Preparation,characterization and biomedical applications of electrospun cellulose acetate nanofiber dressing fabricated with silver and bioglass nanoparticles for efficient wound healing in streptozotocin induced diabetic rat

Abstract

Diabetic wound healing remains a significant challenge due to impaired and delayed healing processes. Recently, nanoscaffold dressings with their intricate architectures gained remarkable attention in regenerative medicine. Herein, electrospun cellulose acetate (CA) nanofiber dressings incorporated with various concentrations of bioglass nanoparticles (BGNPs) and silver nanoparticles (AgNPs) were prepared as novel nanocomposites for possible healing of diabetic wound healing. The prepared dressings were physico-chemically characterized using scan electron microscopy (SEM), Fourier Transform Infra-Red Spectroscopy (FTIR), Energy-dispersive X-ray spectroscopy (EDX) and Thermogravimetric analysis (TGA). The antimicrobial activities for the prepared dressings were firstly evaluated in-vitro and then in-vivo against streptozotocin-induced diabetic rats. FTIR and EDX elemental analyses confirmed the chemical and the structural composition of the prepared electrospun CA/BGNPs/AgNPs nanofiber dressings. SEM analysis revealed uniform, smooth and continuous nanofiber (40–180 nm diameter) that showed higher thermal stability as indicated by TGA analysis. The 3% BGNPs and 5% AgNPs loaded CA nanofibers showed maximal antimicrobial activity specifically against the gram positive Staphylococcus aureus (42 ± 1.9 mm) and the gram negative Escherichia coli (43 ± 2.2 mm) which are the main two bacteria infecting wounds. In vivo study revealed remarkable acceleration in wound healing process with 3% BGNPs and 5% AgNPs combination with maximal efficient wound closure by Day 6 without induction of skin irritation. Therefore, the newly designed CA/BGNPs/AgNPs nanofiber dressing hold promising potential for the management of diabetic wounds.

Introduction

Recently, nanofiber and nanoscaffold dressings with their enhanced physico-chemical properties and intricate architectures have attracted researchers and gained remarkable attention in several fields such as regenerative medicine, tissue engineering and biomedical applications, wound healing and controlled drug delivery.^{1 –3} The nanosized biomaterials had novel physicochemical criteria which were not found in their bulk forms. Owing to their higher surface area to volume ratio, increased adsorptive capacity, and higher interconnected porosity, they could be easily incorporated and reinforced with other nanoparticles (NPs) and/or active biomaterials of interest. This in fact assisted in attaining various dressing patches with unique architecture and a mosaic of mixed biomaterials to resemble the extracellular matrix morphological structure to be used in various applications.^4,5 Those dressings have great advantages over other conventional dressings prepared by the traditional methods.

Various techniques such as template synthesis, lyophilization, force spinning, solvent casting, melt blowing, 3D printing and electrospinning are used to process nanofibers and to develop dressing patches with nanopolymeric biomaterials.^{6 –11} In electrospinning, an electrostatic force is applied on a polymeric solution with certain viscosity to be ejected from a tiny capillary under particular voltage and humidity to produce fibrous mat with variable diameters and shapes.^{12 –14} Electrospinning is a highly efficient and sophisticated approach which became one of the most promising techniques to prepare scalable, flexible, reproducible and inexpensive ultrafine polymeric nanofibers with higher length-to-diameter ratiosand also higher surface area to volume. Several parameters such as voltage, humidity, polymer concentration, viscosity, and working distance can be manipulated produces nanofibers with varying morphology and diameters.^15,16

Wide varieties of synthetic and natural polymers were used to produce polymeric nanofibrous mats. However, naturally abundant polymers exhibited more desirable characteristics in addition to sustainability, environmentally friendly, and capability of nanoscale processing. For those reasons, natural polymers grabbed remarkable attention to be electrospun into fibers and films. Cellulose is one of those abundant polymers that have excellent desirable properties such as safety, low-cost, biocompatibility, biodegradability, thermal stability and chemical resistance. The main drawback for cellulose polymer is its insolubility in aqueous and several organic solvents and this in particular makes it extremely difficult to be electrospun. Acetylation of cellulose produces cellulose acetate (CA) derivative which is soluble in various organic solutions and can be easily electrospun into mats and dressings which exhibited excellent intrinsic properties in mechanical strength, biocompatibility, high affinity and water absorption capabilities. Those unique characteristics made CA electrospun mats viable and promising candidates for various biomedical applications^{17 –21} Moisture, pore size and porosity of electrospun CA mats can also be managed to prevent microbial penetration while enabling oxygen permeability across the dressing applied on the wound site. CA-based nanofibers with their nanoscale characteristics, porosity and large surface area have complex and intricate architecture that mimics the morphological structure of the extracellular matrix.^15,22 This kind of architecture offers temporal and spatial control and release of bioactive molecules, drugs or NPs of interest that could be either adsorbed on nanofiber surface or encapsulated within the electrospun mat matrix to address various health disorders such as wounds, dermal substitutes, bacterial growth, neural tissue engineering, and several other diseases.^{23 –32}

Diabetes mellitus is considered a metabolic disease which results from prolonged un-control of blood glucose levels. It is characterized by several complications including polyphagia, polydipsia, frequent urination, neuropathy, nephropathy, retinopathy, arterial damage, and delayed wound repair.^33,34 The latter is attributed to impaired angiogenesis and re-epithelialisation processes due to prolonged inflammation, poor blood circulation in addition to excessive neutrophil infiltration. Moreover, it is common for the wound to be infected and colonized by Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. Coli) microbes that hinder wound healing and could increase mortality rates in developing countries. This represents a real medical and economic burden on patients and health care providers. ³⁵ Nevertheless the tremendous advancement and breakthroughs over the past few decades, healing of diabetic wound remains a real challenge.^{36 –38} Lately, several wound dressings were developed from various biomaterials such as nanofiber,^39,40 sponge, ⁴¹ and hydrogels^42,43 not only to cover the wound and protect it from microbial pathogens but also to moisten the wounded area, absorb wound exudates and to expedite wound healing efficiency and tissue regeneration via incorporating antibacterial ingredients.^44,45

