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The Preparation Of BSA (Bovine Serum Albumin)

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Materials : Bovine Serum Albumin (MW 66.5 KDa, Faction V), Dipentaerythritol pentaacrylate (5Acl, MW 524.21), Propylamine, Pentylamine, Hexylamine, Heptylamine, Octylamine, Decylamine, Octadecylamine, 3-(Dimethylamino)1-propylamine,Rhodamine 6G, Methylene blue, Fluorescein, Tetracycline hydrochloride, Aspirin and Phosphate buffer saline (PBS) capsules (pH 7.4, 0.01 M) were purchased from Sigma-Aldrich (Bangalore, India) and absolute ethyl alcohol (CAS 64-17-5, Lot 17030799) was purchased from TEDIA Company (United States of America). Methanol (CAS 67-56-1) was purchased from RANKEM (Maharashtra, India).Reagent grade THF was purchased from RANKEM (Maharashtra, India). Cotton was purchased from a local medical shop in Guwahati city (Assam, India). Aluminum wire used for submersion of loaded cotton in PBS buffer was purchased from a local electrical shop in Guwahati city (Assam, India).

General considerations : Glass vials (Borosil) used for preparing the solutions were washed with acetone and ethanol prior to use. The Zeta potential(ζ) and Dynamic Light Scattering (DLS) analysis was carried out using Zetasizer Nano ZS90 (model no. ZEN3690). Release profile of Tetracycline and Aspirin was monitored by a PerkinElmer Lambda 25 UV/Vis spectrophotometer. Contact angle measurements were taken using a KRUSS Drop Shape Analyser-DSA25 with an automatic liquid dispenser at ambient conditions. FTIR spectra were recorded with a PerkinElmer instrument at ambient conditions using KBr pellets. Scanning electron microscope images were obtained using a Sigma Carl Zeiss scanning electron microscope (samples were coated with a thin layer of gold prior to imaging).Digital pictures were acquired using a Canon power shot SX420 IS digital camera. Fluorescence microscopic images of the superhydrophobic cotton before and after loading of Rh-6G,Methylene Blue and Fluorescein were acquired using a AX10 observer Z1 & AXio Cam MRCS, Carl Zeiss, Germany. Milli-Q grade water was used for all experiments.

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Experimental : Preparation of BSA nanoparticles BSA nanoparticles were prepared by desolvation method. To 1ml (50mg/ml) of BSA in aqueous solution, 3ml of ethanol was added dropwise under constant stirring (600rpm) at the rate of 1ml/min for 15mins. Thereafter, the resulting solution was centrifuged down (12000rpm for 15mins) and then re-dispered in 5Acl/methanol (1.325g in 10ml) to induce cross-linking and kept for 1hour to stabilize the nanoparticles formed. The resulting nanoparticles were centrifuged down (12000rpm for 10mins), re-dispered in ethanol and subsequently centrifuged down to wash (repeat twice) the nanoparticles off the loosely bound 5Acl molecules. Then the obtained nanoparticles were re-dispersed in Milli-Q water. The size of the nanoparticles and the surface potential was characterized using DLS and the morphology of the nanoparticle was examined using field emission scanning electron microscopy (FESEM) after drop casting the nanoparticle dispersed in water on a glass slide. FTIR of the nanoparticles was carried out after vacuum drying the solid aggregate obtained on centrifugation.

Coating of medical cotton with Bovine Serum Albumin (BSA) nanocomplex followed by post chemical modification The steps involved in the modification of medical cotton to achieve superhydrophobicity can be categorized as follows: (1)Medical cotton of dimension (1.5cm×1.5cm×0.5cm) was dipped in 3ml aqueous solution of Bovine Serum Albumin (BSA) of concentration 10mg/ml and kept undisturbed for over 3 hours in a petri-dish. To this solution containing the medical cotton, 6ml ethanol was added dropwise till the solution turned turbid and kept undisturbed for a period of 1 hour to let the nanocomplex of BSA to deposit on the cotton. Subsequently, the cotton was washed twice with ethanol to remove the loosely bound nanocomplex. (2) The cotton was then further treated with 5Acl in methanol (1.325g in 10ml) for 3hours after which, it was washed twice with methanol to remove the loosely adhered 5Acl molecules. (3) To achieve the liquid water anti-wetting property the cotton was further washed with THF (10mins) following which it was treated with amine containing small molecules like propylamine, pentylamine, hexylamine, octylamine, decylamine and octadecylamine for overnight, after which the cotton was washed with THF to remove the loosely bound amine molecules. The material was left to air dry. The difference in anti-wetting property of the fibrous substrates treated with amine containing small molecules was observed via contact angle measurements. The digital images of the same were also taken to demonstrate the anti-wetting property.

Loading and release of drug molecules : The concerned drugs were loaded from its ethanolic solution onto the superhydrophobic cotton and left to air dry. To monitor the release profile, the dried cotton containing the drug was placed in PBS buffer (pH=7.4) at 37⁰C to allow the release of the drug. The aliquot was collected at different time intervals, which was followed by UV analysis of the aliquot to determine the concentration of the drug released. For aspirin, a piece of cotton (1.5cm×1.5cm×0.5cm) weighing 54mg absorbed 1200µL of aspirin. The UV absorbance was observed at 265nm. In the case of tetracycline, a piece of cotton (1.5cm×1.5cm×0.5cm) weighing 54mg absorbed 1200µL of tetracycline. The UV absorbance was observed at 385nm. The release of aspirin loaded from differently modified cotton with amine containing small molecules was carried out. Also, different concentrations of aspirin (0.1mg/ml, 0.5mg/ml and 1.5mg/ml) release was also carried out from octadecylamine treated superhydrophobic cotton. In additon, octadecylamine treated cotton was also loaded with a mixture of aspirin and tetracycline in 1:1 ratio and the release profile was monitored at 37⁰C and pH 7.4. The UV analysis was obtained at both 265nm for aspirin and 385nm for tetracycline.


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