- Email: [email protected]
PVA Hydrogel Nanocomposites
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 5 (2018) 21314–21321
www.materialstoday.com/proceedings
ICSEM 2016
In vitro Biocompatibility and Antibacterial Activity of Gamma Ray Crosslinked ZnO/PVA Hydrogel Nanocomposites. Swaroop K. and Somashekarappa H. M.* Centre for Application of Radioisotopes and Radiation Technology (CARRT), Mangalore University, Mangalagangotri-574 199, Mangalore, Karnataka, India
Abstract This research work describes the gamma irradiation synthesis of ZnO/PVA HNC’s and their potential application in wound dressing. For synthesizing ZnO/PVA HNC’s, the aqueous solution of PVA and ZnO nanoparticles was exposed to gamma radiations. The prepared composites were characterized using XRD, UV, FTIR and FESEM studies. Swelling characteristics of ZnO/PVA HNC’s showed decrease in the swelling behaviour with increase in ZnO nanoparticles concentration. Samples were examined for their antibacterial potential against E. coli and S. aureus bacteria and found good antibacterial activity on both the bacteria. In vitro cytotoxicity study was evaluated on Human Fibrosarcoma cells (HT-1080) using SRB assay and demonstrated good biocompatibility. Our results suggest that, ZnO/PVA HNC’s synthesised using gamma irradiation could be an ideal biomaterial for wound dressing applications. © 2018 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON SMART ENGINEERING MATERIALS (ICSEM 2016). Keywords: Gamma irradiation; Zinc oxide; Hydrogel; Wound dressing; Cytotoxicity.
1. Introduction Hydrogels are the cross-linked polymer structures which swell when brought in contact with solvent [1, 2]. Because of covalently cross-linked network, the material does not dissolve in solvent but holds it within the structure intact [3]. Hydrogels have gained interest by the research community because of its application in drug release systems, tissue engineering, and wound dressing [1, 4, 5]. Any biomaterial to be proposed for contact with living
* Corresponding author. Tel.: +91-824-2284545; fax: +91-824-2287367. E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON SMART ENGINEERING MATERIALS (ICSEM 2016).
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21315
organisms has to be non-toxic, sterilized, functionalized, and biocompatible [5, 6]. One of the prime functions of a wound dressing material is to provide a moist environment and prevent the harmful bacteria to the wound exudates [7, 8]. Polyvinyl alcohol (PVA) is a well known biocompatible material with optical transparency, gel forming ability, low toxicity and biodegradability [9, 10]. Transition metal oxide like zinc oxide (ZnO) possesses antimicrobial activity due to stimulation of reactive oxygen specious with the cell which results in the damages the cell membrane [11]. The combination with PVA and ZnO in a hydrogel can be used for tissue engineering, biosensing, bio-actuators, diagnostic, and wound dressing applications [12]. The specific objective of this study is to develop ZnO/PVA hydrogel nanocomposites (HNC’s) using gamma irradiation technique for possible biomedical applications like wound dressing. The antibacterial potential of ZnO/PVA HNC’s was studied against Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacteria. In vitro cytotoxicity studies were performed on Human Fibrosarcoma cells (HT-1080) cells. 2. Experimental 2.1. Materials Zinc acetate dihydrate (Zn(CH3COO)2.2H2O) was purchased from Central Drug House, Delhi. PVA with an average molecular weight of 130,000 (Degree of hydrolysis: 99+ %) was procured form Sigma Aldrich, USA. Double distilled water (DDW) (Resistivity: >5 MΩ. cm @ 250 C) was used for synthesis of ZnO NP’s and ZnO/PVA HNC’s and for all other experiments. The gamma irradiation was done using Gamma Irradiator (GC 5000; BRIT, Mumbai) equipped with 60Co source which delivers a dose rate of 5.7 kGy/hr. 2.2. Synthesis of ZnO nanoparticles Co-precipitation method was adopted for synthesising ZnO NP’s. A solution of 0.1 M Zn(CH3COO)2.2H2O was prepared in DDW. Sodium hydroxide (NaOH) of 1 M concentration was added to the Zn(CH3COO)2.2H2O solution under constant stirring at room temperature. It results in a white precipitate which was allowed to settle down and washed several times with DDW and dried at 1100 C until dried compactly. The dried sample was then calcined at 5000 C for 3 hours. The resulted ZnO powder was further used for the synthesis of ZnO/PVA HNC’s. 2.3. Synthesis of ZnO/PVA hydrogel nanocomposites Aqueous solution of PVA was obtained by dissolving 5 gm of PVA in 100 ml DDW at 800 C under constant mechanical stirring until PVA completely dissolved in water. Four different weight concentration of ZnO namely, 0 % (pure PVA), 1 % (PZ1), 2% (PZ2), and 3% (PZ3) was added to four replicates of 5 % PVA aqueous solution separately. All the four solutions were sonicated for 15 min for uniform distribution of ZnO followed by bubbleing with argon gas for 15 min to remove the O2 content in the solution. These solutions were poured into glass containers separately and exposed to gamma irradiation of 25 kGy dose under ambient conditions. The HNC’s obtained were dried at room temperature and taken for further characterization. 2.4. Characterization Fourier Transform Infrared spectroscopy (FTIR) (Shimardzu, Prestige-21) data were recorded over a wavenumber range of 400 to 4000 cm-1 at room temperature for identifying the functional groups associated with the samples. Microstructural characterization was carried out using powder X-ray diffraction analysis (XRD) (Rigaku Miniflex X-ray diffractometer). This technique was used for identification of PVA and ZnO crystallite phases, for calculating ZnO crystallite size and crystallinity of ZnO/PVA HNC’s. The surface morphology of the ZnO/PVA HNC’s was characterized using Field Emission Scanning Electron Microscopy (FESEM) (ULTRA 55 FESEM, Karl Zeiss, Germany).