Silver has long been used safely as wide spectrum antimicrobial agent against bacteria, fungi and viruses and has been implemented for the treatment of cuts, burns and wounds.^46,47 Its mechanism of action is believed to be through inhibition of ATP synthesis and also through blocking the respiratory chains. ⁴⁸ Presence of silver in nanoparticles (AgNPs) exhibited better antimicrobial activity owing to the gradual release of Ag ions over prolonged time span which enables microbial cell wall disintegration, dimerization of microbial DNA and blocking their respiratory chains leading to microbial death.^{49 –51} Smaller sized AgNPs show greater antimicrobial activity.^52,53 Functional performance of AgNP/nanofiber composites depends on shape, size, spatial distribution of AgNPs on the nanofiber matrix and its content.^54,55

Silver nanoparticles (AgNPs) have gained significant attention due to their unique biological properties, which make them promising candidates in various therapeutic areas such as analgesics, antivirals, anticoagulants, and anticancer agents. Silver nanoparticles exhibit potential analgesic properties due to their ability to reduce inflammation and modulate pain pathways and pro-inflammatory cytokines and oxidative stress. ⁵⁶ AgNPs have also shown considerable promise as antiviral agents due to their ability to interact with viral components and inhibit multiple stages of viral infections. ⁵⁷ Moreover, silver nanoparticles have recently attracted attention owing to their anticoagulant activity in reducing or preventing blood coagulation, strokes and heart attacks. ⁵⁸ AgNPs were also reported to selectively target cancer cells through diverse actions involving oxidative stress, induction of cell cycle arrest, apoptosis, and disruption of mitochondrial function. ⁵⁹

Recently, researchers have been interested in modifying surface properties of nanoparticles such as their shape, size, charge and chemical composition through simple, cost effective and eco-friendly bioengineering processes with attachment of various biomolecules such as peptides, antibodies and drugs. The latter has significantly improved their physicochemical properties, biocompatibility, stability, and targeting capability with the ability to interact more selectively with improvement of overall efficacy in various biological and biomedical applications such as different lines of cancerous cells.^60,61 NPs with their surface coating, charge, energy and size-dependent properties could act as super materials that can be utilized in multi- and fascinating activities. However, a recent concern was raised owing to their high reactivity, higher surface to volume ratio and agglomeration tendency which could make their interactions with biological entities sophisticated, unknown, or dependent on other multifactorial parameters. The latter could pose a real challenge or threat that might have an undesirable or adverse effect. For those particular reasons, NPs have been refereed as double edged swords and it is therefore crucial to comprehensively assess the nongenotoxic and the genotoxic potential of using NPs overwhelmingly in all our daily life activities. ⁶² Although our understanding of the biocompatibility of biosynthesized inorganic nanoparticles remains limited, the concept of biocompatibility is essential when evaluating their potential in biomedical applications. The biocompatibility of nanoparticles can be influenced by several factors, including their size, shape, surface charge, and coating. However, the challenge lies in understanding how these nanoparticles behave in complex biological environments. More comprehensive studies are required to evaluate their long-term effects, distribution, degradation, and potential toxicity in living organisms to ensure they can be safely used in medical treatments. ⁶³

Bioactive glasses (BGs) are ceramics biomaterials that have reactive surface composing mainly of calcium oxide, silicon dioxide, sodium dioxide, and phosphorous. ⁶⁴ As novel biocompatible scaffold biomaterials, BGs have attracted great attention in plenty of biomedical applications such as regenerative medicine, scaffold, and tissue engineering.^65,66 It has been reported that BGs have variable sizes that range from nano to micro scale owing to the highly interconnected pore structure. It is therefore essential to optimize pore architecture and morphology of BGs to manage and fit textural properties for each specific biomedical use. The porosity of BGs can be tailored by controlling reagent concentration, reaction temperature, chemical constituents and also by implementing several mechanical and synthetic strategies such as electro-spinning, 3D scaffold structures, ice-templating, sol-gel foaming and/or foam replication.^{67 –70} Integrating BGs nanostructured biomaterials with various polymer matrices such as Poly(glycerol-sebacate) (PGS) or poly(epsilon caprolactone) (PCL) led to development and design of various composite scaffolds with improved biological and mechanical properties that had been implemented in several biomedical applications such as wound healing, bone remodeling and tissue engineering.^71–73 This could indeed remarkably improve performance in existing biomedical applications with development and design of novel structures for various systems and applications. A previous study has reported that incorporating 3% BGNPs to CA electrospun nanofibers had maximal wound healing effect on diabetic wounded rats with almost a full recovery of wounds within the first 15 days. ²¹

Herein, we prepared a novel electrospun CA nanofibrous dressing/mat fabricated with nanocomposite of 3% BGNPs with different concentrations of AgNPs and investigated its antimicrobial properties and its wound healing capabilities for tissue engineering against streptozotocin induced diabetic rats. We hypothesized that the combination of cellulose acetate electrospun mat with its unique characteristics along with BGNPs and AgNPs loaded nanoparticles might produce better synergistic antibacterial effect against streptozotocin induced diabetic rats. Morphological, structural and biological properties of the fabricated dressing web were intensively evaluated. This study aims at designing a novel dressing mat that could enhance wound healing efficiency in animals/individuals affected with diabetes.

Materials & experimental methods

Materials

Cellulose acetate (CA) used in this study was purchased from Merck (USA). The organic solvents (N,N-Dimethylacetamide and acetone) were obtained from Sisco Research Laboratories (Mumbai, India). All other chemicals were available commercially.

Electrospinning of cellulose acetate polymer fabricated with nanocomposites of BGNPs and AgNPs

Cellulose acetate (CA with average molecular weight 50,000 Da) was dissolved in 1:3 (DMAC/Ac) mixed solvent and stirred for continuous 2 h. After that, three different concentrations of silver nanoparticles (1%, 3%, and 5%) were then mixed individually with CA solution for 2 h using a magnetic stirrer to prepare three different concentrations of Ag loaded CA solutions. To each of the Ag loaded CA solution, 3% BGNP (45% SiO₂, 24.5%CaO, 24.5% Na₂O, and 6% P₂O₅) was added dropwise with continuous stirring until formation of homogenous and clear solutions. For the preparation of BGNPs loaded CA nanofibers (without AgNPs), the same steps mentioned above were follows but without the addition of AgNPs at all. Elecrospinning was carried out using 25 kV DC voltage and 0.3 ml/h flow rate at 22 cm distance where the nanofibers were generated on a rotating drum. The electrospun nanofibrous web was dried in a vacuum oven then kept in the desiccator.

