg mL−1) was added to the sponge in order to measure degradation rate, as given in eq 2. The samples were removed from the solution at variable time and their weights were measured to monitor the weight loss until each sample completely degraded. % = 100 ×

$% &$'

(2)

%$10 ACS Paragon Plus Environment

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where mi and me are the weight of samples before and after degradation at selected time intervals, respectively. Each measurement was carried out in triplicate. In vitro Wound Scratch Assay NIH-3T3 fibroblast cells were seeded into 24–well plates and grown to confluence. Cells were treated with 10 µM of gelatin and G-ONp after 24 h of serum starvation (DMEM supplemented with 1% FBS). The cell monolayer was then damaged by scratching with a sterile 200 µL pipette tip. Cells were then cultured for a period of 10 h in a serum-free basal medium in the continued presence of samples. The scratched areas were visualized using light microscope (Axioscope A1, Carl Zeiss) with magnification 10 X at 0, 2, 4, 6, 8 and 10 h. The photographic images were analyzed using Image J software and migration of cells was reported in terms of % of reduction in area of lesion. Excisional Wound Healing Model Twenty four, 3-month-old female albino Wistar rats, weighing about 150 – 200 g, were randomly divided into three equal groups. Following the surgical procedure, the rats were individually housed in standard plastic cages and fed with a palette complete rat diet and water ad libitum. All the experiments were carried out under the Institutional Animal Ethics Committee, which is recognized by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The rats were anaesthetized by one intraperitoneal injection of a mixture of ketamine hydrochloride (40–80 mg kg−1 body weight) and xylazine (5–10 mg kg−1 body weight). The hair of the dorsal area was removed using an electrical shaver and the skin was cleaned with 70% of ethanol. Open excision wound with 4 cm2 area was created on the back of each rat by the means of a surgical blade. On each group, the wound area of one animal was kept as control without



applying any material. The animals of second and third groups were covered with sterilized sponges of gelatin and G-ONp, which was cut into pieces with areas equivalent to wound. On 4, 8 and 12 days of post operation, the wound site of the rats was photographed using a digital camera (Nikon D5300). Granulation tissues were collected from the wound site and immersed in 10% buffered formalin, embedded in paraffin and then sectioned. The sections were stained with Hematoxylin and Eosin (H&E) and Masson's Trichrome. The images were visualized using light microscope at 20X magnifications. The histological examination of the slides was carried out blindly by a pathologist who was unaware of the treatment groupings. Tensile Strength Analyses The skin samples from each group (control, gelatin and G-ONp) of wound created rats were subjected for the analysis of tensile strength at the end of healing process. An intact skin from the dorsal area was removed and taken as reference. The width and thickness of the wound strips were measured by a digital caliber. The skin strips were clamped into small tensile jigs and tensile loads were applied via a universal testing machine (Instron, model 4502, High Wycombe, UK) using a 100 N load cell and a 5 mm min−1 crosshead speed, up to rupture. The maximum force developed on the sample as the point of failure at which the tensile strength dropped sharply to zero was marked. The tensile strength was measured by dividing the maximum force by the cross-sectioned area of the specimen (about 10 mm × 2.5 mm).


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RESULTS AND DISCUSSIONS

The concnetration of oleylamine protected cerium oxide nanoparticles (ONp) in the lyophilized sample (G-ONp) was optimized using biological measurement, MTT assay. As shown in Figure 2, gelatin has the cell viability 94 ± 0.8% whereas G-ONp has the same as 93 ± 0.4%, 91 ± 0.3%, 89 ± 0.3%, 87 ± 0.9%, 86 ± 1.4%, 78 ± 1.1%, 69 ± 0.8%, and 61 ± 1.2%, on varying concentration of ONp 50, 100, 150, 200, 250, 300, 400, 500 µg/mL in G-ONp, respectivley. We have opted for G-ONp containing 250 µg/mL nanoparticles as it possesed cell viability more than 80% and the difference in cell viability between gelatin and G-ONp containing 250 µg/mL of nanoparticles is not significant. At the same time, significant difference in cell viability has been noted in the composites when the concentration of CeO2 has increased above 250 µg/mL. Though functional cerium oxide nanoparticles are aiding for enhanced wound healing, the excessive concentration of nanoparticles would cause toxic effects to the cellular environment of wound tissue as cerium containing nanoparticles are considered to be foreign antigens to human body. Hence, the concentration of cerium oxide nanoparticles in G-ONp has been optimized to have at least 80% cell viability.