21316
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
2.5. Gel content The net gel content of the HNC’s were estimated by extracting the un-dissolved fraction of gel. The prepared HNC’s were immersed in hot DDW at 1000 C for 24 hours and dried at 400 C until it reaches constant weight. The percentage gel content (%GC) was calculated using equation 1. %GC
Wd X 100 Wi
…… (1)
Where ‘Wd’ is the dried weight after extraction and ‘Wi’ is the initial weight of the HNC’s. 2.6. Swelling studies The completely dry weight of HNC’s sample was recorded as ‘Wi’. The sample was immersed in the DDW at room temperature. The HNC’s were weighed at every predetermined time intervals after removal of excess surface water with filter paper (Wt). The samples were periodically weighed until the sample reaches equilibrium. The swelling degree of the HNC’s were calculated using equation 2.
%S 2.7. Antibacterial activity
Wt Wi X 100 Wi
..... (2)
Antibacterial activity of ZnO/PVA HNC’s in its equilibrium swollen state was evaluated against gram negative E. coli and gram positive S. aureus bacteria using well diffusion method. ZnO/PVA HNC’s were cut in cylindrical disks of 6 mm. Nutrient agar of ~10 ml was poured into the sterilized plates and allowed to solidify. Six mm diameter wells were punched to the agar media using sterilized cork borer followed by inoculation of spore suspension of E. coli and S. aureus. The samples were loaded to the wells of the culture plates and incubated at 370 C for 24 h. After the incubation period, the inhibition zone appeared around the samples was recorded. The experiment was repeated thrice and the average value of the inhibition zone appeared around the sample was considered as antibacterial activity of the samples.
2.8. In vitro cytotoxicity studies In vitro cytotoxicity evaluation of ZnO/PVA HNC’s was assessed to Human Fibrosarcoma cells (HT-1080) using Sulforhodamine B (SRB) assay. HT-1080 cells were grown in RPMI 1640 medium containing 10 % bovine serum and 2 mM L-glutamine. The cells were seeded in 96 well plates and incubated at 370 C, 5 % CO2, 95 % air and 100 % relative humidity for 24 hours prior to addition of the ZnO/PVA HNC’s. Extracts of the sample was solubilised in DMSO and added in four different concentration namely, 10 μg/ml, 20 μg/ml, 40 μg/ml, and 80 μg/ml to the appropriate wells and incubated at standard conditions for 48 hours. The assay was terminated by addition of 30 % (w/v) TCA and incubated for 60 min at 40 C. After washing the plates 50 µl of SRB solution was added to each of the wells and incubated for 20 min at room temperature. After staining, bound stains were subsequently removed with trizma base followed by the spectrometric determination of absorbance at a wavelength of 540 nm in a plate reader. Percentage growth of inhibition of the cells was calculated using equation (3).
%GI
Ti X 100 C
….. (3)
Where, ‘Ti’ test growth in the presence of drug at the four concentration levels and ‘C’ is control growth.