Viscosity and conductivity determination for the electrospun polymer solutions

The intrinsic viscosity (ŋ) and conductivity parameters for the prepared electrospinning solutions were determined using a Brookfield viscometer and a conductivity meter at different shear rates (s⁻¹). Polymer solution viscosity was evaluated at room temperature by a rotation viscometer (Brookfield-DVBT). Polymeric electrical conductivity of prepared with various concentrations of AgNPs were determined using Myron L Ultrameter II, Model 6P.

Physico-chemical characterization of BGNP and AgNP loaded CA nanofibers

Scan electron microscopy (SEM) for surface morphology characterization

Surface morphology and microscopical analysis for the prepared electrospun mat/fiber were evaluated using SEM (Quanta 250 FEG). Samples were electrospinned into mats on aluminum sheet coated with gold layer in sputter coater vacuum machine (S150A Edwards-England) for 200 s. Surface morphology for the prepared samples was scanned at 5–10 kV. ⁶⁶ ImageJ software (https://imagej.nih.gov/ij/download.html) was used to calculate fiber diameters imaged in SEM micrographs.

Fourier transform infra-red spectroscopy (FT-IR)

FTIR instrument (Bruker VERTEX 80 model) combined with Platinum Diamond ATR disk as internal reflector was used to validate and analyze the IR spectra of the prepared samples. Using DTGS (Globar) laser source, different wave number (ν) (4000–400 cm⁻¹) were generated through the interferometer. The background was scanned at certain number of scans (64) with high resolution (4 cm⁻¹) and 2.4 refractive index with nearly 15 min taken to attain clear spectra without interference from carbon dioxide and moisture.

Thermogravimetric analysis (TGA)

Thermal stability and thermal decomposition temperature for the prepared nanofibers (unfabricated CA, BGNPs loaded CA and BGNPs/AgNPs loaded CA) were evaluated using thermagravemitric analysis (TGA). This was operated on TA Instruments Inc (New Castle, DE, USA) with mass loss recording during the heating process from 0°C to 700°C (10°C/min) under nitrogen atmosphere.

EDX for chemical structure characterization

Identification of elemental composition and their quantitative determination within the prepared polymers (unfabricated CA, BGNPs loaded CA and BGNPs/AgNPs loaded CA) were characterized using energy dispersive X-ray (EDX) with an EDX spectrometer (EDX) at a voltage of 20 kV.

In vitro measurement for antimicrobial activity

The agar well diffusion method (Oxoid, UK) was implemented to measure the antimicrobial activities of the fabricated nanofiber mats. This in-vitro assay was conducted to evaluate antifungal and antibacterial properties against various microorganisms including gram positive (Bacillus subtilis, Staphylococcus aureus, and Bacillus cereus) and gram negative bacteria (Escherichia coli and Salmonella tyhimurium). In a laminar flow, a mixture of autoclaved broth and nutrient agar dissolved in distilled water (pH 7.2) was cast into autoclaved petri dishes. The latter was inoculated with 100 colony-forming units of bacteria (A particular microbe type at a time). Afterward, a fabric sample of 300 cm² was aseptically cut into few circles of 5 mm diameter and was then planted onto the on the center of the Mueller Hinton agar (Oxoid, UK). The dish plate was incubated at 37°C for 24 h. Growth free zone around the placed fabric appears as antibacterial agent travels from the fabric spot toward the agar plate periphery. Inhibition zone formation around the placed fabric/mat sample was then calculated based on the AATCC 100 test method. ⁷⁵ The in vitro test for antimicrobial activity was done three times for against each microbe.

In vivo testing of wound healing efficiency

Wound healing capability for the prepared electrospun nanofibers was assessed on Sprague Dawley rats (120–140 g/average weight). Animal experiments were conducted according to the NIH international guidelines for the care and use of Laboratory animals. This study was ethically approved by the animal ethics committee. All rats participating in this study were kept controlled in polypropylene cage at 22°C ± 2°C temperature with 12 h photoperiod and were fed water and standard diet on a daily basis. Prior to the start of the animal experiment, rats were kept for 1 week under observation for adaptation purposes. To induce diabetes mellitus in rats, they were intraperitoneally injected for six consecutive days with streptozotocin (40 mg/kg). Blood glucose levels were measured using (Spectrum, Egypt) respectively to ensure occurrence of diabetes mellitus in rats. As a thumb of rule, rats are labeled diabetic if blood glucose levels exceeded 300 mg/dl for three consecutive days. Rats were then anesthetized generally with ketamine intraperitoneal injection prior to wound induction. Following anesthesia, rat back hair was clipped with a razor and a hair clipper using shaving cream. This was followed by 70% alcohol disinfection to avoid any microbial contamination. In order to obtain a uniform wound with equal depth for all the injured rats, a 9 mm biopsy puncher was applied on the rat dorsal side. After that, rats were then categorized randomly into five groups (three rats each) to overcome any possible biological difference if any. After that, each rat within each group was then punched at two distant locations to create two wound circles in an attempt to minimize any technical differences. The first group acted as a negative group as the created wounds were dressed precisely with unfabricated electrospun CA nanofiber fiber. The second group was covered with CA electrospun nanofiber fabricated with 3% BGNPs. The third, fourth and fifth rat groups were all dressed with CA/3%BGNPs/AgNPs with varying concentrations of AgNPs (1%, 3%, and 5% AgNPs, respectively). To guarantee adequate air exposure and to secure the wound dressing perfectly on the wound sire, an adhesive cover was applied and changed frequently on a daily basis. A digital caliper was then used to measure whether there were changes in in the wound circle diameter for each rat over 9 days of treatment. The following equation was implemented to calculate the reduction in wound area ⁷⁵

$$\mathrm{Wound}\mathrm{area}\%=\left(Wt/Wi\right)\times 100.$$

Where, Wi is the initial wound area are at day 0 and Wt is the wound area at certain time period.

Skin irritation test

Normal unwounded rats were recruited to investigate whether any of the three prepared CA nanofibers (unfabricated, BGNPs and BGNPs/AgNPs loaded mats/dress) would cause skin irritation or would be safe. Each rat had a hair clip in three different sites (one site for each mat/dress). The three produced nanofibers were then topically applied on assigned site of normal unwounded rats for three consecutive days. The following symptoms were closely noticed as skin irritation, erythema, edema, itching, Escher, blisters, and/or scaring.

