RESUMO
Ability of the cells to adhere to an extracellular material is central to successful tissue genesis. Arg-Gly-Asp (RGD) sequences found in extracellular matrix proteins are well known for cell adhesion, however, enzymatic degradation and lack of specificity have limited their widespread use. Besides, a multifunctional material with inherent antimicrobial ability would help in invigorating the practical tissue engineering applications. Here, we report novel modified RGD (MR) and RGD mimic [R(K)] peptides (MOH and MNH2) which were synthesized post-in-silico screening, based on their interactions with integrin protein αVß3 using HEX 8.0 docking server. These mimics, containing hydrophobic Phe-Phe (FF) moiety which has been specifically introduced to initiate the self-assembling process of ß-sheet structures, were characterized thoroughly using various physicochemical and spectroscopic techniques. Under physiological conditions, these mimetics displayed thixotropic behavior rendering them highly suitable as injectable hydrogels having an added advantage of site-specific targeting abilities. Electron microscopy further revealed the formation of nanofibers upon self-assembly of these peptides. Besides, enhanced cell adhesiveness by these peptides compared to the commercial Poly l-lysine coated surfaces as well as the inherent antimicrobial potential against both sensitive and antibiotic-resistant pathogens (Methicillin-resistant Staphylococcus aureus and multi-drug resistant Salmonella enteritidis) substantiated the applicability of these unique injectable hydrogels wherein the porous fibrous framework offered a favorable environment for drug entrapment and 3D cell culture. Altogether, these properties render these novel RGD mimic peptides as promising multifunctional candidates for various tissue regenerative applications.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Peptidomiméticos , Materiais Biocompatíveis , Hidrogéis/química , Oligopeptídeos/farmacologia , Peptídeos/química , Peptidomiméticos/farmacologia , Conformação Proteica em Folha betaRESUMO
Low molecular weight branched polyethylenimines (LMW bPEIs) are almost nontoxic but display poor transfection efficiency due to lack of adequate complexation ability with nucleic acids followed by transportation across the cell membrane. Here, a series of amphiphilic retinoyl-bPEI conjugates (RP-1, RP-2 and RP-3) has been synthesized by allowing the reaction between bPEI (1.8 kDa) and a bioactive and hydrophobic vitamin A metabolite, all-trans-retinoic acid (ATRA), in varying amounts. In aqueous medium, these conjugates self-assembled into core-shell RP nanocomposites with size ranging from ~113-178 nm and zeta potential from ~ +15-35 mV. Evaluation of pDNA complexes of RP nanocomposites revealed that all the complexes exhibited significantly enhanced transfection efficiency without compromising on the cytocompatibility. RP-3/pDNA complex, with the highest content of retinoic acid, exhibited the best transfection efficiency. Further, due to anticancer properties of ATRA, these nanocomposites significantly reduced the viability of cancer cells (HepG2 and MCF-7 cells) without affecting the viability of non-cancerous cells (HEK 293 cells) demonstrating the cell-selective nature of the formulated nanocomposites. The intracellular trafficking and co-localization studies involving RP-3 nanocomposites also showed their higher uptake with intracellular and nuclear accumulation properties. Altogether, the results demonstrate the promising potential of the RP conjugates that can be used in future hepatocellular carcinoma targeted gene delivery applications.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Nanocompostos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Plasmídeos , Polietilenoimina , Transfecção , Transgenes , Tretinoína/farmacologiaRESUMO
The main aim of the current study is to compare the physicochemical properties, cytotoxicity and gene-transfer ability of electrostatically and covalently linked nanocomposites of polyethylenimine (PEI) and polyacrylic acid (PAA) on mammalian cells. Two series of nanocomposites, ionic PEI-PAA (iPP) and covalent PEI-PAA (cPP), were synthesized by varying the amounts of polyacrylic acid (PAA). Physicochemical characterization revealed that iPP nanopcomposites were of bigger sized than cPP nanocomposites with zeta potential almost comparable. Nucleic acid binding assay displayed that iPP and cPP nanocomposites, having sufficient cationic charge, efficiently interacted with plasmid DNA and completely retarded its electrophoretic mobility on agarose gel. In vitro MTT assay showed slightly higher cell viability of cPP/pDNA complexes over their ionic counterparts. Both the series of nanocomposite/pDNA complexes exhibited considerably higher transfection efficacy compared to pDNA complexes of native bPEI and the standard transfection reagent, Lipofectamine, with cPP/pDNA complexes performed much better than iPP/pDNA complexes. Flow cytometry further confirmed these findings where cPP-4/pDNA complex showed transfection in â¼ 85% HEK293 cells, while iPP-2/pDNA complex transfected â¼ 67% HEK293 cells. Lipofectamine/pDNA and bPEI/pDNA complexes could transfect just â¼ 35% and â¼ 26% HEK293 cells. All these results demonstrate the superiority of covalently linked nanocomposites (cPP) which could be used as efficient carriers for nucleic acids in future gene delivery applications.