1. 1. 1 PREPARATION AND CHARACTERIZATION OF BANDAGE GAUZE USING COLLAGEN/ALGINATE INCORPORATED WITH GRAPHENE OXIDE. R.Pujitha Lakshmi 310811214034 B.Tech Biotechology BT2452 INTERNAL GUIDE Dr.J.Helan Chandra, Professor, Dept. of Biotechnology, Jeppiaar Engineering College, Chennai-600119. EXTERNAL GUIDE Dr.T.P.Sastry, Head, Principal Scientist, Bio-Products Laboratory, CLRI, Adyar, Chennai-600025.
2. 2. INTRODUCTION Wound dressings before the 1960s were considered to be only the so-called passive products having a minimal role in the healing process . Though much effect has been focused, it still remains a challenging clinical problem, so correct and efficient wound management is essential. An ideal wound dressing should be biocompatible , stable and prevent bacterial infection (Brett D, 2008). Collagen, constituting one third of the bodily proteins acts as a effective ingredient in wound healing by restoring scar tissues (Lee et al, 2001). Graphene oxide has excellent mechanical strength, thermal strength and electrical conductivity thereby promotes cell viability (Ying et al, 2011). Alginate is an excellent absorber and helps in faster healing by proving moisture around the wound area (Thomas, 2000). 2
3. 3. AIM : To prepare and characterize bandage gauze using Collagen/Alginate incorporated with Graphene oxide. OBJECTIVES : 1. To extract collagen from Chrome Containing Leather Wastes. 2. To synthesize graphene oxide using modified hummers method (2010). 3. To optimize collagen and alginate for film formation. 4. Preparation of an optimized film followed by analysis of its physicochemical properties. 5. To optimize collagen, alginate, graphene oxide and prepare a wound dressing material (bandage gauze). 3
4. 4. Synthesis of GO using modified Hummers method Optimization of Collagen and Alginate ratio Preparation of a film using the optimized ratio Analyzing the Physicochemical properties Fabrication of final Collagen/Alginate/Go gauze Physicochemical characteristics and in vitro study of the gauze Extraction of Collagen from CCLW O V E R A L L W O R K P L A N 4
5. 5. 100g of CCLW. Soak in 100 ml of 5% NaOH solution for 24 hours. Wash until pH 7. Treat with concentrated sulfuric acid (10 to 15ml) for 15-20 minutes. Wash until pH 7. Add 100 ml of hydrogen peroxide solution . (Collagen is the supernatant). Store at 4C. 1. Extraction of Collagen- (Sastry et al, 2014)
6. 6. 1. COLLAGEN EXTRACTION (a) CCLW (b) Soaked in NaOH (f) Collagen (c) Dechromed with H2SO4 (d) Washed with water until pH 7 (e) Addition of H2O2 6
7. 7. 7 Characterization results of collagen 230 KDa 150 KDa 100 KDa 215 KDa
8. 8. 2. Synthesis of Graphene oxide- (Leila et al, Modified Hummers Method, 2010) Add 1g of graphite and 1g of NaNO3 in 46ml of concentrated sulfuric acid (00C) and stirred in ice bath for 4 hrs. Add 6g of KMnO4 in an ice bath with continuous stirring for 2 hrs. The temperature should be maintained below 350c for 35 minutes. Add 92ml of distilled water slowly and raise the temperature to 98oC. After that add 200ml distilled water and 20ml of 30% of H2O2 and left overnight. Collect the residue and wash with distilled water, 5% HCl and acetone by repeated centrifugation at 6000rpm. Dry the residue at 500C overnight. 8
