Novel Dendrimers And Polyphosphoramidates As Gene Delivery Carriers

Gene therapy can be generally defined as a method to provide a patient's somatic cells with the genetic materials required for producing specific proteins to correct or modulate disease. Efforts in gene therapy have grown dramatically in recent years. Basic research as well as clinical activity has made exciting progress and are beginning to offer renewed hope that gene therapy may be a promising approach to the treatment of inherited as well as acquired diseases, such as cardiovascular disease and cancer.Since the sequencing of the human genome is completed, the bottleneck of gene therapy has shifted from gene cloning to gene delivery. Gene delivery systems are used in the field of gene therapy to introduce foreign DNA encoding therapeutic protein sequences into cells. Several gene delivery systems have been developed to promote gene expression either in vitro or in vivo. Among them, viral methods are well known and can be extremely efficient (viral vectors were used in the first human gene therapy test), but the safety (including the immunogenicity and the risk associated with replication-competent viruses) and production issues of viral vectors have stimulated efforts toward the development of nonviral gene delivery systems such as cationic lipids, polymers and other mechanical and electrical methods. Among the nonviral gene delivery systems, novel biocompatible polymers have gained increasing attention and been examined for their properties as gene carriers.Although the use of polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity and mutagenesis, they are usually inefficient and toxic. Inefficient endosomal release, cytoplasmic transport and nuclear entry of plasmids are currently the limiting factors in the use of polymers for effective plasmid-based gene therapy. The chapter 1 focuses on the exploration of the barriers along the plasmid DNA delivery pathway and presents a detailed review of the recent progresses achieved in polymer-based gene delivery. Finally, we point out the new directions in designing safer and more efficient polymeric gene carriers.Polyamidoamine (PAMAM) dendrimers are a class of nanoscopic, spherical, well defined, highly branched, and monodisperse polymers that carry primary amino groups on the surface. They were first used as gene carriers in 1993 by Haensler et al., and could mediate high efficiency transfection of a variety of suspension and adherent cultured mammalian cells. Their characteristics of precise control of structure, favorable pKa's, and relatively low toxicity have fueled more studies on new molecular designs and the characterization of transfection efficiency both in vitro and in vivo. In chapter 2, a series of five polyamidoamine (PAMAM) dendrimers with trimesyl core at different generations (G4 to G8) were synthesized and evaluated as gene carriers, and the effects of the core structure as well as generation of dendrimers on the complex formation and transfection efficiency were also investigated. The structures of all the dendrimers were confirmed by ~1H-NMR, FT-IR and Gel Permeation Chromatography (GPC). DNA binding capacities of these dendrimers were characterized by gel retardation assay and ethidium bromide exclusion assay. All five dendrimers retained DNA completely at or above N/P ratio of 1, suggesting that they were able to condense negative charged DNA through electrostatic interaction. Their cytotoxicity was evaluated in HeLa cells using MTT assay, and the results suggested that it increased with the polymer generation. The LD50 values of G4through G8 were 628, 236, 79, 82 and 77 jag/mL, respectively, which were higher than those of PEI and PLL. Particle size measurement showed that dendrimers of generation six or higher (G6, 7 or 8) could condense DNA into complexes with an average diameter ranging from 100 to 300 run, but dendrimers of the 4th and 5th generations (G4 and G5) formed severe aggregates. The in vitro transfection efficiency was evaluated in HeLa cells, COS-7 cells and primary rat hepatocytes. It decreased in the order of G6>G7>G8>G5>G4. The G6 showed the highest gene transfection efficiency, reaching a level comparable to that of PEI. The transfection mediated by G6 was significantly inhibited in the presence of bafilomycin Al. The acid-base titration curve for G6 snowed high buffer capacity in the pH range from 5.5 to 6.4 (pKa ~ 6). This permits G6 to buffer the pH change in the endosomal compartment, suggesting the high transfection