Potential Use Of Non-viral Vector(CyD-DABs) In Digestive Malignancy

Target therapy is the new approach to the treatment of malignant disease.The therapeutic gene or low-molecular drugs were transferred to the target site directly, inducing apoptosis of tumor cells directly or indirectly by mutant gene correction,immune response activation and so on.The key-point of target therapy is an appropriate vector which can deliver the optimal gene or drug to the certain site effectively.Most ongoing human gene therapy protocols rely on recombinant retroviral and adenoviral vehicles,whose risk encountering infection and immunological problems with large scale or repeat use,besides of their limited carrying capacity.Synthetic vectors,such as cationic polymers,have gained more and more interest as alternatives to viral vectors because of their safty,including the avoidance of potential immunogenecity and toxicity,the possibility of repeated admimistration,and the ease of the establishment of good manufacturing practice. However,the non-viral vectors order more work to increase their transfection efficiency and targetability,as they were mostly phagocytosed by cells through charge interaction.The object of successful treatment to cancer is to transport the therapeutic gene or drugs to the malignant cells directly,without effect to normal cells.An effective strategy is to link tumor-specific ligands to the vectors,induce receptor-ligand mediated phagocytose.Another hinder to the development of non-viral vector is the cytotoxicity,which depends on the molecular weight and concentration of the vector. DABs contained up to 64 terminal amino groups and were studied as pH-sensitive controlled release systems for drug delivery,it contained 100% protonable nitrogens,making it ideally suited as DNA binding and possibly DNA transportting agents.According to both in vitro and in vivo studies,cyclodextrins (CyDs)may have enhancer activity on transport through cell membranes,and usually act as a degradable drug cartier.Targeting of non viral vectors with folate(FA) is an attractive approach to deliver chemotherapy agents or therapeutic gene to folate receptor(FR)expressing tumor.In this study,low molecular DABs(DAB-4,DAB-8,DAB-16)were conjugated toα-CyD,β-CyD andγ-CyD,and the transfection efficiency of the conjugates were increased obviously.Then we link folate to the the polymer,and identify the cytotoxicity and transfection efficiency in vitro.This study was divided into three parts:1)conjugation and chemical character identification of CyD-DABs.2)In vitro identification of the biological properties of CyD-DABs,and selectα-CyD-DAB-8,β-CyD-DAB-8,γ-CyD-DAB-8 as the vector with low toxicity and high transfection efficiency.3)Conjugation and in vitro characterization of CyD-DAB-FA.Partâ… :Conjugation and Chemical Character Identification of CyD-DABsObjective:In this part,a new cationic polymer was developed,then the chemical characters as well as the capacity of DNA condensing of the polymer were identified.Methods:We graft low molecular DABs onα-CyD,β-CyD,γ-CyD with CDI.The chemical characters of the vector were investigated and confirmed by ¹H NMR, FT-IR,TGA.The potential of DNA condensing were identified with agarose gel electrophoresis assay,TEM,particle size and zeta potential analysis.Results:¹H NMR spectra showed the linkage ofβ-CyD and DAB-8,approved the molar ratio of CyD and DAB in the copolymer.FT-IR present a new peak in 1700 cm^-1,indicated that a band of O=C-N was appeared in the final product.Agarose gel electrophoresis indicated that DNA condensing ability of CyD-DAB was much better than DABs.TEM image explored that particle size of the CyD-DAB/DAB complexes were 150-200nm.The surface charge of the omplex was positive, indicating that the CyD-DABs would provide a strong ability for DNA binding.Conclusion:CyD-DABs can bind DNA effectively,and the complex of CyD-DAB/DNA was suitable for cell taking according to its size and surface charge.Partâ…¡:In vitro Identification of the Biological Properties of CyD-DABsObjective:In this part,The biological properties of the CyD-DABs were identified by MTT assay and gene transfer efficiency in various cell lines,in order to select the polymer with low toxic and high delivery ability。