9. 9. 2. SYNTHESIS OF GO USING MODIFIED HUMMERS METHOD (a) 1g of graphite and 1 g NaNO3 (b) 46 ml of H2SO4 (c) 6g of KMNO4 (d) 200mL distilled water and 20ml of 30% of H2O2 (e) Centrifuge at 6000rpm and wash with H2O, 5% HCl and acetone (f) Graphene oxide9 92 ml of H20
10. 10. Characterization results of Graphene oxide FT-IRFTRAMAN SEMTGA 10
11. 11. 3. Optimization of Collagen and Alginate Different ratios of collagen and alginate were tried by varying one another : 1.Volume in ml (1,2,3,4,5), (0.1,0.2,0.3,0.4,0.5) 2.Concentration in % (1,2,3,4,5), (0.1,0.2,0.3,0.4,0.5) In the process of optimization of collagen for electro spinning after varying the ratios and incubation which did not yield satisfactory results, the end process of washing the wells used for the experiment gave us an unexpected outcome of a transparent film. INFERENCE OF THE TRIALAND ERROR : Varying the volume of alginate resulted in an improper mixture and varying the volume of collagen resulted in a gel like substance. It was found that increasing the concentration of alginate increased the viscosity and the role of collagen got reduced due to more amount of alginate. In case of film, varying alginate concentration gave better results of thin, flexible and a strong film. In the vice versa case, increasing the collagen concentration stiffened the film. 11
12. 12. 1 2 3 4 5 0.1 0.1:1 0.1:2 0.1:3 0.1:4 0.1:5 0.2 0.2:1 0.2:2 0.2:3 0.2:4 0.2:5 0.3 0.3:1 0.3:2 0.3:3 0.3:4 0.3:5 0.4 0.4:1 0.4:2 0.4:3 0.4:4 0.5:5 0.5 0.5:1 0.5:2 0.5:3 0.5:4 0.5:5 1 2 3 4 5 1 1:1 1:2 1:3 1:4 1:5 2 2:1 2:2 2:3 2:4 2:5 3 3:1 3:2 3:3 3:4 3:5 4 4:1 4:2 4:3 4:4 4:5 5 5:1 5:2 5:3 5:4 5:5 COLLAGEN VS ALGINATE Conc. Const C O L L A G E N ALGINATE C O L L A G E N ALGINATE ml ml ml ml 12
13. 13. COLLAGEN VS ALGINATE Volume Constant 1 2 3 4 5 0.1 0.1:1 0.1:2 0.1:3 0.1:4 0.1:5 0.2 0.2:1 0.2:2 0.2:3 0.2:4 0.2:5 0.3 0.3:1 0.3:2 0.3:3 0.3:4 0.3:5 0.4 0.4:1 0.4:2 0.4:3 0.4:4 0.5:5 0.5 0.5:1 0.5:2 0.5:3 0.5:4 0.5:5 1 2 3 4 5 1 1:1 1:2 1:3 1:4 1:5 2 2:1 2:2 2:3 2:4 2:5 3 3:1 3:2 3:3 3:4 3:5 4 4:1 4:2 4:3 4:4 4:5 5 5:1 5:2 5:3 5:4 5:5 C O L L A G E N C O L L A G E N % % % ALGINATE ALGINATE 13
14. 14. 4. Preparation of collagen alginate film-(Deepachitra et al, 2014) 25g of Collagen 10g of Alginate Made to 200ml Mixed well using stirrer 2ml of Ethylene glycol Stirred for 15mins Made into a film 14
15. 15. Characterization results of Col/Al film Tensile strength TGA Sample no. C:A Elongation break (%) Tensile strength (MPa) 1 C:A (1:0.1) 36.28 21.92 2 C:A (1:0.3) 39.98 19.83 3 C:A (1:0.5) 23.65 13.42 4 C:A (2:0.1) 19.25 6.75 5 C:A (2:0.3) 28.25 20.42 6 C:A (2:0.5) 22.98 8.33 7 C:A (2:0.7) 14.31 6.08 8 C:A (2.5:1) 45.55 11.58 9 C:A (1:1) 38.05 21.17 10 A:C (1:0.1) 1.88 1.92 11 A:C (1:0.3) 29.50 17.67 12 A:C (1:0.5) 36.16 18.33 13 A:C (1:0.7) 8.26 10.2515