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21317
3. Results and discussion The gamma irradiation crosslinking of PVA in its aqueous state is well known. Interaction of gamma radiation with PVA aqueous solution will leads to the production of free radicals like hydroxyl radicals (OH·) and hydrogen atoms (H·) as a major fraction of energy absorbed by the solvent. The presence of OH· and H· will lead to the removal of hydrogen atom from the main polymer chain to form PVA macro-radical. The reaction of these macroradicles will leads to the formation of PVA crosslinking to form PVA network [13]. Since the ZnO nanoparticles were well dispersed at the time of irradiation the ZnO NP’s will stay intact within the PVA network. Figure (1) shows the FTIR spectra of ZnO/PVA HNC’s. PVA exhibits several characteristic bands, which has shown considerable differences in the peaks with addition of ZnO NP’s to the PVA matrix. FTIR spectra of PVA shows a absorption band appeared at ~3340 cm-1 is attributed to O-H stretching vibrations; at ~2918 and ~2852 is attributed to C-H symmetric stretching vibrations; at ~1641 cm-1 corresponds to C=C stretching vibration; at ~1388 and ~1344 cm-1 was assigned to the combination frequencies of CH-OH; at 1091 cm-1 relates to the stretching vibration of C-O group. A narrow peak appeared at 1145 cm-1 (at shoulder of 1091 cm-1) indicates the occurrence of crystalline phase of PVA. With addition of ZnO NP’s, PVA peak has showed shift in the O-H stretching vibration and decrease in the intensity of C-O stretching vibrations due to defects induced by ZnO. New peaks observed at ~1537 cm-1 and 459 cm-1 are due to C=O stretching (corresponding to metal acetate bonding) and Zn-O stretching vibrations respectively. ZnO/PVA composites has shown increase in the intensity of the 1145 cm-1 peak indicating increase in the crystalline phase of the composites with increase in concentration of ZnO [14].
Fig. 1. FTIR spectra of ZnO/PVA HNC’s.
XRD analysis of ZnO NP’s and ZnO/PVA HNC’s within the scan range of 100 to 800 2θ value was shown in figure (2). XRD spectrum of ZnO nanoparticles prepared by co-precipitation method (figure 2 (a)) shows the bragg’s diffraction plane at 2θ = 31.68, 34.04, 35.5, 48.12, 56.76, 63.52, 65.92, and 68.4 represents the hexagonal wurtzite structure of ZnO NP’s (ICPDS card No.89.1397). The ZnO/PVA HNC’s has shown (figure 2 (b)) diffraction peak at 200 representing the intermolecular hydrogen bonding of PVA along with the ZnO crystallite plane confirming the ZnO NP’s in PVA matrix. The crystallite size of the ZnO NP’s was calculated using DebyeSchrrer equation and found to be ~26 nm. The intensity of PVA crystalline plane has shown increase in the intensity with addition of ZnO NP’s indicating increase in crystallinity of ZnO/PVA HNC’s. XRD results pertaining crystallinity of ZnO/PVA HNC’s is well in agreement with the FTIR data which also shows the increment in crystallinity with ZnO NP’s concentration.
21318
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
Fig 2. XRD diffraction pattern of (a) ZnO NP’s and (b) ZnO/PVA HNC’s.
FESEM analysis was carried out to observe the morphological changes on PVA films occurred due to addition of ZnO NP’s (figure (3)). A clear and uniform morphology was observed for pure PVA hydrogel (figure 3(a)). ZnO/PVA HNC’s showed a well dispersed ZnO NP’s all along the PVA matrix (figure 3(b)). The magnified view of ZnO/PVA HNC’s (figure 3(c)) illustrates the aggregation of ZnO NP’s on the surface of PVA matrix. a
b
c
Fig 3. FESEM images of (a) pure PVA hydrogel, (b) ZnO/PVA HNC’s, and (c) magnified view of ZnO/PVA HNC’s.
Gel fraction of a hydrogel material describes the degree of cross-linking between the polymeric chains. Gel fraction of a ZnO/PVA HNC’s has shown decrement with increase in ZnO NP’s concentration. The gel fraction of pure PVA hydrogel was 86 % and tend to decrease up to 69 % for higher concentration of ZnO NP’s. The swelling properties of ZnO/PVA HNC’s were studied in water medium at room temperature for 24 hours. The swelling degree of a hydrogel attains equilibrium when the hydration force and elastic force of the cross-linking saturates. Equilibrium degree of swelling (% EDS) of ZnO/PVA HNC’s were observed after one day of immersion. The plot of ‘% S’ versus ‘time’ is shown in figure (4). According to the results obtained, it could be seen that the pure PVA hydrogel possess high swelling degree and decreases significantly with addition of ZnO NP’s. This is may be due to defects induced by ZnO NP’s to the hydrogel matrix. Swelling behaviour of hydrogel material is an important aspect for many bio-applications. Though the results show decrease in the swelling capability of ZnO/PVA HNC’s with addition of ZnO NP’s, EDS of ~300 % is sufficient for its applicability in wound dressing [15].
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21319
Fig 4. Swelling ratio of ZnO/PVA HNC’s in water medium at room temperature.
The antibacterial activity of HNC’s containing ZnO NP’s presented significant inhibition against E. coli and S. aureus bacteria (figure 5). Pure PVA hydrogels did not show antibacterial activity against both bacteria. Higher inhibition zone was observed for sample with high ZnO concentration for both the bacteria. ZnO has already been proven as a good antibacterial agent against large spectrum of gram positive and gram negative bactericides. The possible mode of cell death is may be due to the direct or electrostatic interaction between ZnO and cell surface may damage the membrane. The obtained results suggest that the antibacterial activity of ZnO/PVA HNC’s is mainly due to the presence of ZnO nanoparticles in the PVA matrix.