Statistical analysis

Experimental results were expressed as mean value with standard deviation (mean ± SD). For each set of experiment, there was at least three different replicates/samples. Analysis of variance (one way ANOVA) and Tukey tests were utilized for comparison with p-value ⩽0.05 considered to be significant. SPSS software v-15 (SPSS, USA) and GraphPad Prism software V5 (GraphPad, California, USA) were used to analyze data.

Results and discussion

Preparation and characterization of unfabricated CA nanofibers, CA nanofibers loaded with BGNPs and CA nanofibers loaded with both BGNPs and AgNPs

Cellulose acetate dissolved in DMAC/Ac mixture was used to prepare unfabricated CA solution, 3% BGNPs loaded CA solution and three other solutions of CA each was loaded with 3% BGNPs and either 1%, 2%, or 3% of AgNPs. It is reported that density, electrospun nanofiber diameter and morphology depend on various factors such as electrical conductivity, solution concentration, solution viscosity, dipole moment and surface tension. Moreover, other processing parameters such as electric field strength, flow rate, tip-to-collector distance, temperature, collector composition, air flow and needle shape could greatly influence electrospinning and mat formation. Achieving desirable fiber diameter and morphology requires perfect optimization of all those parameters. In the current study, it was observed that the optimal processing parameters for electrospinning the prepared samples as follows; 0.3 ml/h flow rate using 25 kV DC voltage at 22 cm distance.

All the prepared solutions were chemically and physically characterized prior to evaluating their biological properties. It is well known that solution electrical conductivity and viscosity are the main key players that could affect the electro-spinnability of a solution. For instance, fiber would not be formed for solutions of low viscosity and on the contrary solutions with high viscosity might not be jet ejected. ⁷⁶ On the other side, electrical conductivity is another variable which counteracts viscoelastic force to initiate the eletrospinning process. Higher conductivity can lead to non-continuous fiber formation. ⁷⁷ Therefore optimal viscosity and conductivity would guarantee sufficient viscoelastic force for electrospinning and would hinder polymer breakdown and bead fiber formation. The conductivity, viscosity and nanofiber formation for the produced admixture solutions prepared in this study were first characterized. As seen in Table 1, incorporating 3% BGNPs slightly increased polymer solution viscosity from 425 to 543 cP. This was also accompanied by slight increase in electrical conductivity from 4.7 to 6.59 µS/cm along with fiber diameter increase from 50 to 90 to 100–200 nm. CA solution loaded with 3% BGNPs and 1% AgNPs exhibited remarkable increase in both viscosity and conductivity to be 978 cP and 10.9 µS/cm, respectively with 150–180 nm fiber diameter. The presence of 3% AgNPs resulted in dramatic increase in viscosity and conductivity to be 1685 cP and 16.14 µS/cm, respectively. Increasing AgNPs concentration led to decrease in fiber diameter to be 40–180 nm. Further increase in AgNPs concentration (5%) resulted in slight increase in viscosity and conductivity to be 1842 cP and 42.48 µS/cm, respectively with the same fiber diameter range 40–180 nm. It is worth mentioning that uniformity and homogeneity of nanofiber diameter was observed in all prepared nanofibers as confirmed by SEM data. SEM was carried out to investigate surface morphology of the BGNPs/AgNPs fabricated CA nanofibers (Figure 1). The BGNPs/AgNPs fabricated CA nanofibers were straight, uniform, continuous with smooth and flexible interconnected porous mats with no deformation or beads at all. Incorporation of BGNPs/AgNPs did not exhibit negative effect on the morphology of the fabricated CA nanofibers. Incorporating BGNPs and AgNPs into CA nanofiber did not change the morphology of the fabricated CA nanofiber significantly. It is worth mentioning that the small particle size of BGNPs and AgNPs particles enabled them to be smoothly and successfully incorporated within the wider porous structure of the CA nanofiber scaffold. The incorporated NPs exhibited homogenous and uniform morphological structures. The BGNPs/AgNPs loaded CA nanofibers showed continuous, smooth and clear random oriented beadles morphology. Analyses of images indicated that the majority (52%) of the 3% BGNPs fabricated CA nanofibers had average diameters of 100–200 nm. On the other side, nearly 80% of all the three concentrations of BGNPs-AgNPs fabricated CA nanofiber (1%, 3%, and 5%) had an average fiber diameter of 40–180 nm. The reduction in fiber diameter might be related to improved conductivity of the polymeric solution following incorporation of increasing amount of AgNPs. The increased conductivity might be related to the increased charge density of the solution which improved jet self-repulsion and stretching forces. The 3% BGNPs-5% AgNPs incorporated CA solution exhibited the highest conductivity with the formation of the most finest and spinnable nanofiber nanocomposite and was further selected in biological and chemical investigations. Consequently, it was apparent that all fabricated nanofibers were in nano size and could be suitable for wound dressing applications.

Table 1.

The effects of incorporating 3% BGNPs and various concentrations of AgNPs (1%, 3%, and 5%) on polymer solution conductivity, viscosity, and nanofiber diameter.

Samples	Viscosity (cP)	Conductivity (µS/cm)	Nanofiber diameter (nm)
CA	425 ± 25	4.70 ± 0.4	50–90
CA/3% BGNPs	543 ± 47	6.59 ± 0.6	100–200
CA/3% BGNPs/1%AgNPs	978 ± 48	10.9 ± 0.8	150–180
CA/3% BGNPs/3%AgNPs	1685 ± 54	16.14 ± 1.3	40–180
CA/3% BGNPs/5%AgNPs	1842 ± 73	42.48 ± 1.7	40–180

Figure 1.

SEM and histogram of cellulose acetate (CA) nanofiber containing 3% BGNPs and different concentrations of AgNPs (1%, 3%, and 5%).

