اصلاح انتقال ژن توسط سامانه كيتوزان با استفاده از رتينوئيك اسيد و پكتين به سلول HepG2

چكيده انگليسي :

Modification of Chitosan Mediated Gene Delivery System with Retinoic acid and Pectin Ali Fattahi a fatahi@ma iut ac ir 7 03 2012 Department of Material Engineering Isfahan University of Technology Isfahan 84154 83111 Iran Isfahan University of Medical Sciences Isfahan 81746 734611st supervisor Mohammad Ali Golozar1 golozar@cc iut ac ir2nd supervisor Jaleh Varshosaz varshosaz@pharm mui ac irAdvisor Mohammadhosein Fathi Advisor Hamid Mir Mohammad Sadeghi Department Graduate Coordinator Masoud PanjepourAbstract improve of chitosan mediated gene delivery system with retinoic acid and pectin for gene delivery toHepG2 has been investigated in the current dissertation Initially tragacntic acid TA kind of pectin wasextracted from gum tragacanth Molecular weight MW was determined by gel permeation chromatographyand intrinsic viscosity Desertification degree ED was measured by titration Its cytotoxicity has been checkedon Hela HepG2 and L929 Chitosan tragacanthic acid nanopartilces have been fabricated with ionic gelationmethod in different weight ratios Chitosan retinoic acid RA derivatives were prepared in various molar ratiosvia condensation method and the validity of reaction was confirmed using FTIR and NMR spectroscopy Micelles have been prepared with ultrasonic method Critical concentration of micelle CMC was determinedusing pyrene Loading of RA in the micelles was assayed and loading efficacy loading content and release ratewere investigated using UV spectroscopy Plasmid has been loaded on chitosan TA nanoparticles and chotosan RA micelles and in polyplexes by ionic gelation Pectin has been coated on polyethylenimine PEI polyplexwith same method in different weight ratios Optimized charge ratio of vehicle to plasmid was determinedthrough either gel electrophoresis or Cybergreen 1 fluorometry assay and transfection was studied in chargeratios upper than optimum charge ratio Particle size surface charge and pH effect on surface charge andparticle size of nanoparticles have been determined by light scattering method Morphology was investigated bySEM Finally cytotoxicity of optimum particles were evaluated on Hela and HepG2 cell lines by MTT assay MW and ED of TA were 300 kDa and 94 respectively TA had shown no cytotoxicity on mentioned celllines and its cell adhesion was better than alginate Nano composites of chitosan tragacanthic acid wereconsisted of spherical particles with particle size blow than 200 nm with positive charge Nanoparticles withweight ratio of 3 particle size of 132 5 6 77 nm and charge ratio of 30 45 1 84 mV were chosen for genedelivery study Spherical micelles with CMC of 1 3 10 2 to 2 13 10 2 mg ml were prepared Their size andsurface charge were reneged from 142 14 to 208 4 nm and from 27 25 to 34 48 mv respectively Gelelectrophoresis results showed complete plasmid loading by nanoplexes in upper charge ratios comparing tochitosan polyplexes while it happened in lower charge ratio for miceplexes Transfection of nanoplexes washigher than polyplexes in Hela and HepG2 cell lines It can be attributed to galactose groups in side chain of TAand existing of asialoglicoprotein receptors in HepG2 cell membrane Miceplexes gene expression was lower inHela and HepG2 compared to that of polyplexes and nanoplexes However transfection was increased inpresence of 10 fetal bovine serum Plasmid content was more in this system comparing to other systems Effect of pectin coat on cytotoxicity and stability of PEI polyplexes showed less cell cytotoxicity and moresystem stability comparing to uncoated polyplexes while coated system transfection was not that much lowercomparing to uncoated system Overall nanoplex is a proper choice for liver gene delivery because of targetingto hepatocytes More stability of miceplex and its more gene expression in serum medium make this systemmore suitable for in vivo gene delivery Pectin coat is a good method for gene delivery in in vivo because of lesscell cytotoxicity and more system stability to

فتاحي، علي

اصلاح انتقال ژن توسط سامانه كيتوزان با استفاده از رتينوئيك اسيد و پكتين به سلول HepG2

تراگاكانتيك اسيد , سميت سلولي