Fig 5. Antibacterial screening of ZnO/PVA HNC’s against E. coli and S. aureus bacteria.
Human Fibrosarcoma cells were exposed to pure PVA and PZ3 HNC’s extracts for 24 h and cell response are compiled in figure (6). The control growth percentage was calculated and tabulated in table 1. The cell density of HT-1080 cells after contact with pure PVA and PZ3 extracts remained same for all the concentration. The control growth percentage value of the positive control in considerably lower than the sample. Figure (7) shows the morphology of cells cultured in negative control, positive control, pure PVA and PZ3 extracts. The cell density of
Fig 6. Cytotoxicity evaluation of pure PVA, PZ3, and positive control samples on HT-1080 cells.
21320
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
the positive control was considerably lower than the sample, which are circular in shape. The cells exposed to negative control, pure PVA and PZ3 extracts appeared to show normal morphology and high membrane integrity. Since the studied parameters of cytotoxic evaluation of extracts of PVA and PZ3 samples were similar and showed nearly 100 % control growth, ZnO/PVA HNC’s are considered to be non-toxic.
HT-1080 PVA PZ3 ADR
Drug concentrations (µg/ml) calculated from graph LC50 TGI GI50* NE NE >80 NE NE >80 <10 <10 <10
Table 1. Material toxicity data by SRB assay.
Figure 7: Morphologies of HT-1080 cells cultured in extracts (a) negative control, (b) positive control, (c) pure PVA hydrogel, and (d) PZ3 HNC.
4. Conclusion Biocompatibility and antibacterial applications of ZnO/PVA HNC’s synthesized using gamma irradiation was studied. Gamma irradiation technique was proven to be a clean and effective way for the synthesis of hydrogel samples. FTIR, XRD analysis confirms the presence of ZnO NP’s in PVA matrix. FESEM images show well dispersed ZnO NP’s on the PVA surface. Though the swelling capacity of the samples decreased from ~650 % to ~265 % with increase in ZnO NP’s concentration, it is sufficient for wound dressing applications. In vitro antibacterial activity studies of ZnO/PVA HNC’s and found to be excellent in inhibiting the growth of E. coli and S. aureus bactericides. In vitro cttotoxicity studies show no toxic effect on Human Fibrosarcoma cells. Hence we conclude that the prepared ZnO/PVA HNC’s are potential candidates for biomedical applications like wound dressing. Acknowledgements Authors are thankful to the Coordinator, DST-PURSE program, Mangalore University for permitting to use FESEM facility.
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21321
References [1] A.S. Hoffman, Adv. Drug Deliv. Rev. 64 (2012) 18–23. [2] M. Kokabi, M. Sirousazar, Z.M. Hassan, Eur. Polym. J. 43 (2007) 773–781. [3] A. Martínez-Ruvalcaba, J.C. Sánchez-Díaz, F. Becerra, L.E. Cruz-Barba, A. González-Álvarez, Express Polym. Lett. 3 (2009) 25–32. [4] K.H. Seo, S.J. You, H.J. Chun, C.H. Kim, W.K. Lee, Y.M. Lim, Tissue Eng. Regen. Med. 6 (2009) 414–418. [5] L. Varshney, Nucl. Instruments Methods Phys. Res. Sect. B Beam Interact. with Mater. Atoms. 255 (2007) 343–349. [6] T. Enrica, Hydrogels: Biological Properties and Applications, 2009. [7] S. Kurhade, M. Momin, P. Khanekar, S. Mhatre, Int. J. Drug Deliv. 5 (2013) 353–361. [8] J. Dutta, Am. J. Chem. 2 (2012) 6–11. [9] R.N. Oliveira, G.B. McGuinness, R. Rouze, B. Quilty, P. Cahill, G.D.A. Soares, R.M.S.M. Thire, Interface Focus, 123(2015) 1-11. [10] Y.C. Nho, T.H. Kim, K.R. Park, Radiat. Phys. Chem. 69 (2004) 351–353. [11] J.W. Rasmussen, E. Martinez, P. Louka, D.G. Wingett, NIH Public Accsess. 7 (2011) 1063–1077. [12] A.K. Gaharwar, N.A. Peppas, A. Khademhosseini, Biotechnol. Bioeng. 111 (2014) 441–453. [13] S. Francis, L. Varshney, Radiat. Phys. Chem. 74 (2005) 310–316. [14] O.N. Tretnnikov, N.I. Sushko, S.S. Zagorskaya, Poly. Sci. 55(2013) 91-97. [15] H.L. Abd El-Mohdy, J. Polym. Res. 20 (2013) 1-12.