FT-IR analysis for unfabricated CA nanofibers, CA nanofibers loaded with BGNPs and CA nanofibers loaded with both BGNPs and AgNPs

FT-IR was carried out to validate chemical structures of the generated nanofibers. FT-IR overlaid spectra for unfabricated CA, BGNPs loaded CA and BGNPs/AgNPs loaded CA nanofibers are displayed in Figure 2 over the 4000-400 cm⁻¹ wave number range. The FT-IR spectrum of pure unfabricated CA nanofiber showed characteristics peaks for the CH₂ symmetric stretching and CH₃ asymmetric stretching at 2852 and 2950 cm⁻¹ bands, respectively (Figure 2). Moreover, bands appearing at 3360 and 1730 cm⁻¹ are due to vibrations around the O-H and the C=O groups. C-H (CH₂) scissoring vibration and C-H (CH₃) bending symmetric were observed at 1463 and 1373 cm⁻¹, respectively. Furthermore, Stretching mode around C-C and C-O backbone for CA polymer along with CH₂ bending were noticed at 1160–1290 cm⁻¹ band range which corresponds to the main CA scaffold structure. The same pattern, without presence of any skewed peaks, was also noticed in BGNPs and BGNPs/AgNPs loaded CA nanofibers. This also indicated that the main backbone composition for the fabricated CA mats (BGNPS and BGNPs/AgNPs) did not change as there was no chemical reactions that resulted from the loading/incorporation process of BGNPs and AgNPs. Addition of BGNPs to CA nanofiber was noticed through the appearance of 2 bands located at 459 and 676 cm⁻¹ which are related to bending vibration of Si-O-Si and Si-O-B, respectively (Figure 2). The characteristic 805 cm⁻¹ band related to the Si-O symmetric stretch of oxygen bridging between tetrahedrons was also noticed. Moreover, the characteristic Si-O- bond in (2NBO) was also observed at 955 cm⁻¹ absorption band. The ~1093 cm⁻¹ absorption band for Si-O bonds in (1NBOS) tetrahedral units appeared in FT-IR spectra. Furthermore, two bands at 560 and 1028 cm⁻¹ were noticed for the P-O bending vibration and the P-O stretching vibration of amorphous calcium phosphate (ACPs), respectively. It is believed that the 546 cm⁻¹ band could be related to the glass structure itself. Additionally, the 1152 cm⁻¹ band was attributed to the P=O stretching vibration (Figure 2). Based on Luz and Mano ⁷⁸ report, the the N=O bending of calcium nitrate precursor showd bands at 1400–1300 cm⁻¹. The FTIR spectrum for AgNPs/BGNPs loaded CA nanofiber exhibited also characteristic peaks for silver nanoparticles at 1631, 1383, and 2927 cm⁻¹. The sharp peak at 1631 cm⁻¹ corresponds to the (NH) C=O stretching vibration meanwhile the 1383 cm⁻¹ band is assigned to the C-C and C-N stretching. The C-H and C-H (methoxy compounds) stretching vibration was observed at 2927 cm⁻¹. It was also noticed that there was a lower peak intensity in the 3360 cm⁻¹ band in AgNPs/BGNPs loaded CA as compared to unfabricated CA nanofiber. This might be due to the reduction of Ag⁺ ions through the hydroxyl groups on the cellulosic structure leading to AgNPs adsorption on the hydroxyl groups of CA structure with reduction in the 3360 cm⁻¹ band intensity. ⁷⁹

Figure 2.

FT-IR spectrum for the electrospun nanofibers of unfabricated CA, 3% BGNPs loaded CA and 3%BGNPs/AGNPs loaded CA nanofibers.

EDX analysis for unfabricated CA nanofibers, CA nanofibers loaded with BGNPs and CA nanofibers loaded with both BGNPs and AgNPs

EDX spectra with qualitative and quantitative elemental composition for BGNPs/AgNPs loaded CA nanofibers are shown in Figure 3. As seen in Figure 3, the EDX of the prepared CA/BGNPs nanofibers with various concentrations of AgNPs (1%, 3%, and 5%) are characterized by various unique peaks for the elements Si, C, O, and Ag indicating the effective incorporation of BGNPs and AgNPs on CA nanofibers. Moreover, new additional peaks were observed that were assigned to Ag-Lα, Ag-Lβ, and Ag-Lβ2 of AgNPs (Figure 3), in addition to the parent CA peaks (C and O). The highest weight percentages was observed for the C element in all CA fabricated nanofibers with 1%, 3%, and 5% of incorporated AgNPs with percentages of 57.59%, 52.59%, and 50.38%, respectively. The second highest weight percentages was observed for the O element in all CA fabricated nanofibers with 1%, 3%, and 5% of incorporated AgNPs with percentages of 36.44%, 28.7%, and 27.7%, respectively. The weight percentages for AgL element incorporation in CA fabricated nanofibers with 1%, 3%, and 5% of incorporated AgNPs were found to be 0.67%, 4.28%, and 4.7% respectively. These results show that the immobilization of BGNPs and AgNPs on the CA surface was successful. Hence, EDX analysis confirmed the presence of BGNPs and AgNPs onto the surface of cellulose substrates.

Figure 3.

EDX spectra for CA electrospun nanofibers loaded with 3% BGNPs and various concentrations of AgNPs (1%, 3%, and 5%).

