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In-Silico Designing, Synthesis and Modification of Papilloma based CPPs in Drug Delivery

KrishiKosh

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Title In-Silico Designing, Synthesis and Modification of Papilloma based CPPs in Drug Delivery
 
Creator Saxena, Shikha
 
Contributor Ramteke, P. W.
 
Subject farmyard manure, planting, wheats, nutrients, fertilizers, grain, yields, crops, nutrient management, vegetative propagation
Cell-penetrating peptides, DNA molecules, papilloma
 
Description Thesis titled “In-Silico Designing, Synthesis and Modification of Papilloma based CPPs in Drug Delivery submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy in Biotechnology by Shikha Saxena.
(CPPs) have proven their potential as an efficient delivery system due to their intrinsic ability to traverse biological membranes and transport various cargoes into the cells. In this study two papilloma based cell penetrating peptides; one from L1 protein of human papilloma virus type-16 and other from the bovine papilloma virus type-1(HPV and BPV) were designed by cell penetrating peptide prediction software and the chimericpeptides from these CPPs by conjugation of R9 (HPV-R9 and BPV-R9) were alsosynthesized by solid phase Fmoc methodology. These peptides were labelled with FITC andpurified by RP-HPLC and characterized for their conformation using circular dichroismspectroscopy in water and different concentration of TFE. It was observed that these peptidesadopted the beta sheet structure and also adopted the random coil structure as the concentration of TFE increased. The conformational studies of DNA in peptide/DNA complexindicated that the normal B structure of DNA was not altered significantly. The gel retardation,nuclease protection and DNA release assays indicated that 1:20 N/P ratio found to bemost effective ratio for transfection of DNA molecules. Dye exclusion assay as determined by spectrofluorimetry showed more than 25% DNA compaction. Internalization ability of these peptides (FITC labelled) was observed by fluorescence microscopy, confocal microscopyand flow cytometry. By quantitation of flow cytometry it was observed that the FITClabelled peptides ranging from 40-90% found to be internalized. The chimeric CPPs (HPVR9and BPV-R9) were found to be more efficiently deliver into the cells as compare to plainpeptide as well as the well known CPP oligoarginine (R9). Toxicity of peptides/DNA complexeswere assessed by viable count of cells and it was found that all these four FITC labelledpeptides as well as the peptide/DNA complex showed more than 70-80% viability invitro. The cojugated CPPs were able to deliver cargo (pDS Red) into the Vero cells. The transfection ability to deliver the cargo was found to be more than 25-30% as determined byFACS analysis. In summary, the present study provides evidence that an integrated in- silico and experimental approached an effective strategy for the identification of novel CPPs and CPPs identified in the present study have promising perspectives for future drug deliverya pplications.
 
Date 2017-01-16T14:23:07Z
2017-01-16T14:23:07Z
2016
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/96484
 
Language en_US
 
Format application/pdf
 
Publisher Sam Higginbottom Institute of Agriculture, Technology & Sciences (SHIATS)