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Silk fibroin coated with poly 2-hydroxyethyl methacrylate impregnated with silver nanoparticles coupled with ciprofloxacin as a biomaterial for biomedical applications.

Introduction

Nanofibers are porous and have high specific surface area which has resulted in significant interest for various biomedical applications. Silk fiber produced by the silkworm Bombyx mori possesses excellent mechanical properties [1]. Recently, silkworm is being used as biofactory for the production of useful protein using silk gland, which has promoted the technological development in sericulture [2]. Silk protein is proteinous in nature and can be digested by the action of proteolytic enzymes present in body. i.e., it is biodegradable and this property makes it a biocompatible material [3]. The use of silk proteins as bandage materials, surgical sutures is due to its better mechanical properties such as flexibility and tensile strength. Moreover, the good biocompatibility and the ability to resist infection have made it a novel wound coagulant material [4]. Silk fibroin is an FDA approved product used in many medical applications such as surgical sutures, drug delivery and tendon tissue engineering [5, 6]. Moreover, silk fibroin finds its use as a smart structural fabric for a range of applications and a variety of composites which impart magnetic and electrical properties [7].

Nanoparticles have a special role in targeted drug delivery [8]. Silver nanoparticles has bactericidal properties and has low toxicity towards human cells, this has resulted in the usage of silver in various forms and is ideally suited for consumer and medical applications [9, 10]. Hence, it is important to modify, manipulate and fabricate the materials at nano scale for their use in medical and technological applications. Specifically, the materials used in medical applications should be non toxic and biocompatible besides being active agents for particular purpose [11]. It is well documented that silver nanoparticles has strong inhibitory bactericidal effects and exhibit a broad spectrum of antimicrobial activities for bacteria, fungi and virus [12]. Antibacterial activity of the materials which contain silver nanoparticles has been utilized in medicine to reduce infections as well as to prevent bacterial colonization on prostheses, catheters, vascular grafts, dental materials and human skin [13, 14].

Hydrogels are three-dimensional, cross-linked networks of water soluble polymers such as Poly 2-hydroxyethyl Methacrylate which enables them to absorb large amount of water. The swelling properties of pHEMA are mainly related to the elasticity of the network, the presence of hydrophilic functional groups in the polymer chains, the extent of cross linking and porosity of the polymer [15]. Their porosity permits loading of drugs into the gel matrix and subsequently drug is released at a rate dependent on the diffusion coefficient of the small molecule or macromolecule through the gel network [16].

Therefore, the present investigation describes fabrication and characterization of silk fibroin fibers coated with Poly 2-hydroxyethyl Methacrylate, impregnated with silver nanoparticles coupled with ciprofloxacin as biomaterial for wound healing and tendon reconstruction purposes. Moreover, the antimicrobial activities of the biomaterial against both gram positive and negative bacterial strains were performed. Furthermore, biocompatibility of the biomaterial was conducted on 3T3 fibroblast cell lines. A complete representation of this study is shown in the graphical abstract.

Experimental section

Bombyx mori silk cocoons were kindly supplied by department of sericulture, Govt. of Andrapradesh, India. Fibroblast 3T3 cell line was purchased from (NCCS, Pune), Pune, india, poly (2-Hydroxy ethyl metha acrylate), sodium meta bisulphate, potassium persulphate, silver sulfate, sodium borohydrate, PBS 10%, Dulbecco's Modified Eagle Medium (DMEM) containing serum was purchased from Sigma-Aldrich. Muller Hinton medium was purchased from Hi-media Laboratories Pvt. Ltd, Mumbai, India. All other chemicals used were of analytical grade.

Isolation of Silk Fibers from the Cocoons of Silk Worm (SF)

Raw silk cocoons obtained were boiled at 55[degrees]C for one hour until the color of the water turns golden yellow in color, the color change indicates that the sericin gum is dissolved in water [17]. Later the cocoons containing silk fibroin fibers were cooled and dried at room temperature. The cocoons were cut into discs (0.5cm in diameter) and individual fibers were also removed manually (SF) and stored in glass container till further use.