ScienceDirect Materials Today: Proceedings 5 (2018) 21314–21321
www.materialstoday.com/proceedings
ICSEM 2016
In vitro Biocompatibility and Antibacterial Activity of Gamma Ray Crosslinked ZnO/PVA Hydrogel Nanocomposites. Swaroop K. and Somashekarappa H. M.* Centre for Application of Radioisotopes and Radiation Technology (CARRT), Mangalore University, Mangalagangotri-574 199, Mangalore, Karnataka, India
Abstract This research work describes the gamma irradiation synthesis of ZnO/PVA HNC’s and their potential application in wound dressing. For synthesizing ZnO/PVA HNC’s, the aqueous solution of PVA and ZnO nanoparticles was exposed to gamma radiations. The prepared composites were characterized using XRD, UV, FTIR and FESEM studies. Swelling characteristics of ZnO/PVA HNC’s showed decrease in the swelling behaviour with increase in ZnO nanoparticles concentration. Samples were examined for their antibacterial potential against E. coli and S. aureus bacteria and found good antibacterial activity on both the bacteria. In vitro cytotoxicity study was evaluated on Human Fibrosarcoma cells (HT-1080) using SRB assay and demonstrated good biocompatibility. Our results suggest that, ZnO/PVA HNC’s synthesised using gamma irradiation could be an ideal biomaterial for wound dressing applications. © 2018 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON SMART ENGINEERING MATERIALS (ICSEM 2016). Keywords: Gamma irradiation; Zinc oxide; Hydrogel; Wound dressing; Cytotoxicity.
1. Introduction Hydrogels are the cross-linked polymer structures which swell when brought in contact with solvent [1, 2]. Because of covalently cross-linked network, the material does not dissolve in solvent but holds it within the structure intact [3]. Hydrogels have gained interest by the research community because of its application in drug release systems, tissue engineering, and wound dressing [1, 4, 5]. Any biomaterial to be proposed for contact with living
* Corresponding author. Tel.: +91-824-2284545; fax: +91-824-2287367. E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON SMART ENGINEERING MATERIALS (ICSEM 2016).
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21315
organisms has to be non-toxic, sterilized, functionalized, and biocompatible [5, 6]. One of the prime functions of a wound dressing material is to provide a moist environment and prevent the harmful bacteria to the wound exudates [7, 8]. Polyvinyl alcohol (PVA) is a well known biocompatible material with optical transparency, gel forming ability, low toxicity and biodegradability [9, 10]. Transition metal oxide like zinc oxide (ZnO) possesses antimicrobial activity due to stimulation of reactive oxygen specious with the cell which results in the damages the cell membrane [11]. The combination with PVA and ZnO in a hydrogel can be used for tissue engineering, biosensing, bio-actuators, diagnostic, and wound dressing applications [12]. The specific objective of this study is to develop ZnO/PVA hydrogel nanocomposites (HNC’s) using gamma irradiation technique for possible biomedical applications like wound dressing. The antibacterial potential of ZnO/PVA HNC’s was studied against Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacteria. In vitro cytotoxicity studies were performed on Human Fibrosarcoma cells (HT-1080) cells. 2. Experimental 2.1. Materials Zinc acetate dihydrate (Zn(CH3COO)2.2H2O) was purchased from Central Drug House, Delhi. PVA with an average molecular weight of 130,000 (Degree of hydrolysis: 99+ %) was procured form Sigma Aldrich, USA. Double distilled water (DDW) (Resistivity: >5 MΩ. cm @ 250 C) was used for synthesis of ZnO NP’s and ZnO/PVA HNC’s and for all other experiments. The gamma irradiation was done using Gamma Irradiator (GC 5000; BRIT, Mumbai) equipped with 60Co source which delivers a dose rate of 5.7 kGy/hr. 2.2. Synthesis of ZnO nanoparticles Co-precipitation method was adopted for synthesising ZnO NP’s. A solution of 0.1 M Zn(CH3COO)2.2H2O was prepared in DDW. Sodium hydroxide (NaOH) of 1 M concentration was added to the Zn(CH3COO)2.2H2O solution under constant stirring at room temperature. It results in a white precipitate which was allowed to settle down and washed several times with DDW and dried at 1100 C until dried compactly. The dried sample was then calcined at 5000 C for 3 hours. The resulted ZnO powder was further used for the synthesis of ZnO/PVA HNC’s. 2.3. Synthesis of ZnO/PVA hydrogel nanocomposites Aqueous solution of PVA was obtained by dissolving 5 gm of PVA in 100 ml DDW at 800 C under constant mechanical stirring until PVA completely dissolved in water. Four different weight concentration of ZnO namely, 0 % (pure PVA), 1 % (PZ1), 2% (PZ2), and 3% (PZ3) was added to four replicates of 5 % PVA aqueous solution separately. All the four solutions were sonicated for 15 min for uniform distribution of ZnO followed by bubbleing with argon gas for 15 min to remove the O2 content in the solution. These solutions were poured into glass containers separately and exposed to gamma irradiation of 25 kGy dose under ambient conditions. The HNC’s obtained were dried at room temperature and taken for further characterization. 2.4. Characterization Fourier Transform Infrared spectroscopy (FTIR) (Shimardzu, Prestige-21) data were recorded over a wavenumber range of 400 to 4000 cm-1 at room temperature for identifying the functional groups associated with the samples. Microstructural characterization was carried out using powder X-ray diffraction analysis (XRD) (Rigaku Miniflex X-ray diffractometer). This technique was used for identification of PVA and ZnO crystallite phases, for calculating ZnO crystallite size and crystallinity of ZnO/PVA HNC’s. The surface morphology of the ZnO/PVA HNC’s was characterized using Field Emission Scanning Electron Microscopy (FESEM) (ULTRA 55 FESEM, Karl Zeiss, Germany).