Figure 2. Cell viability of NIH−3T3 cell line observed after 24 h incubation with the samples gelatin and G-ONp, each with concentration 5%, w/v. The concentration of cerium oxide nanoparticles in G-ONp varied from 50 – 500 µg/mL. The control was the cell line without any additives. * Statistically significant as compared to control (p ˂ 0.05, Tukey). From Figure 3, it can be seen that gelatin has its characteristic UV-Vis peak at 278 nm corresponding to tyrosine residue.28 ONp has a peak at 312 nm indicating that nanoparticles have been adsorbed by oleylamine.29 The appearance of blue color because of genipin dimeric cross linkage in G-ONp has displayed an additional absorbance peak at 603 nm.


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Figure 3. UV-Vis spectra of gelatin, oleylamine protected cerium oxide nanoparticles (ONp) and cross linked composite of gelatin/cerium oxide nanoparticles (G-ONp). The UV-Vis peak at 603 nm indicates genipin dimeric cross linkage in G-ONp.

Gelatin is generally amorphous with little crystalline nature and hence displays a broad peak around 20° (Figure S1) in X-ray diffratogram.30 ONp has shown lattice planes of cubic fluorite structured CeO2 at (111), (200), (220), (311), (222), (400), (331) and (420).31 X-ray diffractogram of G-ONp has possessed the peaks of gelatin as well as high intensive peaks of ONp confirming dispersion of coated nanoparticles in gelatin scaffold. As observed in Figure S2a, DLS measurement has revealed that hydrodynamic size (DH) of oleylamine coated CeO2 nanoparticle was 195 ± 3 d. nm with polydispersity index (PI) 0.194. Further, the value of zeta potential (ζ) was found to be 22.4 mV. The lesser DH and PI and the larger value of ζ proves colloidal stability of the particles in solution. While visualizing TEM image of ONp, individual



spherical nanoparticles seem to assemble to a spherical form in dried state (Figure S2b). The corresponding histogram of nanoparticle show that the size of nanoparticles is ranged 2.5 – 6.5 nm (Figure S2c). Size of the nanoparticle plays an important role and has significant impact on its application. When the nanoparticles are monodispersed with smaller size, they can express more activity due to large surface area to volume. The literature reports reveal that 3.8 nm sized CeO2 nanoparticles are more reactive against free radicals investigated than 8.2 nm sized CeO2 nanoparticles.18 Capillary flow porometry is a simple and non-destructive technique that allows rapid measurement of pore size distribution by tracing the gas pressure and flow rates between dry and wet samples. The dry curve corresponds to the permeability of gaseous molecules over a wide range of pressure. It is evident from Figure 4a and Figure 4c, the flow rate of gas into the porous gelatin was 2.60 L min−1 at its maximum pressure while that of porous G-ONp accounted 1.42 L min−1. Further, the mean flow pressure, which is determined from the intersection of wet-flow and half-dry flow curves, of G-ONp was 1.1 Psi, which has been found to be greater than that of gelatin (0.5 Psi). These results indicate that porous network of G-ONp sponge is more complicated with irregular paths and cross sections, when compared to gelatin sponge.


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Figure 4. Gaseous flow rate Vs pressure of gelatin (a) and G-ONp (c) by capillary air flow porometry are shown. Dry, wet and half dry curves are indicated by square, diamond and triangle dotted lines, respectively. The corresponding pore size distribution histograms are shown in (b) and (d), respectively.

The literature reveals that three kinds of pores are available in any porous material namely closed, blind and through pores. Through pore can permeate fluids through its path and hence is relevant for the porosity determination.32 The Figure 4b and Figure 4d reveal that pore size distribution of gelatin and G-ONp, has been extended to the range of 1-140 µm and 1-19 µm, respectively. G-ONp has shown a decreasing order of pore size from 3 to 19 µm. From porosity



pattern, it is understood that G-ONp sponge contains more constricted pores than gelatin. The same has been reflected in their morphology of SEM images (Figure S3). Free water content transforms into bound water content when hydrogen bonds of water molecules are engaged in interaction with polymeric chains of hydrogel. They may be further classified into weakly bound and strongly bound water molecules.33 When cross linkage occurs in gelatin by genipin, the cross linking density between polymeric chains increases resulting in increase in bound water content. Strongly bound water is considered to be integral part of hydrated polymers. However, weakly bound water molecules disrupt geometrical arrangement of free water molecules in a hydrogel system. As a result, lowering in transition melting point takes place depending on the availability of bound water content.34 As shown in Figure 5a, DSC endotherm of gelatin and G-ONp hydrogels had transition melting point (Tm) at −0.48 ± 0.11 °C and −1.68 ± 0.05 °C, respectively. The lowering in transition melting point clearly indicates that G-ONP has experienced large degree of cross linkage to accommodate more bound water content. As shown in Figure 5b and Figure 5c, DSC pore size distribution plot investigates nanosized pores in the range of 7-100 nm. An increase in number of intensive peaks at lower radii range indicates a high porosity pattern. The hydrogel composite, G-ONp had more intensive peaks at the range of 7-24 nm reflecting a better porosity pattern when compared to gelatin, which contains the peak in the range of 7-48 nm. In brief, the porosity patterns determined by capillary air flow porometry and thermoporometry demonstrate that G-ONp has better porosity with increased number of pores in lower radii of microsized pores in solid state and nanosized pores in liquid state than non-cross linked gelatin.