Preparation of pHEMA

The procedure followed to prepare pHEMA from its monomer is based on a free radical initiated polymerization reaction. Sodium meta bisulphate (0.15 g) and potassium persulphate (0.15 g) were dissolved in 100 mL of distilled water and heated upto 55[degrees]C for 10 mins. To this solution 10 mL of monomer HEMA was added and stirred continuously for 30 mins. The jelly like compound attains white color which was filtered using nylon mesh followed by repeated washing in distilled water and air dried. The obtained pHEMA crystals (~1 g) were dissolved in acetone: water in the 80:20 ratio followed by continuous stirring for 20 mins to ensure the completion of the reaction [18].

Preparation of Silver Nanoparticles

Silver nanoparticles were prepared using a modified procedure previously described in the literature [19]. In brief; 20 mL of aqueous silver sulfate solution (1*10[conjunction]4) was stirred for 1 h. To this solution, 4 mL of 1M sodium borohydrate dissolved in 0.3M NaOH was added dropwise with stirring until the golden brown color is obtained. At this stage the addition of sodium borohydrate was stopped but the stirring process was continued for another 20 mins in ultra sonicater to ensure the complete formation of AgNPs. This solution containing AgNPs was stored at 10[degrees]C till further use.

Coating of pHEMA onto SF (SF-P)

1 g of dried pHEMA was dissolved in 10 ml of acetone water mixture (80:20) solution. This solution was stirred vigorously for 15 mins invortex mixer till the solution attains the milky white color. The prepared individual fibers and discs (SF) were treated in dissolved pHEMA solution for 1 h and air dried at 30[degrees]C (SF-P).

Coating Silver Nanoparticles into SF-P (SF-P-Ag)

10 mL of AgNPs solution was taken in a petridish and the prepared SF-P individual fibers and discs were treated in the solution for 1 h. Later, the samples (SF-P-Ag) were dried at room temperature (30[+ or -]2[degrees]C) for 2 h and stored in polythene cover for further analysis.

Incorporation of drug into SF-P-Ag (SF-P-Ag-D)

2 mg of ciprofloxacin was dissolved in 50 mL of water and SF-P-Ag individual fibers and discs were treated in this solution for 1 h. Later the sample was dried at room temperature (30[+ or -]2[degrees]C) for 2 h. The dried samples of SF and SF-P-Ag-D were sterilized for 4 h at 45[degrees]C with 100% ethylene oxide (ETO) and stored in polythene covers till further analysis.

Characterization

The prepared biomaterials were characterized for their FITR, UV, SEM, EDX, TGA, Anti-bacterial screening and in vitro biocompatibility. The UV-VIS absorption spectra of the samples were recorded on a Jasco UV-VIS-V530 spectrometer with a spectral resolution of 2 nm. IR spectroscopy of samples was taken using Nicollet impact 400 FTIR spectroscopy by preparing a 500mg KBr pellet containing 2-6mg of the sample. SEM measurements were carried out on a Leica stereo scan-440 scanning electron microscope equipped with phoenix EDX attachment. The EDX spectrum was recorded in the spot profile mode by focusing the electron beam onto the specific regions of the bio-composites. The thermal stability of the samples was determined with a thermo gravimetric (TG) analyzer (Perkin-Elmer TGA) over a temperature range of 37[degrees]C to 585[degrees]C at a heating rate of 20[degrees]C / min under nitrogen atmosphere. To find out the antibacterial properties of the samples, the bacterial cells were cultured aerobically in 25 mL of nutrient broth at 37[degrees]C for 24 hrs by disc diffusion method. Nutrient agar for bacteria plates were inoculated with 5ml of an appropriate dilution of the tested cultures Escherichia coli (A.T.C.C. 8739) and Bacillus subtilis (A.T.C.C. 6633). The SF disc (0.5cm) alone was used in the control plate. The SF-P-Ag, SF-P-Ag-D discs (each 0.5cm) were tested to establish the antibacterial activity of silver particles and drug present in the discs. The fabricated discs were placed on the surface of the culture plates in aseptic condition. Then the plates were incubated for 24 hrs at 37[degrees]C. The diameter of inhibition zone cm including the disc diameter was measured in the culture plates[20].