16. 16. The ratio obtained was again varied with either collagen or alginate constant at a time. The following ratios were tried: Collagen : Alginate (1:0.1, 1:0.3, 1:0.5, 1:1) Alginate : Collagen(1:0.1, 1:0.3, 1:0.5, 1:1) Among the above mentioned ratios, Collagen : Alginate (1:0.3) was proven to have the best properties. Sample no. Collagen Alginate GO 1 1 0.3 0.1 2 1 0.3 0.3 3 1 0.3 0.5 4 1 0.3 0.7 5 1 0.3 1 5. Optimization of Collagen/Alginate and Graphene oxide for preparation of wound dressing material. 16
17. 17. Characterization results of Col/Al/GO film FT-IR TGA SEM 17
18. 18. Tensile strength Antibacterial activity Sample no. C:Al:GO Elongation break (%) Tensile strength (MPa) 1 1:0.3:0.1 37.80 26.83 2 1:0.3:0.3 45.77 28.50 3 1:0.3:0.5 27.05 24.00 4 1:0.3:0.7 20.89 28.50 5 1:0.3:1 13.08 8.17 E.coli S.aureus18
19. 19. Water uptake ability Sample no. C:Al:GO Before water uptake (mg) After water uptake (24hrs) (mg) 1 1:0.3:0.1 3.5 21.8 2 1:0.3:0.3 3.5 44.9 3 1:0.3:0.5 3.5 25.4 4 1:0.3:0.7 3.5 25 5 1:0.3:1 3.5 22.5 0 5 10 15 20 25 30 35 40 45 50 1 2 3 4 5 Before water uptake (mg) After water uptake (24hrs) (mg) 1)1:0.3:0.1 2)1:0.3:0.3 3)1:0.3:0.5 4)1:0.3:0.7 5)1:0.3:119
20. 20. Collagen-Alginate Film Collagen /Alginate Bandage gauze Collagen/Alginate/GO bandage gauze 20
21. 21. 21 CONCLUSION The goal of the project is to prepare bandage gauze with properties like biodegradability, biocompatibility, good gelling properties, high mechanical strength, non toxicity and thermally insulating capacity. Both collagen and alginate proved to be biodegradable, biocompatible, good tensile strength, water absorbing capacity and they help in faster wound healing. In this scaffold, a novel material (Graphene oxide) was incorporated into bandage gauze made of collagen/alginate. In vitro and In vivo studies would confirm its compatibility with human beings.
22. 22. REFERENCES 1. Ying Wang, Zhaohui Li, Jun Wang, Jinghong Li and Yuehe Lin (2011). Graphene and GO: biofunctionalization and applications in biotechnolog. Trends in Biotechnology, Vol. 29, No. 5, pp: 205-212. 2. Compton. O.C, Nguyen. T, Small, 2010, Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials ,Volume 6, 711723. 3. Chi Lee. H , Anuj Singla , Yug yung Lee ,2001, Biomedical applications of collagen, International Journal of Pharmaceutics 221, Vol.122. 4. Ramani, Deepachitra, Varadarajan, Ramnath, 2014, Graphene oxide incorporated collagen-fibrin bioflim as a wound dressing material, RSC Advances, Vol 107. 5. Biji Balakrishnan, M. Mohanty, P.R. Umashankar, A. Jayakrishnan(2005). Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials 26. pp 63356342. 6. Brett D (2008). A review of collagen and collagen based wound dressings. Wounds 20(12). pp: 347-53. 7. Daniela C. Marcano, Dmitry V. Kosynkin, Jacob M. Berlin, Alexander Sinitskii, Zhengzong Sun, Alexander Slesarev, Lawrence B. Alemany, Wei Lu, and James M. Tour (2010). Improved Synthesis of GO. ACS NANO, VOL. 4, NO. 8. pp 48064814 8. Hummers, W & Offeman, R (1957). Preparation of Graphitic oxide 9. Kim, H. and Kim W.J.(2013). Photothermally controlled gene delivery by reduced GOpolyethylenimine nanocomposite. 10. Thomas,S (2000). Alginate dressings in surgery and wound management part 1. Journal Of Wound Care, VOL 9, NO 2, pp:10-15. 22
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