efficiency of G6 might be partially due to its buffer capacity or endosomal escape property. This study demonstrates the potential of PAMAM dendrimer with trimesyl core as a non-viral gene carrier.A thorough exploration of relationship between chemical structure and transfection efficiency will be very helpful to design safer and more efficient polymeric gene carriers. The pentavalency of a phosphorus atom in the backbone of polyphosphoesters makes it possible to conjugate different functional groups as side chains. Polyphosphoesters bearing charged groups through a phosphate (P-O) or a phosphoramide (P-N) bond as side chains have been proved efficient gene carriers. In chapter 3, we choose the polyphosphoesters as molecular model to systemically study the structure-function relationship because of their structural versatility. Therefore, a series of polyphosphoramidates (PPAs) with identical backbone, consistent molecular weight but variable side chains were synthesized and investigated as non-viral gene carriers. The obtained PPAs can be categorized into two types based on their pendent groups, including linear-PPAs with linear side chains and branched-PPAs with branched side chains. Their chemical structures were confirmed by "H- and 31P-nuclear magnetic resonance (NMR). The average molecular weight of polymers were determined by Gel Permeation Chromatography (GPC), which were in the range of 21.2~27.lkDa corresponding to the degree of polymerization about 100. These cationic polymers showed good DNA binding capacity at N/P ratio of I and above, and they could condense DNA into nanoparticles with the diameter less than 150 nm at charge ratio higher than 10. PPAs-mediated transgene capacity was evaluated in vitro and they showed good gene transfection efficiency in cell lines, i.e. COS-7 and HeLa cells, as well as primary rat hepatocytes. It has been demonstrated that the transfection efficiency of PPAs depended on their side chain structure rather than spacer length, and branched-PPAs exhibited higher transgene expression than linear-ones. The most effective gene carriers among these PPAs series, namely PPA-DEA, exhibited comparable gene transfection efficiency level as PEI (widely used cationic gene carriers) without any helper molecules, and showed lower cytotoxicity. The LD50 of PPAs were in the range of 100-120 ug/ml in Hela cells determined by MTT assay, in contrast to 23 and 45 ug/ml for PEI and PLL, respectively. In addition, PPAs also could enhance luciferase expression following intramuscular injection of PPA/DNA complexes at N/P ratio of 0.5, which was up to 16-fold higher than naked DNA. Another administration route, bile duct injection, was performed to evaluate in vivo transfection efficiency of PPA-DEA. The results on the 3rd day revealed that PPA-DEA mediated luciferase expression in liver was 225 times higher than naked DNA.An ideal gene delivery vector should not only be safe, stable, cost-effective to produce in clinical relevant quantities, but also capable of efficient and tissue-specific gene delivery. Since the galactose moiety can be specifically recognized by asialoglycoprotein receptors (ASGPR) on hepatocytes, galactose is the most extensively studied to target genes to liver parenchymal cells. Gene transfer efficiency can be improved as a result of the enhanced cellular uptake via the ASGPR mediated endocytosis. In chapter 4, Galactosylated polyphosphoramidate (Gal-PPA) with different ligand substitution degrees (4%, 6.5%, 12.5% and 21.8%, respectively) were synthesized and evaluated as hepatocyte-targeted gene carriers. MTT assay revealed that in vitro cytotoxicity of these gene carriers decreased significantly with the increase of galactose substitution degree. More importantly, the affinity of Gal-PPA/DNA nanoparticles to galactose-recognizing lectin increased with the galactose substitution degree. However, no enhancement effect was observed in transfection mediated by these galactosylated PPAs in HepG2 cells. Based on the results of gel retardation assay and polyanion competition assay, we hypothesized that the reduced transfection efficiency of Gal-PPA/DNA nanoparticles was due to the decreased stability of these nanoparticle. We therefore prepared nanoparticles by precondensing DNA with PPA at a charge ratio of 0.5, yielding nanoparticles with negative surface charge, followed by coating with Gal-PPA, resulting in a Gal-PPA/ DNA/PPA ternary complex. Such a ternary nanoparticle formulation led to significant size reduction in comparison with