Thermal stability and decomposition

Thermal stability and thermal decomposition were examined by thermogravimetric analysis (TGA) under nitrogen atmosphere and wide range of temperature (0°C–700°C). Unfabricated CA, BGNPs and BGNPs/AgNPs loaded CA nanofibers were characterized by unique thermal degradation curves (Figure 4). Minor weight loss noticed in all TGA curves as endothermic peaks below 150°C refers to the evaporation and/or desorption water molecules attached to the nanofiber surface of adsorbed moisture. As temperatures increased, there were two distinctive thermal events at different temperature ranges with different mass losses detected in the thermogram curves. Unfabricated CA polymer was found to be highly stable up to 249.5°C with two steps of degradation. The first degradation step was initiated at around 249.5°C with the loss of 12.5% of molar mass. The first degradation event reached maximal weight loss of 82.5% at around 387.5°C where CA chains were ruptured with the release of CO₂ and H₂O. The second degradation event occurred at around 485.5°C with remaining molar mass of 14.5%. Upon increasing the temperature above 485.5°C, the TGA curves became flat indicating that the composite nanofibers had almost burned. At the end of the thermal degradation experiment at 700°C temperature, nearly 86.4% of the CA polymer and its functional groups were completely degraded and the remaining 13.6% of unburned inorganic residues were deposited as ash content on the crucible chamber. Despite the thermal events did not change dramatically upon incorporation of BGNPs or BGNPs/AgNPs mixture to unfabricated CA nanofiber, the thermal stability for the loaded nanocomposites has greatly enhanced with remarkable decrease in thermal decomposition as noticed from the measured residual ash mass (Figure 4). Both BGNPs and BGNPs/AgNPs loaded CA nanofibers remained stable up to 277°C with molar mass loss of 12% and 11.5%, respectively. Moreover, maximal weight losses recorded at the end of the first degradation event at 390°C were 79.5% and 68% for BGNPs and BGNPs/AgNPs loaded CA nanofibers, respectively. The residual ash mass obtained for BGNPs and BGNPs/AgNPs loaded CA nanofibers at 700°C were 14.2% and 23.4%, respectively and this could betaken as an indication for loading the CA polymer with the nanoparticles. TGA curves indicate that fabricating the porous structure of CA nanofibers with either BGNPs alone or BGNPs and AgNPs had significantly reinforced its structure with greater enhancement of its stability leading to remarkable shift in its maximal mass loss toward higher temperatures. As expected, BGNPs and BGNPs/AgNPs loaded CA nanofibers exhibited higher stability with less degradation and decomposition capabilities leading to more residual ash weight as compared to unfabricated CA nanofiber. BGNPs/AgNPs loaded CA nanofibers showed the highest residual ash weight and the highest stability among all investigated nanofibers.

Figure 4.

TGA curves for electrospun nanofibers of unfabricated CA, 3% BGNPs loaded CA and 3%BGNPs/AGNPs loaded CA nanofibers (Black: unfabricated CA; Yellow: 3% BGNPs loaded CA; Blue: 3% BGNPs/AgNPs loaded CA).

Antibacterial activity

Table 2 show the antimicrobial activity of unfabricated CA, BGNPs loaded CA, and BGNPs/AgNPs loaded CA. The antimicrobial activity was evaluated using the agar well diffusion method against wide range of human pathogenic microbes (bacteria and fungi). The microbes tested included Candida albicans 10231, Candida tropicals 750, Sacctaromyces Chevalier 9804, and Aspergillus niger EM77 (KF774181) fungi in addition to Escherichia Coli 8739, Bacillus subtilis 6633, Salmonella tyhemurum 14028, Staphylococcus aureus 25923 bacteria, and Bacillus cereus 33018. Microbial strains were cultured on agar plates, then the prepared nanofibers were placed on that agar plate for 1 day. The inhibition zone which reflects the potency of the antimicrobial activity against tested human pathogens was then determined for each nanofiber against each type of microbe. Table 2 displays the inhibition zones diameters recorded for the explored nanofibers around the disk placed against tested human pathogenic bacteria and fungi. Unfabricated CA nanofiber did not show any kind of antimicrobial activity against all tested microbes. For antifungal activity, the inhibition zone reported for BGNPs loaded CA against Candida albicans 10231 was 25 meanwhile BGNPs/AgNPs loaded CA exhibited higher antifungal activity with increasing AgNPs concentration to be 28, 35, and 41 for 1%, 3%, and 5% AgNPs concentration, respectively. The same antifungal activity pattern was also noticed against Candida tropicals 750 as shown in Table 2 with the highest antifungal activity recorded in treatment with 5% AgNPs concentration. For Sacctaromyces Chevalier 9804, there was no difference between BGNPs and 1% AgNPs/BGNPs preparations as both of them had 17 zone of inhibition. Increasing AgNPs concentration to 3% and 5% led to increase in inhibition zone to be 24 and 29, respectively. BGNPs loaded CA had slight antifungal activity against Aspergillus niger EM77 (KF774181) with an inhibition zone of 15. Incorporation of increasing concentration of AgNPs to the BGNPs loaded CA led to significant increase in its antifungal activity reaching the highest inhibition zone of 27 in case of 5% AgNPs concentration Concerning the antibacterial activity, it was evident that all the fabricated CA nanofiber (BGNPs and BGNPs/AgNPs loaded CA) had better antibacterial activity over the antifungal activity. For instance, antibacterial activity reported for BGNPs loaded CA against Escherichia Coli 8739 exhibited a zone of inhibition of 28. On the other hand, increasing silver nanoparticles concentration remarkably increased the inhibition zone to be 33, 37, and 43 in case of 1%, 3%, and 5% AgNPs concentration. For Salmonella tyhemurum 14028, BGNPs loaded CA showed a zone of inhibition of 29 meanwhile addition of silver nanoparticles increased inhibition zone to 30. About 34 and 39 in case of 1%, 3%, and 5% AgNPs loaded CA. The BGNPs loaded CA nanofiber had antibacterial activity against Bacillus subtilis 6633 & Bacillus cereus 33018 with a zone of inhibition reaching 27 and 24, respectively. BGNPs/AgNPs loaded CA nanofiber showed better antibacterial activity than BGNPs loaded CA with inhibition zone reaching 30, 33, and 37 against Bacillus subtilis 6633 with 1%, 3%, and 5% AgNPs concentrations, respectively. Moreover, the antibacterial activity recorded for BGNPs/AgNPs loaded CA against Bacillus cereus 33018 had inhibition zone of 25, 29, and 35 for 1%, 3%, and 5% AgNPs concentrations, respectively. The highest antimicrobial activity was noticed against Staphylococcus aureus 25923 with zones of inhibition of 32, 34, 37, and 42 for BGNPs loaded CA, 1%, 3%, and 5% AgNPs loaded CA nanofiber. Despite all the fabricated CA nanofibers exhibited antibacterial and antifungal activities, it was evident that the antibacterial activity was much more greater than the antifungal activity.

Table 2.

Antimicrobial (antibacterial and antimycotic) activity of unfabricated CA, 3% BGNPs loaded CA, and 3% BGNPs/AgNPs loaded CA with different concentrations (1%, 3%, and 5%) of AgNPs.