In vitro study

Cell proliferation MTT assay was performed according to the earlier procedure [21]. Briefly, SF control and SF-P-Ag-D composites were cut out with punch (0.5cm in diameter) and put onto the 98-well culture plates (MTT). The discs were coated with 300 ml/well of 3T3 fibroblast cells, in PBS by overnight adsorption at room temperature, the wells were then washed with PBS buffer and cells were detached by trypsin digestion, the cells were allowed to settle/adhere for 1hr at 37[degrees]C in an atmosphere of 5% C[O.sub.2] incubator. The wells were washed once with PBS, and the remaining bound cells were fixed with 10% formalin in PBS for 15mins. The fixative was aspirated; the wells were washed twice with PBS. The discs were examined under Phase contrast microscope. To ensure a representative count, each disc sample was divided into

quarters. Average percentages and standard deviations were calculated from three independent experiments.

Results and Discussion

Normally, in clinics, hospitals and diagnostic laboratories patients often acquire infections associated with bacteria and viruses present on the surfaces of medical devices and other equipments. Therefore, there is a need and high demand for biomaterials which has antibacterial agents on its surface. Nanoparticle based agents applied as coatings have shown promise in delivering the improved sterility against variety of microbes. Hence, the present study attempts at fabricating a biomaterial where the silk fibers have AgNPs and ciprofloxacin drug on its surface to provide antibacterial property required for clinical wound healing applications.

UV Spectroscopy

The UV-Vis absorption maxima of prepared AgNPs dispersed in the sodium chloride solution to avoid agglomeration of the nanoparticles was obtained in the visible range at 401 nm (Fig. 1). The observed absorption band confirms the formation of silver nanoparticles and is in agreement with that reported in the literature [22].

Infra Red Spectroscopy

FTIR spectra of SF, SF-P, SF-P-Ag are shown in Figure 2. In the IR spectrum of SF (Fig. 2a), the peaks observed at 1637 and 1550 [cm.sup.-1] are attributed to amide I and amide II bands of protein respectively. Broad hydroxyl peak is observed from 3100 to 3500 [cm.sup.-1] and -C[H.sub.2] stretching vibration band is noticed at 2923 [cm.sup.-1]. This spectrum reveals the protein nature of the sample. In the IR spectrum of SF-P (Fig. 2b), a sharp band at around 1723 [cm.sup.-1] representing carbonyl peak (-CO[O.sup.-]) in addition to the bands corresponding to SF is observed. This confirms the successful coating of pHEMA on the SF. The spectrum of SF-P-Ag (Fig. 2c) shows the shifting of amide and carboxyl bands to 1652 and 1710 [cm.sup.-1] respectively. This shift in the bands is probably due to the presence of silver nanoparticles in the biomaterial [23].

Scanning Electron Microscope

SEM images of SF, SF-P, SF-P-Ag are depicted in Fig. 3. Smooth surface can be observed in SEM images of SF (Fig. 3a) and the diameter of the fiber is around 20.5 [micro]m. Coating of PHEMA is clearly seen on the surface of SF (Fig. 3b) and diameter of the fiber is around 30 [micro]m, the increase in diameter of the fiber is due to the coating of PHEMA. Incorporation of AgNPs on the SF-P is observed in Fig. 3c and diameter of SF-P-Ag fiber is found to be 35 [micro]m. The EDX spectrum of SF-P-Ag (Fig. 3d) clearly shows the signal of silver atoms which confirms the presence of AgNPs in the nanocomposite [24].

Thermo Gravimetric Analysis

Thermograms of SF, SF-P and SF-P-Ag are illustrated in Fig. 4. For SF sample exhibited (Fig. 4a), an initial weight loss of 11% till 237[degrees]C which could be attributed to the loss of water, both in free as well as in bound form. At higher temperatures, a two step weight loss of 45% at 237-339[degrees]C and 16% at 339-443[degrees]C respectively was noticed. The weight loss observed at 237-339[degrees]C is assigned to the denaturation of beta sheet into random coil structure of silk protein. The second weight loss is attributed to the degradation of the denatured products into C[O.sub.2], NOx and [H.sub.2]O. In the sample SF-P (Fig. 4b), four step weight losses was observed from 235 to 450[degrees]C. The first weight loss (9%) upto 235[degrees]C is due to the loss of water and bound water. The second and third weight losses of 11% each are the result of denaturation of the protein coil structure and its degradation respectively. The final weight loss of 24% is ascribed to the decomposition of PHEMApolymer. Similar pattern was observed in SF-P-Ag (Fig. 4c). However, in this sample, 28.43% residue was still present at 522[degrees]C. The presence of AgNPs in the sample results in increased thermal stability and might be beneficial in specific applications such as biosensors.