Methods:COS7,HepG2,SGC7901and RKO cell lines were cultured in DMEM and RPMI 1640 supplemented with 10%FCS at 37℃,5%CO2.Different concentrations of CyD-DABs were added to each well,and MTT assay were taken 24h later.The transfection efficiency was tested on COS7,HepG2,SGC7901,SMMC7721 and RKO cell lines with pCAG-Luc plasmid.We use 10%FCS to simulate in vivo environment.Results:CyD-DABs displayed low cytotoxicity in all cell lines.After DAB conjugated with CyD,the toxicity was not increased until the concentration of 50μg/ml,except for DAB-16.CyD-DAB-4 and CyD-DAB-8 present low toxicity and high transfection efficiency in COS7,HepG2,SGC7901,SMMC7721 and RKO ceil lines,α-CyD-DAB-8,β-CyD-DAB-8,γ-CyD-DAB-8 also have repectiveiy higher transfection efficiency in serum,better than PEI 25KD.Conclusion:α-CyD-DAB-8,β-CyD-DAB-8,γ-CyD-DAB-8 displayed low cytocoxicity and high gene transfer efficacy in various cell lines,and also implied a potential use for in vivo gene delivery.Thus,we chooseα-CyD-DAB-8,β-CyD-DAB-8,γ-CyD-DAB-8 to link folate in the next step.Partâ…¢:Conjugation and in vitro Characterization of CyD-DAB-FA.Objective:FR targeting vectors with FA ligand were produced.The chemical character and biological properties ofα-CyD-DAB-8-FA,β-CyD-DAB-8-FA,γ-CyD-DAB-8-FA were identified,and the potential in vivo use of the new compound were estimated.Methods:α-CyD-DAB-8,β-CyD-DAB-8,γ-CyD-DAB-8 and FA were linked by CDI.After that,¹H NMR and FT-IR were taken to identify the chemical character of the new compound,MTT assay as well as transfection efficiency were identified in KB cell line.After the FR targeting vector condensd pCMV-luc plasmid,added to the cell for 48hr,the luciferase expression was tested.We use 10%FCS to simulate in vivo environment.Results:The FR targeting vector display a weak proliferative effect at low dose, when the concentration was up to 100μg/ml,cell viability was still over 90%.The transfection in KB cell line suggested that the new compound has high transfer efficacy in FR(+)cell line,when tested in serum containing culture medium,the compound has an obvious increase of transfection efficiency,and higher than PEI 25KD with respect toα-CyD-DAB-8-FA,β-CyD-DAB-8-FA.Conclusion:The new compound ofα-CyD-DAB-8-FA,β-CyD-DAB-8-FA has a low cytotoxicity and high transfection efficiency even in the presentence of serum, implying a potential use in the treatment of FR(+)tumor in vivo.Major Conclusion:1.The research produced a series of CyD-DABs compounds,and the chemical characterization of the compounds were determined.2.A series ofα-CyD-DAB-8,β-CyD-DAB-8,γ-CyD-DAB-8 compound was selected as with low cytotoxicity and high gene transfer efficiency in vitro.3.Sucessfully link FA toα-CyD-DAB-8,β-CyD-DAB-8,γ-CyD-DAB-8,produced water soluble FR targeting vector.4.We find that the compound ofα-CyD-DAB-8-FA,β-CyD-DAB-8-FA,γ-CyD-DAB-8-FA has lower cytotoxicity and higher transfection efficiency in FR(+)cell line,compared to PEI 25KD orα-CyD-DAB-8+FA, β-CyD-DAB-8+FA,γ-CyD-DAB-8+FA complex.5.The cytotoxicity of CyD-DABs has the trend ofγ-CyD-DAB-16＞β-CyD-DAB-16＞α-CyD-DAB-16＞γ-CyD-DAB-8＞β-CyD-DAB-8≥α-CyD-DAB-8≥γ-CyD-DAB-4≥β-CyD-DAB-4≥α-CyD-DAB-4.After linked to FA,the new compound display a weak proliferative effect at low dose,and high cell viability even at much higher dose.6.We implied that the new compound ofα-CyD-DAB-8-FA,β-CyD-DAB-8-FA,γ-CyD-DAB-8-FA has the targetability to FR(+)cell,and with high transfection efficiency in serum-containing medium,suggesting the potential use of the vector in the treatment of FR(+)tumor.