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Novel Cationic Polymer And Liposome For Gene And SiRNA Delivery

Posted on:2013-02-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y ZhangFull Text:PDF
GTID:1261330395987483Subject:Chemical Biology
Abstract/Summary:PDF Full Text Request
Gene therapy provides potential method for some diseases caused by genes.There are lots of stuides on non-viral gene vectors for their safety and lowimmunogenicity. However, the transfection efficiency of non-viral vectors is usuallylower, and they also have poor stability in vivo, compared with viral gene vector. Inorder to improve the transfection efficiency of non-viral vectors and the in vivostability, we designed several novel cationic polymers and liposomes as carriers forefficient gene and siRNA delivery.The thesis is divided into four parts:Part I. Study of PEGylated supramolecular assembly as polyplexes in non-viral genedelivery systemIn order to improve the stability of polyplexes in physiological environment, inthis research, PEGylated gene assembly was constructed by adding the PEGcontaining amphiphilic polymers, named LD0-PEG, into PEI/DNA complexes. It wasinteresting to find that the addition sequence of LD0-PEI had great effect on thestability of Polyplexes. The PEI/LD0-PEG/DNA formed by adding LD0-PEI and PEIinto DNA solutions, showing great antiaggreration effect in physiologicalenvironment. Whereas PEI/DNA/LD0-PEG complexes formed by the addition ofLD0-PEI into PEI/DNA complexes had no effect to improve the stability ofpolyplexes. It was shown that PEI/LD0-PEG/DNA was stabile in high saltconcentration and with the presence of serum, zeta potential of such complexes waslower than PEI/DNA complexes indicating that LD0-PEG could shield excess positivecharge on the surface of complex. In vitro transfection experiments showed that underthe conditions of the presence of serum, transfection of PEI/LD0-PEG/DNAcomplexes was still very high. In conclusion, the PEGylated polyplexes could beconstructed to improve the stability and transfection efficiency by entrapment of LD0-PEI. It also provided a simple and facile approach to fabricate the PEGylatedsupramolecular assembly in gene delivery.Part II.Study of polyamidoamine dendrimers with modified pentaerythritol core assuicide gene vector With the studies of cancer molecular mechanisms, more and more researchesfocused on the suicide gene therapy. However, few studies were carried onpolyamidoamine dendrimer (PAMAM) as suicide gene carrier. We previouslysynthesized a novel PAMAM with a pentaerythritol core, and the fifth generation PD-PAMAM (G5PD-PAMAM) is an effective gene carrier. Here, we used G5PD-PAMAM as suicide gene vestor, and the suicide gene we used was cytosinedeaminase(CD). Agarose gel electrophoresis result showed that G5PD-PAMAMcould combine with pCD-EGPF-N1to form complexes, and the particle size wasabout100nm measured by DLS. There were high transfection efficiency in humantumor cells KB, HepG2and A549cells transfected by G5PD-PAMAM/pCD-EGPF-N1complexes.5FC sensitivity test showed that the high expression of cytosinedeamniase gene mediated by G5PD-PAMAM, leading to very low amount of5-fluorocytosine was induced to5-fluorouracil (5FU) in a short time. Compared with5FU, the G5PD-PAMAM mediated CD/5FC system could induce cell death moreeffectively. These results indicate that the G5PD-PAMAM mediated suicide genesystem could induce CD gene to highly express in human tumor cell lines.Part III. Study of polyamidoamine dendrimers with modified pentaerythritol core assiRNA vector.RNAi has great prospect potential applications in the treatment of disease.However, efficient siRNA delivery still remains elusive. Here, we used G5PD-PAMAM as siRNA vectors, in order to provide a new way for siRNA vector. Agarosegel electrophoresis retardation assay showed that G5PD-PAMAM and siRNA couldform nanoparticles at a low weight ratio (0.5:1), demonstrating strong complexationbetween G5PD dendrimer and siRNA molecules. The atomic force microscopyshowed that the particle size of G5PD-PAMAM/siRNA nanoparticles was40-70nm.There was no significant cytotoxicity and high gene silencing efficiency when SK-Hep1cells were treated with G5PD-PAMAM/siRNA nanoparticles. Cell biologymethods showed that G5PD-PAMAM/siRNA nanoparticles could enter into cellsmainly by clathrin-mediated endocytosis. Using molecular beacons to study therelease of siRNA showed that siRNA can be released from the G5PD-PAMAMeffectively. Part IV. Study of novel lipoidal amine-based nano liposome as siRNA vectorDevelopment of lipid-based formulations is ongoing. For example, novel lipid-like materials, called lipidoids, Avridine with immune activity, shares structuralsimilarity with the lipidoids, has been used in the preparation of liposomes, howeverhas yet to be incorporated in cationic nanocarrier formulation for siRNA delivery.Avridine, DOPE or Span80as well as the TPGS were used to build into the novelLANC formulations for siRNA delivery. The LANC-DOPE with5%TPGS wasselected as the optimal LANC formulation for its favorable particle size, zetapotential and siRNA complexation efficiency. In addition, the LANC-DOPE with5%TPGS mediated efficient cellular uptake and delivery of siRNA into the cytoplasmusing the fluorescent labeled siRNA and MB, thus resulting in significant theluciferase gene knockdown. Our study provides a new strategy for the design of newimmunoliposomes as a siRNA vector.In summary, efficient and safe gene vector needs a breakthrough in materialsdesign. Base on the requirement of improving, interaction between vector and gene orsiRNA, moderating the cell compatibility, enhancing the cellular uptake, we designeddifferent novel cationic polymers and liposome for efficent gene and siRNA delivery.Our studies provide new strategies for future study on non-viral gene and siRNAvector.
Keywords/Search Tags:Gene Transfection, siRNA delivery, non-viral vectors, Polyamidoaminedendrimer, Polyethylenimine, Immunoliposome
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