21316
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
2.5. Gel content The net gel content of the HNC’s were estimated by extracting the un-dissolved fraction of gel. The prepared HNC’s were immersed in hot DDW at 1000 C for 24 hours and dried at 400 C until it reaches constant weight. The percentage gel content (%GC) was calculated using equation 1. %GC
Wd X 100 Wi
…… (1)
Where ‘Wd’ is the dried weight after extraction and ‘Wi’ is the initial weight of the HNC’s. 2.6. Swelling studies The completely dry weight of HNC’s sample was recorded as ‘Wi’. The sample was immersed in the DDW at room temperature. The HNC’s were weighed at every predetermined time intervals after removal of excess surface water with filter paper (Wt). The samples were periodically weighed until the sample reaches equilibrium. The swelling degree of the HNC’s were calculated using equation 2.
%S 2.7. Antibacterial activity
Wt Wi X 100 Wi
..... (2)
Antibacterial activity of ZnO/PVA HNC’s in its equilibrium swollen state was evaluated against gram negative E. coli and gram positive S. aureus bacteria using well diffusion method. ZnO/PVA HNC’s were cut in cylindrical disks of 6 mm. Nutrient agar of ~10 ml was poured into the sterilized plates and allowed to solidify. Six mm diameter wells were punched to the agar media using sterilized cork borer followed by inoculation of spore suspension of E. coli and S. aureus. The samples were loaded to the wells of the culture plates and incubated at 370 C for 24 h. After the incubation period, the inhibition zone appeared around the samples was recorded. The experiment was repeated thrice and the average value of the inhibition zone appeared around the sample was considered as antibacterial activity of the samples.
2.8. In vitro cytotoxicity studies In vitro cytotoxicity evaluation of ZnO/PVA HNC’s was assessed to Human Fibrosarcoma cells (HT-1080) using Sulforhodamine B (SRB) assay. HT-1080 cells were grown in RPMI 1640 medium containing 10 % bovine serum and 2 mM L-glutamine. The cells were seeded in 96 well plates and incubated at 370 C, 5 % CO2, 95 % air and 100 % relative humidity for 24 hours prior to addition of the ZnO/PVA HNC’s. Extracts of the sample was solubilised in DMSO and added in four different concentration namely, 10 μg/ml, 20 μg/ml, 40 μg/ml, and 80 μg/ml to the appropriate wells and incubated at standard conditions for 48 hours. The assay was terminated by addition of 30 % (w/v) TCA and incubated for 60 min at 40 C. After washing the plates 50 µl of SRB solution was added to each of the wells and incubated for 20 min at room temperature. After staining, bound stains were subsequently removed with trizma base followed by the spectrometric determination of absorbance at a wavelength of 540 nm in a plate reader. Percentage growth of inhibition of the cells was calculated using equation (3).
%GI
Ti X 100 C
….. (3)
Where, ‘Ti’ test growth in the presence of drug at the four concentration levels and ‘C’ is control growth.
