Preparation Of Tumor-targeted Gene Vector Based On Low Molecular Weight Polyetyleneimine And Amphiphilic Chitosan

Based on genetic engineering and molecular genetics, gene therapy is a newmethod to cure cancers. It has become the most promising field because it directlyaims at the biological foundation of the occurrence and development of cancers. Thenature of gene therapy is to feed therapeutic genes into target cells by gene vectors,whose key is to find a reliable and effective gene delivery system. A perfect genevector has to meet the following conditions:(1) condense DNA effectively (2) stablein body fluid (3) target to the specific cells (4) cross membranes and releaseefficiently.Non-viral gene vectors are non-immunogenic, non-infectious, easier to beprepared and capable of carrying large amounts of genetic materials. Non-viral genevectors have attracted more and more attentions.Among non-viral vectors, cationic polymers have been widely explored in genedelivery research. Polyethylenemine (PEI) and its derivatives are the most extensivelyinvestigated because of the “proton sponge effect”. PEI with abundant positivecharges can condense DNA by electrostatic interaction to compact complexes, whichenables breaking through various barriers to the nucleus of target cells. DNA iscondensed as nanoparticles so that it is not easily degradated by nuclease or gatheredas precipitate, which results in high transfection efficiency. However, it has beenreported that molecular weight of PEI has strong influence on its transfectionefficiency and cytotoxicity. PEI with high molecular weight performs not only hightransfection efficiency but high cytotoxicity. On the contrary, PEI with low molecularweight performs low cytotoxicity but poor transfection efficiency. In addition, there isan inconsistency between its stability in body fluid and cellular uptake. It has beenproved that PEI derivatives obtained by crosslinking low molecular weight PEI withdegradable materials display higher transfection efficiency and lower cytotoxicity.Chitosan, obtained by alkaline N-deacetylation of chitin, is one kind of the mostwidely used natural cationic polysaccharides. Chitosan shows particularly highbiocompatibility and fairly low cytotoxicity, therefore in fact it can be used as a gene vector. Nevertheless, chitosan is insoluble at physiological pH, meanwhile it is lack ofcharges. These major drawbacks limit its use for gene delivery.Integrin is an important kind of cell surface receptor. Integrins can recognizedifferent distinct proteins as receptors. Integrins are made of α (120-185kD) and β(90-110kD) subunits. So far over20integrins have been found. Among them αvβ3receptors are highly expressed on tumor cells and tumor blood vessels but rarelyexpressed on normal cells. Therefore αvβ3can be used as a promising target ofanti-tumor therapy, RGD peptide can bind to αvβ3inegrin specially. Cell penetratingpeptides (CPPs) can translocate various molecules across the plasma membranequickly in order transfection efficiency. TAT (49-57) is the smallest fragment as aCPP.Based on the above analysis, we first synthesized amphiphilic chitosan,N-octyl-N-quatenary chitosan (OTMCS), then crosslinked the amphiphilic chitosanwith low molecular weight PEI as OTMCS-PEI. The bifunctional R13is made ofRGD peptide and TAT (49-57). At last R13linked with OTMCS-PEI asOTMCS-PEI-R13. In this way we acquired a new type of degradable amphiphilicchitosan-PEI derivative.Chapter one gave an overview on the application of polyethylenemine andchitosan as gene carriers. Characteristics and derivatives of polyethylenemine andchitosan were introduced. Furthermore the research hotspots of polyethylenemine andchitosan were also analyzed.Chapter two was synthesis and characterization of OTMCS-PEI-R13. At first thebifunctional peptide R13was prepared by the solid phase method. The structure,content and adsorbability of R13to Hela cells and B16cell (αvβ3positive cells) wereperformed respectively by MS, HPLC and HRP labeling. Then wesynthesizedamphiphilic chitosan, N-octyl-N-quatenary chitosan (OTMCS). After that wecrosslinked OTMCS activated by bis-(trichloromethyl)-carbonate and solidN-hydroxysuccinimide to PEI2KDa to get a high molecular weight PEI derivateOTMCS-PEI. At last OTMCS-PEI was modified by R13and the new type genevector was obtained as OTMCS-PEI-R13. Every product was characterized by IR and1H-NMR. The results indicated that R13was synthesized successfully with the correctsequence of RGDCRKKRRQRRR and its content was as high as95%and ability ofbinding to αvβ3positive cells in vitrowas good.OTMCS-PEI has been also preparedsuccessfully. The bifunctional peptide R13was also binded to OTMCS-PEI by SMCC successfully.Chapter three was the characterization of the new gene vectors OTMCS-PEI andOTMCS-PEI-R13. Degradation of P123-PEI-R13was estimated by gel permeationchromatography. Buffering capacities of them was measured by acid-base titration.The particle sizes and zeta potentials of these polymer/DNA complexes were alsomeasured. The DNA condensation capacity, the resistance to DNase I,FBS andsodium heparin digestion of OTMCS-PEI/DNA and OTMCS-PEI-R13/DNA weredetermined by agarose gel electrophoresis. The cytotoxicity of these polymers on theHela cells were measured by the MTT assay. The results indicated thatOTMCS-PEI-R13was degraded slowly and the degradation was nearly completedafter about60h. The particle sizes of these complexes were about100-250nm, andzeta potential were proper. OTMCS-PEI was able to condense DNA effectively andneutralized its charge at w/w ratio of0.6. OTMCS-PEI-R13-l can condense DNAcompletely at w/w ration of1, OTMCS-PEI-R13-h can condense DNA completely atw/w ration of3. All these polymers can protect DNA from being digested by DNase I,FBS and sodium heparin at high concentrations. Furthermore, OTMCS-PEI andOTMCS-PEI-R13showed significantly lower cytotoxicity in Hela cell line comparedwith PEI25kDa.Chapter four was the measurement of gene transfection efficiency of the vectorsin vitro and in vivo. We examined the ability of OTMCS-PEI and OTMCS-PEI-R13to transfect in Hela cells and B16cells using the plasmid pEGFP-N2andpGL3-Control as reporter genes.GFP transfection and luciferase activity wererespectively determined using inverted microscope and a luminometer so that we canthe quantitatively and qualitatively studied the transfection of OTMCS-PEI/DNA andOTMCS-PEI-R13/DNA complexes in vitro. In addition, we established B16animaltumor model and investigated distribution and transfection efficiency of thesecomplexes using the plasmid pGL3-Control as reporter gene. The transfectionexperiments in vitro showed that transfection efficiency of OTMCS-PEI/DNA andOTMCS-PEI-R13/DNA complexes increased as the w/w ratio increasing butdecreased as R13modification degree increasing. All synthesized complexes showedmuch higher gene transfection efficiency compared with PEI2KDa, OTMS-PEI-R13was also much higher than OTMCS-PEI. In vivo ExperimentOTMCS-PEI-R13showed higher gene transfection efficiency compared with OTMCS-PEI and PEI25KDa. The distribution of the complexes in different tissures was changed because of the modification of R13. However, we also found that the transfection efficiency invivo was lower than that in vitro.