binary nanoparticles, and achieved enhanced gene transfection in asialoglycoprotein receptor (ASGPR)-rich cell types like HepG2 cells and primary rat hepatocytes but not in ASGPR-absent cell type like HeLa cells and HEK 293 cells. The enhancement was compatible with the enhanced cellular uptake after galactosylation. In addition, the fact that Gal-PPA/DNA/PPA ternary nanoparticles mediated transfection could be inhibited in the presence of free D-galactose was consistent with the result of RCA 120 assay, suggesting that the cellular uptake is through ASGPR-mediated endocytosis. After intraportal vein injection, the 4%Gal-PPA-DEA ternary nanoparticles mediated luciferase expression in liver was 30-fold higher than unmodified PPA-DEA. The galactosylation of PPA-DEA altered the distribution of transgene expression in mice's main organs as well.The grafted PEG moieties are generally believed to prevent the serum components from precipitating onto the surface of the complexes, which could in part form large aggregates and finally result in the loss of the biological activity. Furthermore, the attachment of the ligand on the tethered chain end of the shell-forming PEG segment of a PEGylated polyplex system should allow better accessibility of the ligand to its cellular receptor due to the spacer effect of PEG. In chapter 5, in order to increase the stability in physiological fluid as well as in vivo transfection efficiency of PPA/DNA complex, we conjugated PPA with terminally galactose-grafted PEG (Gal-PEG-PPA), and compared its physicochemical properties and transfection efficiency with Gal-PPA and unmodified PPA. MTT assay and the tissue response study revealed lower cytotoxicity and better tissue compatibility after the conjugation of PEG segment. The Gal-PEG-PPA/PPA/DNA ternary nanoparticles also showed a reversible interaction with RCA 120 and enhanced cellular uptake in HepG2 cells. In addition, Gal-PEG-PPA showed the highest transgene expression in the primary rat hepatocytes in the presence of 10% serum compared with those polymers without PEG segment. After intraportal vein injection, the significantly enhanced transgene expression mediated by Gal-PPA and Gal-PEG-PPA ternary nanoparticles was observed in liver compared with unmodified PPA. Incorporation of PEG segment would also enhance geneexpression in spleen, lung, kidney and heart, which is likely due to the prolonged circulation of the nanoparticles. The luciferase expression in rats' liver at the 3rd day after bile duct injection of PPAs/DNA nanoparticles was also evaluated. Generally, transfection efficiency in liver by the bile duct was about 10-fold higher than by the portal vein. Enhanced transgene expression mediated by both modified PPAs was observed in all liver lobes, and Gal-PEG-PPA ternary nanoparticles mediated the highest transgene expression in liver, which was 4-fold higher than Gal-PPA ternary nanoparticles and 33-fold higher than PPA-DEA/DNA complexes.Gene delivery depends on the ability to preferentially target specific cell types with limited toxic side effects and allow release of the DNA from endocytic compartments into the cytoplasm. The latter one is believed to be the limiting step in poor gene delivery mediated by many cationic polymers. To overcome this obstacle, chapter 6 develop a series of imidazole-containing polyphosphoramidates (PPA) as gene carriers, which were designed to condense DNA through protonated primary amines and mediate endosomal escape with the ionizable imidazole groups via the hypothesized proton sponge effect. Their chemical structure and the amount of imidazole groups were confirmed by 'H NMR. The incorporation of imidazole groups improved PPAs' buffer capacity in the pH range of 5 to 7 as well as reduced cytotoxicity of PPA carriers. However, gel retardation assay demonstrated that the incorporation of imidazole groups interfered with the electrostatic interaction between PPA and DNA. Based on the theoretical calculation, we found the net positive charge of PPA carriers decreased significantly after conjugation of imidazole groups, which accounted for the poorer DNA binding ability. The complexes prepared with imidazole-containing PPAs were observed to increase in size and decrease in zeta potential, which consequently resulted in significant decrease of transfection efficiency. It seems necessary to optimize the design of imidazole-containing PPA conjugate, which should maintain the favorable buffer capacity and low cytotoxicity without the loss of DNA compaction ability.