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21317
3. Results and discussion The gamma irradiation crosslinking of PVA in its aqueous state is well known. Interaction of gamma radiation with PVA aqueous solution will leads to the production of free radicals like hydroxyl radicals (OH·) and hydrogen atoms (H·) as a major fraction of energy absorbed by the solvent. The presence of OH· and H· will lead to the removal of hydrogen atom from the main polymer chain to form PVA macro-radical. The reaction of these macroradicles will leads to the formation of PVA crosslinking to form PVA network [13]. Since the ZnO nanoparticles were well dispersed at the time of irradiation the ZnO NP’s will stay intact within the PVA network. Figure (1) shows the FTIR spectra of ZnO/PVA HNC’s. PVA exhibits several characteristic bands, which has shown considerable differences in the peaks with addition of ZnO NP’s to the PVA matrix. FTIR spectra of PVA shows a absorption band appeared at ~3340 cm-1 is attributed to O-H stretching vibrations; at ~2918 and ~2852 is attributed to C-H symmetric stretching vibrations; at ~1641 cm-1 corresponds to C=C stretching vibration; at ~1388 and ~1344 cm-1 was assigned to the combination frequencies of CH-OH; at 1091 cm-1 relates to the stretching vibration of C-O group. A narrow peak appeared at 1145 cm-1 (at shoulder of 1091 cm-1) indicates the occurrence of crystalline phase of PVA. With addition of ZnO NP’s, PVA peak has showed shift in the O-H stretching vibration and decrease in the intensity of C-O stretching vibrations due to defects induced by ZnO. New peaks observed at ~1537 cm-1 and 459 cm-1 are due to C=O stretching (corresponding to metal acetate bonding) and Zn-O stretching vibrations respectively. ZnO/PVA composites has shown increase in the intensity of the 1145 cm-1 peak indicating increase in the crystalline phase of the composites with increase in concentration of ZnO [14].
Fig. 1. FTIR spectra of ZnO/PVA HNC’s.
XRD analysis of ZnO NP’s and ZnO/PVA HNC’s within the scan range of 100 to 800 2θ value was shown in figure (2). XRD spectrum of ZnO nanoparticles prepared by co-precipitation method (figure 2 (a)) shows the bragg’s diffraction plane at 2θ = 31.68, 34.04, 35.5, 48.12, 56.76, 63.52, 65.92, and 68.4 represents the hexagonal wurtzite structure of ZnO NP’s (ICPDS card No.89.1397). The ZnO/PVA HNC’s has shown (figure 2 (b)) diffraction peak at 200 representing the intermolecular hydrogen bonding of PVA along with the ZnO crystallite plane confirming the ZnO NP’s in PVA matrix. The crystallite size of the ZnO NP’s was calculated using DebyeSchrrer equation and found to be ~26 nm. The intensity of PVA crystalline plane has shown increase in the intensity with addition of ZnO NP’s indicating increase in crystallinity of ZnO/PVA HNC’s. XRD results pertaining crystallinity of ZnO/PVA HNC’s is well in agreement with the FTIR data which also shows the increment in crystallinity with ZnO NP’s concentration.
21318
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
Fig 2. XRD diffraction pattern of (a) ZnO NP’s and (b) ZnO/PVA HNC’s.
FESEM analysis was carried out to observe the morphological changes on PVA films occurred due to addition of ZnO NP’s (figure (3)). A clear and uniform morphology was observed for pure PVA hydrogel (figure 3(a)). ZnO/PVA HNC’s showed a well dispersed ZnO NP’s all along the PVA matrix (figure 3(b)). The magnified view of ZnO/PVA HNC’s (figure 3(c)) illustrates the aggregation of ZnO NP’s on the surface of PVA matrix. a
b
c
Fig 3. FESEM images of (a) pure PVA hydrogel, (b) ZnO/PVA HNC’s, and (c) magnified view of ZnO/PVA HNC’s.
Gel fraction of a hydrogel material describes the degree of cross-linking between the polymeric chains. Gel fraction of a ZnO/PVA HNC’s has shown decrement with increase in ZnO NP’s concentration. The gel fraction of pure PVA hydrogel was 86 % and tend to decrease up to 69 % for higher concentration of ZnO NP’s. The swelling properties of ZnO/PVA HNC’s were studied in water medium at room temperature for 24 hours. The swelling degree of a hydrogel attains equilibrium when the hydration force and elastic force of the cross-linking saturates. Equilibrium degree of swelling (% EDS) of ZnO/PVA HNC’s were observed after one day of immersion. The plot of ‘% S’ versus ‘time’ is shown in figure (4). According to the results obtained, it could be seen that the pure PVA hydrogel possess high swelling degree and decreases significantly with addition of ZnO NP’s. This is may be due to defects induced by ZnO NP’s to the hydrogel matrix. Swelling behaviour of hydrogel material is an important aspect for many bio-applications. Though the results show decrease in the swelling capability of ZnO/PVA HNC’s with addition of ZnO NP’s, EDS of ~300 % is sufficient for its applicability in wound dressing [15].
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21319
Fig 4. Swelling ratio of ZnO/PVA HNC’s in water medium at room temperature.
The antibacterial activity of HNC’s containing ZnO NP’s presented significant inhibition against E. coli and S. aureus bacteria (figure 5). Pure PVA hydrogels did not show antibacterial activity against both bacteria. Higher inhibition zone was observed for sample with high ZnO concentration for both the bacteria. ZnO has already been proven as a good antibacterial agent against large spectrum of gram positive and gram negative bactericides. The possible mode of cell death is may be due to the direct or electrostatic interaction between ZnO and cell surface may damage the membrane. The obtained results suggest that the antibacterial activity of ZnO/PVA HNC’s is mainly due to the presence of ZnO nanoparticles in the PVA matrix.