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Figure 5. DSC melting endothermic curves of water molecules of gelatin and G-ONp hydrogels (a). Pore size distribution pattern featuring pore radii (Rp) and pore volume with respect to pore radii (dV/dRp) of gelatin (b) and G-ONp (c) has been shown.

As far as swelling ratio is concerned, a gradual increase has been accounted for both gelatin and G-ONp on increasing time (Figure S4a). However, the value of swelling ratio of G-ONp has been, significantly, higher than gelatin at regular interval of time studied. At the end of process, swelling ratio of G-ONp (25.3 ± 1.2) has been found to be three fold to gelatin (9.2 ± 1.4). It is well known that enzymatic action of trypsin is to cleave carboxyl terminal side of lysine and arginine and thus hydrolyze gelatin into peptides.35 A complete degradation (≥ 98 %) of gelatin has taken place at 120 min while the same of G-ONp has lasted until 180 min, as observed in Figure S4b. Owing to chemical cross linkage, G-ONp has shown an increase in swelling ratio and a decrease in degradation rate (%), when compared to non-cross linked gelatin.



Wound scratch measurement was carried out in order to evaluate the effect of samples on in vitro wound healing. The rate of scratch wound closure in G-ONp has been found higher than gelatin in the culture medium (Figure 6a and Figure 6b). At 10 h of the assay, the order of total % of reduction in lesion area was as follows, G-ONp (49.4 ± 0.2%) > gelatin (47.3 ± 0.3%) > control (44.8 ± 0.2%). CeO2 nanoparticles present in G-ONp might have acted as chemoattractant to enhance cell migration, which is essential for skin integrity. The previous studies revealed that CeO2 nanoparticles have caused a persistent increase in the concentration of cytokine-induced neutrophil chemo-attractants CINC-1 and CINC-2, when applied for intratracheal instillation and inhalation.36, 37 The difference in population of migrated cells at 10 h has been compared with the number of cells at 0 h among control, gelatin and G-ONp.

Figure 6. Wound scratch model (in vitro) of NIH-3T3 cell line by adding gelatin and G-ONp. Control was the cell line without samples (a). The difference in lesion areas among the samples has been shown at t = 0 and 10 h. The remaining lesion area of wound, with scale bar 100 µm, has been presented in percentage with the function of time (b).


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The period of wound healing process usually depends on seriousness of the wound, age and physical condition of the animal.38 There were significant changes observed in wound area of gelatin and G-ONp treated groups when compared to control (Figure 7). However, complete healing has been observed for G-ONp treated group when compared to gelatin treated group on 12th day. Ulubayram et al. has reported that epidermal growth factor (EGF) containing gelatin based wound dressing material has taken 14 post day operations to cover epidermis layer of wounded skin leading to complete healing.13 Naseri-Nosar et al. has reported that cerium oxide (1.5%, w/v) nanoparticle functionalized poly (ε-caprolactone)/ gelatin electrospun fiber has helped for wound healing with wound closure area 98.80 ± 1.07% on 14th day of post operation.39 In our present work, the optimized genipin cross linked gelatin containing cerium oxide nanoparticles (250 µg/mL) has healed the wound within 12 days of post operation. Cerium oxide nanoparticle act as chemo-attractant to help migration of fibroblast cells on the gelatin matrix. It has also been reported that gelatin contains Arg-Gly-Asp (RGD) sequence, which is responsible for cell attachment via αvβ3 and α5β1 integrins.40 Hence, the combined properties of cerium oxide nanoparticles and gelatin macromolecules are helpful for proliferation of fibroblast cells, which plays crucial role in the formation of epidermis layer in the wound tissue. In addition to it, genipin dimeric cross linkage between polymeric chains formed during reaction has been reported to have anti-inflammatory property by inhibiting the expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α.41 Hence, genipin cross linked gelatin-CeO2 composite has more advantageous properties to promote significant enhanced wound healing.