Microorganism	Inhibition zone (mm)
	CA	CA/3%BGNPs	CA/3%BGNPs/1%AgNPs	CA/3%BGNPs/3%AgNPs	CA/3%BGNPs/5%AgNPs
Candida albicans 10231	-	25 ± 1.9	28 ± 2.1	35 ± 1.8	41 ± 2.2
Candida tropicals 750	-	21 ± 1.4	24 ± 1.3	30 ± 1.7	33 ± 1.6
Sacctaromyces Chevalier 9804	-	17 ± 1.2	17 ± 1.4	24 ± 1.3	29 ± 2.3
Aspergillus niger EM77 (KF774181)	-	15 ± 0.9	18 ± 1.1	22 ± 1.4	27 ± 1.5
Escherichia Coli 8739	-	28 ± 1.5	33 ± 1.4	37 ± 1.8	43 ± 2.2
Salmonella tyhemurum 14028	-	29 ± 1.5	30 ± 1.4	34 ± 1.7	39 ± 1.8
Bacillus subtilis 6633	-	27 ± 1.7	30 ± 0.9	33 ± 1.2	37 ± 1.5
Bacillus cereus 33018	-	24 ± 1.3	25 ± 1.2	29 ± 1.1	35 ± 1.7
Staphylococcus aureus 25923	-	32 ± 1.4	34 ± 1.7	37 ± 1.6	42 ± 1.9

Those results indicated that both BGNPs and BGNPs/AgNPs loaded CA nanofibers exhibited promising antimicrobial activity in comparison to unfabricated CA mats. Furthermore, increasing silver nanoparticles concentrations incorporated in the CA nanofiber showed higher antibacterial and antifungal activities with wide range of activity against gram negative (Escherichia coli and Salmonella tyhimurium) and gram positive (Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus) bacteria. The highest antimicrobial activity was noticed in BGNPs/AgNPs incorporated CA nanofiber loaded with 5% AgNPs concentration. It was also obvious that the 5% AgNPs loaded CA nanofiber was highly effective against the most common bacteria that exist in burns and wounds namely Staphylococcus aureus and Escherichia coli.^{80 –83}

The antimicrobial activity reported for BGNPs loaded CA nanofiber might be attributed to the ionic compound release over time once in touch with aqueous medium resulting in increasing the environmental pH and killing of the microbes. ⁸⁴ Moreover, silica release from the bioactive glass into the medium has been associated with antimicrobial activity. ⁸⁵ Several mechanisms have been proposed for the antimicrobial activity caused by the aqueous silver ion (Ag⁺) released from the fabricated nanofiber. One of those mechanisms is attributed to the interference of Ag⁺ cation ion with the electron transport chain through binding to various electron donor groups such as nitrogen, oxygen, phosphorous and sulfur groups which exist on various biological molecules. Another suggested mechanism is through interaction of silver ions with nucleophilic amino acid residues on cell membrane proteins leading to their inactivation/denaturation and cell death. The third mechanism is proposed to be through Ag⁺ cation ion binding to negative charge on DNA molecules which results in disabling cellular replication and cell death. ⁸⁷

Despite development of various antibiotic classes against microbial infections, multi-drug-resistant (MDR) microbes have arisen due to repetitive usage of medications, mistreatment and carelessness. This led to increase in number of nosocomial infections that are resistant to several antibiotics causing a real threat to the global human community.^87,88 The latter is a real challenge as it might lead to a lengthy hospital stay which is critical and have economic burden. Some species causing infections in biomedical environments with MDR include S. aureus, P. aeruginosa, and L. monocytogenes.^{89 –91} BGNPs/AgNPs loaded CA nanofibrous scaffold tested in this study were proven highly effective and extremely promising as antimicrobial agents against various microbe classes including also the most common bacteria that exist in burns and wounds namely Staphylococcus aureus and Escherichia coli.

In vivo wound healing experiment on streptozotocin induced diabetic rats

Diabetes mellitus (DM) is a major health disorder that has acute and chronic complications. The acute complications include hyperglycemia, glucosuria, polyphagia and polydipsia and frequent urination. However chromic complications affect other organs such as eye retina, nerves, kidney nephron and others leading to retinopathy, neuropathy and nephropathy, respectively. DM also leads to cardio- and cerebro-vascular disorders and delayed wound healing capability due to uncontrolled and disrupted blood glucose level management. DM is therefore a multifaceted process that depends on blood circulation, re-epihelialization and angiogenesis. ³⁴ Effective healing of diabetic wounds remains a major and great challenge that needs novel interventions. ³⁶

In a previous study, it was demonstrated that incorporating 3% BGNPs to CA electrospun nanofibers had promising wound healing effect on diabetic wounded rats with almost a full recovery from wounds within the first 15 days. ²¹ In the current study, we prepared a novel electrospun CA nanofibrous dressing/mat fabricated with nanocomposite of the most spinnable and efficient solution of 3% BGNPs and different concentrations of AgNPs (1%, 3% and 5%) and investigated its wound healing capabilities for tissue engineering against streptozotocin induced diabetic rats. Streptozotocin induced diabetic rats of this study (over three successive days of intraperitoneal induction) exhibited extremely high blood glucose levels (exceeding 350 mg/dl).

It was noted that wound created with the 9 mm biopsy puncher induced uniform wounds for all rats on the rat dorsal side with equal depth and without necrosis, infection, or exudates near the wound area (Figure 5). Unfabricated electrospun CA nanofiber was applied to the first rat group. The 3% BGNPs loaded CA electrospun nanofiber mat was applied to the second rat group. The third, fourth, and fifth rat groups were all dressed with CA/3% BGNPs/AgNPs with varying concentrations of AgNPs (1%, 3%, and 5% AgNPs, respectively). To ensure adequate air exposure, the wound dress was changed on a daily basis. Wound healing process was monitored for all recruited rats over 9 days. Figure 5 illustrates the daily progress in the wound healing process over days (1, 3, 6, and 9).

Figure 5.

Effect of unfabricated CA, 3% BGNPs loaded CA, and 3%BGNPs/AgNPs loaded CA with different concentrations (1%, 3%, and 5%) of AgNPs on wound healing for streptozotocin induced diabetic rats over 9 days.