Fig 5. Antibacterial screening of ZnO/PVA HNC’s against E. coli and S. aureus bacteria.
Human Fibrosarcoma cells were exposed to pure PVA and PZ3 HNC’s extracts for 24 h and cell response are compiled in figure (6). The control growth percentage was calculated and tabulated in table 1. The cell density of HT-1080 cells after contact with pure PVA and PZ3 extracts remained same for all the concentration. The control growth percentage value of the positive control in considerably lower than the sample. Figure (7) shows the morphology of cells cultured in negative control, positive control, pure PVA and PZ3 extracts. The cell density of
Fig 6. Cytotoxicity evaluation of pure PVA, PZ3, and positive control samples on HT-1080 cells.
21320
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
the positive control was considerably lower than the sample, which are circular in shape. The cells exposed to negative control, pure PVA and PZ3 extracts appeared to show normal morphology and high membrane integrity. Since the studied parameters of cytotoxic evaluation of extracts of PVA and PZ3 samples were similar and showed nearly 100 % control growth, ZnO/PVA HNC’s are considered to be non-toxic.
HT-1080 PVA PZ3 ADR
Drug concentrations (µg/ml) calculated from graph LC50 TGI GI50* NE NE >80 NE NE >80 <10 <10 <10
Table 1. Material toxicity data by SRB assay.
Figure 7: Morphologies of HT-1080 cells cultured in extracts (a) negative control, (b) positive control, (c) pure PVA hydrogel, and (d) PZ3 HNC.
4. Conclusion Biocompatibility and antibacterial applications of ZnO/PVA HNC’s synthesized using gamma irradiation was studied. Gamma irradiation technique was proven to be a clean and effective way for the synthesis of hydrogel samples. FTIR, XRD analysis confirms the presence of ZnO NP’s in PVA matrix. FESEM images show well dispersed ZnO NP’s on the PVA surface. Though the swelling capacity of the samples decreased from ~650 % to ~265 % with increase in ZnO NP’s concentration, it is sufficient for wound dressing applications. In vitro antibacterial activity studies of ZnO/PVA HNC’s and found to be excellent in inhibiting the growth of E. coli and S. aureus bactericides. In vitro cttotoxicity studies show no toxic effect on Human Fibrosarcoma cells. Hence we conclude that the prepared ZnO/PVA HNC’s are potential candidates for biomedical applications like wound dressing. Acknowledgements Authors are thankful to the Coordinator, DST-PURSE program, Mangalore University for permitting to use FESEM facility.
Swaroop et.al / Materials Today: Proceedings 5 (2018) 21314–21321
21321
References [1] A.S. Hoffman, Adv. Drug Deliv. Rev. 64 (2012) 18–23. [2] M. Kokabi, M. Sirousazar, Z.M. Hassan, Eur. Polym. J. 43 (2007) 773–781. [3] A. Martínez-Ruvalcaba, J.C. Sánchez-Díaz, F. Becerra, L.E. Cruz-Barba, A. González-Álvarez, Express Polym. Lett. 3 (2009) 25–32. [4] K.H. Seo, S.J. You, H.J. Chun, C.H. Kim, W.K. Lee, Y.M. Lim, Tissue Eng. Regen. Med. 6 (2009) 414–418. [5] L. Varshney, Nucl. Instruments Methods Phys. Res. Sect. B Beam Interact. with Mater. Atoms. 255 (2007) 343–349. [6] T. Enrica, Hydrogels: Biological Properties and Applications, 2009. [7] S. Kurhade, M. Momin, P. Khanekar, S. Mhatre, Int. J. Drug Deliv. 5 (2013) 353–361. [8] J. Dutta, Am. J. Chem. 2 (2012) 6–11. [9] R.N. Oliveira, G.B. McGuinness, R. Rouze, B. Quilty, P. Cahill, G.D.A. Soares, R.M.S.M. Thire, Interface Focus, 123(2015) 1-11. [10] Y.C. Nho, T.H. Kim, K.R. Park, Radiat. Phys. Chem. 69 (2004) 351–353. [11] J.W. Rasmussen, E. Martinez, P. Louka, D.G. Wingett, NIH Public Accsess. 7 (2011) 1063–1077. [12] A.K. Gaharwar, N.A. Peppas, A. Khademhosseini, Biotechnol. Bioeng. 111 (2014) 441–453. [13] S. Francis, L. Varshney, Radiat. Phys. Chem. 74 (2005) 310–316. [14] O.N. Tretnnikov, N.I. Sushko, S.S. Zagorskaya, Poly. Sci. 55(2013) 91-97. [15] H.L. Abd El-Mohdy, J. Polym. Res. 20 (2013) 1-12.