Anti-Bacterial Screening

The anti-bacterial property of the biomaterial has been studied on two different bacterial strains: Escherichia coli and Bacillus subtilis using disc diffusion technique and are illustrated in Fig. 5. In control SF no zone of inhibition is observed Fig. 5a (i), & 5b (1). The diameter of inhibition zones around each disc with different samples are represented in table 1. In the plate of Escherichia coli, 6 mm diameter zone of resistance was observed, in SF-P-Ag disc Fig. 5a (ii), the zone diameter has increased to 13 mm in the case of SF-D Fig. 5c. The highest antibacterial activity was observed in SF-P-Ag-D disc which shows a zone of 24 mm Fig. 5a (iii). The plate containing Bacillus subtilis shows 11 mm diameter zone in SF-P-Ag disc Fig. 5b (iii) and the zone diameter has increased to 16 mm in the case of SF- D disc (Fig. 5d). 21 mm zone of inhibition was observed in the SF-P-Ag-D disc Figure 5b (iii). The enhanced antibacterial effects could be attributed to the presence of AgNPs and the ciprofloxacin drug in the composite. The positively charged silver atoms adhere to bacterial cell walls because of the overall charge on the cell surface at biological pH is negative. These oppositely charged electrostatic interactions might be the reason for the bactericidal effect of AgNPs to enter into bacteria, inhibit the ATP synthesis, denature DNA and blocking the respiratory chain. These studies indicate that the material could be utilized for the repair of infected defective sites, in skin and tendons [20].

In vitro study

MTT assay was carried out to assess the biocompatibility of 3T3 fibroblasts in presence of SF used as control and SF-P-Ag-D biocomposite as scaffolds (Fig. 6a, b and c). Hincal et al. in his study has stated that ciprofloxacin increased cell viability in human fibroblasts at lower concentrations [24]. The results of our study revealed an increase in percentage of cell viability (69.3%) in presence of SF-P-Ag-D discs, when compared with 52.4% for SF discs. This increase in cell viability due to the effects of ciprofloxacin incorporated in the scaffolds is in agreement with that reported by Hincal et al [25].

Conclusion

In this work, the development of a biomaterial of silk fibroin coated with Poly 2-hydroxyethyl Methacrylate impregnated with silver nanoparticles has been described. The biomaterial prepared has exhibited enhanced antibacterial properties against both Gram positive (Bacillus subtilis) and Gram negative (Escherichia coli) bacterial strains. Also, better cell viability was observed in SF-P-Ag-D (69.3%) due to the presence of AgNPs and the drug, ciprofloxacin incorporated in the biocomposite compared to the control SF (52.4%) disc. Hence, it could be clearly stated that SF-P-Ag-D discs might be an ideal candidate as biomaterial for biomedical purposes especially in wound dressing and tissue engineering applications.

Acknowledgements

Dr.S.Sekar would like to thanks CSIR for funding. The authors would like to thank Dr. Krishna Kumar and Saranya, JRF, Sankaranethralya, Chennai, India, their help in in vitro studies.

References

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Santhanam Sekar, Ramasamy Manikandan, Samickanu Sankar, Thotapalli Parvathaleswara Sastry *

Bioproducts Lab, Central Leather Research Institute, Adyar, Chennai 20, Tamil Nadu, India.

* Corresponding Author: T.P. Sastry, sastrytp@hotmail.com

Received 15 July 2014; Accepted 5 December 2014; Available online 10 December 2014

Table 1: Antimicrobial activities of the biomaterial

              Zone of Inhibition (mm)

S.No         Strain         Control (SF)

1            E.coli              Nil
2       Bacillus subtilis        Nil

            Zone of Inhibition (mm)

S.No    SF-P-Ag    SF-P-Ag-D     SF-D

1         6 mm       24 mm      13 mm
2        11 mm       21 mm      16 mm
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Title Annotation:Original Article
Author:Sekar, Santhanam; Manikandan, Ramasamy; Sankar, Samickanu; Sastry, Thotapalli Parvathaleswara
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Oct 1, 2014
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