Figure 7. Excisional wound healing model using albino Wistar rats treated with gelatin and GONp has been shown. The wound area of the control group was left without any treatment. Digital photographs taken on 4, 8 and 12 post day operations of wound creation has been shown with scale bar 1 cm. Healing of closed excisional wounds is best determined by tensile strength measurements, which reflect the quality and speed of tissue regeneration. This gross measurement of wound strength has been found to correlate directly with the collagen content of wounds. Stress-stroke relationship curve, as shown in Figure S5, is generated to determine the ultimate tensile strength of skin, which can be inferred from the point, Smax, at which the stress becomes discontinuous and strain rate is switched on. The tensile strength of skin of control, gelatin and G-ONp groups were 5.30 ± 0.22, 5.61 ± 0.41 and 6.09 ± 0.23 MPa, respectively, at 24 days of post operation. The intact skin has tensile strength about 7.85 ± 0.12 MPa. It is concluded that the non-treated


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wound skin have reached at least 68.0% of the tensile strength of intact skin, while the skin treated with gelatin and G-ONp have retained 71.8% and 76.9%, respectively.

Figure 8. Histological examination of granulation tissues of wound from control, gelatin and GONp treated albino Wistar rat groups. H & E staining demonstrates infiltration of leukocytes appearing with blue stained nucleus on 8th day of post operation (a). Masson’s Trichrom staining of the samples collected on 12th of post operation shows collagen deposition with purple streak (b). The scale bar represents 20 x magnifications.

Histological examinations were helpful to provide insight into the cellular mechanism of wound healing. It has been reported that massive degradation products of matrix polymers, in our case gelatin, could stimulate inflammatory cells aggregation, promote epithelial and vascular endothelial cells, which leads to significant wound healing rate of treated groups than control group.42 It is evident from Figure 8a, which shows infiltration of leukocytes in granulation tissues collected on 8th day of post operation from each group. Leukocytes play an important role



in preventing infection and clearing debris, thereby enabling migration of keratinocytes and fibroblasts of the new skin tissue. As expected, number of leukocytes including neutrophil and basophil found in G-ONp and gelatin were excessive than control. However, density of blood cells of G-ONp treated group has been found larger than gelatin exploiting the role of cerium oxide, which effectively eliminate excessive oxidative stress and promote angiogenesis with facile oxygen transport to the wound site.43 Chigurupati et al. has reported that nanoceria attenuate the accumulation of 4-hydroxynonenal produced by lipid peroxidation and nitro tyrosine modified proteins in the damaged wound tissue.21 Collagen deposition in granulation tissue is one of the markers indicating healing of wound. As observed in Figure 8b, the group GONp has large collagen content followed by gelatin treated group and control, on 12th day of post operation. These findings indicate that G-ONp has facilitated the proliferation and migration of fibroblasts and permitted the normal sequence of dynamic events of newly generated skin. As we have reported earlier, number of microsized pores in sponge and nanosized pores in hydrogel of G-ONP has accessed permeability of oxygen molecules to reach the wound site at sustained level, before and after wound exudate come into contact with the dressing material. Further, lift time of matrix of G-ONp has been extended by genipin dimeric cross linkage and hence long term activity has also been feasible to heal excisional wound effectively than noncross linked gelatin without CeO2.

CONCLUSIONS The physico-chemical and biological characterizations have optimized concentration of nanoparticles in the nanocomposite of cross linked gelatin-cerium oxide nanoparticles (G-ONp) to be 250 µg/mL with cell viability 86 ± 1.4% and proved that G-ONp has more bound water


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content, porosity and swelling ratio than the non-cross linked gelatin. In vivo wound healing studies have shown that G-ONp has shown its wound healing efficiency in wound created rat groups to close the wound completely in 12 days, increasing accumulation of leukocytes and collagen deposition than gelatin and control groups. Therefore, we conclude that the cross linked composite of functional optimized CeO2 nanoparticles in gelatin matrix can be attempted for its effective usage as wound healing material in future.

ASSOCIATED CONTENT Supporting Information Powder XRD of gelatin, oleylamine protected cerium oxide nanoparticles (ONp) and cross linked gelatin with dispersed cerium oxide nanoparticles (G-ONp); Hydrodynamic diameter by DLS and TEM micrograph of ONp; SEM micrographs, swelling ratio and degradation rate (%) of gelatin and G-ONp sponges; Stress-stroke relationship demonstrated by tensile strength analysis of skin samples collected from control, gelatin, G-ONp treated groups.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]; Phone: +91 44 24437188 ORCID Nishter Nishad Fathima: 0000-0003-4646-2342 Author contributions IS Raja carried out the experiments and wrote the manuscript. NN Fathima gave critical comments and corrected the manuscript.



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Funding Department

of

Biotechnology,

Government

of

India

(Grant

number-

BT/PR4406/NNT/28/574/2011). Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT We would like to acknowledge surgeon Dr. Jayachandran Dare and research scholars Ms. Sowmya and Mrs. Kavitha, who were helpful during animal handling. Histological staining of the samples was carried out by ‘‘H & E Histology, Senthil Histopath Lab”, India.


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Graphical abstract


Gelatin-Cerium Oxide Nanocomposite for Enhanced Excisional

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