Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Pharmaceutical Sciences

Research Advisor

Ram I. Mahato, Ph.D.


Ramareddy V. Guntaka, Ph.D. Bernd Meibohm, Ph.D. Duane D. Miller, Ph.D. Lawrence M. Pfeffer, Ph.D.


The objectives of the study were to investigate and develop lipid and polymeric carriers for nucleic acid delivery. These included: i) to develop novel cationic lipids for plasmid, oligonucleotide, and siRNA delivery; ii) to develop a novel polymeric delivery system, polyethylene glycol (PEG) based bio–conjugate, for oligonucleotide delivery; iii) to develop a novel bio–conjugate delivery system for siRNA delivery.

In Chapter 2, we discussed the barriers and strategies of nucleic acid delivery, as well as summarized the commonly used lipids, polymers, and the corresponding carriers in terms of their characteristics, applications, advantages and limitations.

Cationic lipids are most commonly used transfection reagents in delivery of nucleic acids to target cells in vitro. In Chapter 3, we synthesized a series of pyridinium lipids which contain a heterocyclic ring and a nitrogen atom. The structure–activity relationship (SAR) and formulation of corresponding cationic liposomes were studied for gene and siRNA delivery. The pyridinium lipids were mixed with a co–lipid, such as 1,2–dioleoyl–sn–glycero–3–phosphoethanolamine (DOPE) and cholesterol, to prepare cationic liposomes by sonication. These liposomes were mixed with plasmid DNA and transfected into CHO cells. Several factors including hydrophobic anchor chain length, anchor chain type, configuration of double bond, linker type, co–lipid type, cationic lipid/co–lipid molar ratio, charge ratio (N/P), concentration of serum, and cell type had significant influence on transfection efficiency and cytotoxicity. Pyridinium lipids with amide linker showed higher transfection efficiency compared to their ester counterparts. Liposomes prepared at a 1:1 molar ratio of pyridinium lipid and co–lipid showed higher transfection efficiency. Pyridinium lipids based on a hydrophobic anchor chain length of 16 showed higher transfection efficiency and lower cytotoxicity. The trans–isomers of pyridinium lipids showed higher transfection efficiency than the cis–isomers at the same fatty acid chain length. In the presence of serum, C16:0 and Lipofectamine significantly decreased their transfection efficiencies, which were completely lost at a serum concentration of 30% and higher, while C16:1 trans–isomer still had high transfection efficiency under these conditions. The optimized formulation was further investigated in delivery of siRNAs and showed equal or higher gene silencing effect at the low dose of siRNAs compared to Lipofectamine 2000.

To avoid use of polycations, in Chapter 4, we conjugated galactosylated poly(ethylene glycol) (Gal–PEG) to ODN via an acid labile ester linkage of β–thiopropionate. The conjugate was purified by RP–HPLC and verified by polyacrylamide gel electrophoresis. To determine the biodistribution and pharmacokinetic parameters of Gal–PEG–ODN, ODN was radiolabeled by 33P before the conjugation reaction. Following tail vein injection into rats, Gal–PEG–33P–ODN rapidly cleared from circulation and

60.2% of the injected dose accumulated in the liver at 30 min post–injection, which was significantly higher than that deposited after injection of 33P–ODN. The plasma concentration versus time profile of Gal–PEG–33P–ODN was biphasic, with 4.38 ± 0.36 min as t1/2 of distribution and 118.61 ± 22.06 min as t1/2 of elimination. Prior administration of excess Gal–BSA decreased the hepatic uptake of Gal–PEG–33P–ODN from 60.2% to 35.9%, suggesting galactose triggers the asialoglycoprotein receptor–mediated endocytosis of Gal–PEG–33P–ODN by hepatocytes. A large proportion of the injected Gal–PEG–33P–ODN was taken up by the hepatocytes as evidenced by determination of radioactivity in the digested liver cells upon liver perfusion and separation by centrifugation on a Nycodenz gradient.

Although the potency and specificity of siRNA was demonstrated, so far, siRNA has not been successfully used as a clinical therapeutic due to its short circulation time in blood stream, non specific tissue or cell targeting, and insufficient intracellular transport. In Chapter 5, a similar strategy was used to design siRNA conjugates. To target to hepatocytes and hepatic stellate cells, galactose and M6P were used as the ligands respectively to synthesize Gal–PEG–siRNA and M6P–PEG–siRNA. In this study the cleavable disulfide bond was introduced between siRNA and PEG to ensure siRNA dissociation from the conjugate in the reducing environment in cytoplasm. After conjugation reaction, the conjugate was purified by ion exchange HPLC and verified by gel retardation assay. After treatment with DTT, the conjugated siRNA was disassociated from its conjugate and verified by gel retardation assay. To evaluate the gene silencing ability of siRNA conjugate, an effective luciferase siRNA sequence was designed and conjugated with Gal–PEG and M6P–PEG. Then Gal–PEG–siRNA and M6P–PEG–siRNA were transfected with luciferase expression HepG2 cells and rat HSCs respectively. We found both conjugates could down–regulate the luciferase gene expression for about 40% without any transfection reagents, while the gene down–regulation level reached more than 98% with the help of cationic liposomes at the same dose.

In conclusion, we synthesized a series of pyridinium lipids and studied their SAR and corresponding liposomal formulations. We found pyridinium lipids showed high transfection efficiency and had the potential to be used as transfection reagents in vitro. The polymeric conjugate delivery systems, Gal–PEG–ODN, Gal–PEG–siRNA and M6P–PEG–siRNA were successfully designed and developed. The in vitro and in vivo studies showed that the conjugate delivery systems could effectively deliver nucleic acids into the target cells, release their cargo, and manipulate the target gene expression. These research works strengthened the development of lipid and polymeric carriers as the effective nucleic acid delivery systems.