As shown in Figure 5, the wound area for diabetic rat group treated with unfabricated CA wound dress did not exhibit wound healing capability at all over the 9 days of treatment but rather the wound area increased with appearance of some exudative, necrotic and abscess spots starting from Day 6. The wound areas observed for diabetic rat group treated with unfabricated CA on Days 1, 3, 6, and 9 were 0.85, 1.85, 2.75, and 5.25 cm², respectively. On the contrary, rat group treated with BGNPs loaded CA electrospun nanofiber dress had a better relieving effect on wound healing from Day 3 with noticeable decrease in wound diameter by Day 9 without formation of any necrotic, exudative or abscess spots. Diabetic rat group treated with BGNPs loaded CA mat exhibited wound areas of 0.90, 1.35, 1.95, and 1.65 cm² on Days 1, 3, 6, and 9, respectively. Nevertheless, by Day 9 there was still smaller marks left without complete wound closure. On the other hand, addition of AgNPs to BGNPs loaded CA electrospun CA nanofiber dressing had significantly improved and accelerated wound cure and healing (Figure 5) with no exudates, abscess or necrosis. In case of addition of 1% AgNPs to BGNPs/CA nanofiber, wound healing started earlier on Day 1 leaving a wound area of 0.80 cm². There was significant reduction in wound spots by Day 3 and Day 6 with a wound area of 0.65 and 0.55 cm², respectively. On Day 9, there was 100% closure of wound spot. Increasing incorporated AgNPs to 3% to BGNPs/CA nanofiber dressing resulted in even more efficient and faster wound closure reaching 0.75 and 0.60 cm² on Days 3 and 6, respectively with 100% closure on Days 6 and 9. Maximal wound healing and closure was observed in case of 5% AgNPs incorporated with BGNPs/CA nanofiber dress with wound areas of 0.65 and 0.45 cm² on Days 1 and 3, respectively. Absolute wound healing and closure was achieved by the sixth day (Figure 5).

It is also worth mentioning that all the three preparations that contained (1%, 3%, and 5%) AgNPs incorporated with the BGNPs loaded electrospun CA nanofiber dress that were topically applied on the surface of the uninduced rats group did not exhibit any signs or marks of itching, erythema, scars, infection, exudates, blisters, or edema over 3 days (Data not shown). The latter indicates that the prepared nanofiber dressings did not cause any skin irritation and were safe. Therefore, the prepared nanifiber dressing are believed to be biocompatible with skin and fibroblast. Furthermore, the water absorption capability of CA electrospun nanofibers, their ability to interact softly with fibroblast and their biocompatibility are considered excellent properties needed for successful wound healing by novel wound dress. ⁹²

Various polymeric materials such as Poly (ε-caprolactone) (PCL), Poly(lactic-co-glycolic acid) (PLGA), Poly (L-lactic acid) (PLA), Polyvinyl Alcohol (PVA), and Polyvinylpyrrolidone (PVP) have been used successfully either alone or in combination with either herbal extracts or nanoparticles (NPs) to prepare nanofiber membranes for efficient treatment of diabetic wounds. ⁹³ For instance, Polycaprolactone/chitosan (PCL/CH) nanofibers loaded with Cordia myxa fruit extract (CMFE) were reported as effective and biocompatible antimicrobial nanofibrous wound dressing. ⁹⁴ Moreover, wound closure was significantly enhanced in the animal group treated with bromelain loaded silver nanoparticles (Br-AgNP). There was induction of dense fibrous connective tissue in the wound area, which was rich in irregular bundles of collagen fibers and interspersed with numerous fibroblasts. ⁹⁵ Herein, we prepared a novel electrospun CA nanofibrous dressing/mat fabricated with nanocomposites containing 3% BGNPs and different concentrations of AgNPs (1%, 3%, and 5%) and investigated the efficiency of wound healing capabilities against in-vivo streptozotocin induced diabetic rats. It was obvious that addition of AgNPs to BGNPs incorporated electrospun CA nanofber had a promising and effective wound healing activity in diabetic rats. This effect was directly proportional to the increasing percentages of incorporated AgNPs with the maximal and efficient effect noticed in case of 5% AgNPs with complete wound closure by the sixth day of treatment.

This promising effect might be related to the presence of both bioactive bioglass and AgNPs nanoparticles. The former was reported to accelerate granulation growth for the vascularization process and also to promote proliferative activity of fibroblast for wound healing. Moreover, bioactive bioglass had the ability to adsorb several growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in addition to epidermal cells and fibroblast for enhancement of wound closure. ⁹⁶ On the other side, Ag has long been used safely as broad spectrum antimicrobial agent against bacteria, fungi and viruses and has been implemented for the treatment of cuts, wounds and burns.^46,47 Its mechanism of action is thought to be through inhibition of ATP synthesis and also through blocking the respiratory chains. ⁴⁸ Presence of Ag in nanoparticles (AgNPs) form exhibited better antimicrobial activity owing to the gradual release of Ag ions over prolonged time span which enables microbial cell wall disintegration, dimerization of microbial DNA and blocking their respiratory chains leading to microbial death.^{49 –51} Smaller sized AgNPs show greater antimicrobial activity.^52,53 Functional performance of AgNP/nanofiber composites depends on shape, size, spatial distribution of AgNPs on the nanofiber matrix and its content.^54,55

Conclusion

This study reports, for the first time, novel fabrication of CA polymeric solution with BGNPs and AgNPs with different concentrations. Their physicochemical properties, structural, and antimicrobial activities along with biological wound healing capabilities were thoroughly investigated on streptozotocin induced diabetic in vivo rats. Analysis with SEM revealed the formation of uniform, thin and continuous bead free nanofibers with 40–180 nm diameter. TGA analysis also exhibited high thermal stability for the prepared nanocomposites (BGNPs and AgNPs) loaded electrospun CA nanofibers. Moreover, Chemical structure validation the prepared BGNPs/AgNPs loaded electrospun CA nanofibers was supported by both FTIR spectroscopy and EDX elemental analysis as well. The prepared BGNPs/AgNPs loaded electrospun CA showed wide range of antimicrobial activity specifically against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) which are the main bacteria infecting wounds. In vivo study revealed remarkable acceleration in wound healing process with 3% BGNPs and 5% AgNPs combination with maximal efficient wound closure by Day 6 and were found to be biocompatible without inducing any sign for skin irritation symptoms. In conclusion, BGNPs/AgNPs loaded CA electrospun nanofibrous novel dressing had maximal wound healing capability in streptozotocin induced diabetic rats. The powerful antimicrobial activity along with the smooth and uniform morphology for the newly fabricated dress mats could possibly make them cost-effective, eco-friendly and promising patches as for wound healing and tissue engineering purposes.