Preeti Yadava

Effect of Lyophilization and Freeze-thawing on the Stability of siRNA-liposome Complexes

The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65–75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA’s intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased (∼50–70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.

Liposomal siRNA Delivery

With the recent discovery of small interfering RNA (siRNA), to silence the expression of genes in vitro and in vivo, there has been a need to deliver these molecules to the cell nucleus. Forming a lipid/nucleic acid complex has become a solution and is explored here. Certain methods and ideas are used, such as: the positive/negative electrostatic interaction with a cationic lipid and an anionic RNA molecule, the size of the lipid vesicle aiding the uptake target tissues, targeted lipoplexes which can increase efficiency, and the protection of the siRNA molecule from the natural defenses of the immune system. Many lipid formulations exist and can be experimented with to achieve varying results depending on the application.

Rationally Designed Synthetic Vectors for Gene Delivery

Vector development is one of the most important challenges facing the successful use of genes for treatment of diseases. Although chemically produced vectors offer distinct advantages over biological systems such as viruses, there are still some hurdles that have to be overcome before synthetic gene delivery vectors can be successfully implemented. This brief review discusses the biological barriers that limit current delivery strategies and reviews currently employed strategies for plasmid delivery. Nanoparticle-based gene delivery is reviewed along with methods for their characteriza- tion, physiochemical properties and toxicity. Finally a prospectus is provided for future development of an ideal synthetic gene delivery vector. GENE THERAPY Gene therapy involves the use of exogenous DNA as the therapeutic agent. Originally it was targeted towards treat- ment of inheritable single-gene disorders caused due to an absent or defective gene. However, applications of gene therapy have expanded to include treatment of acquired and infectious diseases and exogenously administered genes now are used in a wide variety of applications including immu- nomodulation, genetic vaccination and genetic pharmacol- ogy (1-6).

Liposome fluidity alters interactions between the ganglioside GM1 and cholera toxin B subunit.

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.

Sulfhydryl based cationic surfactants and the impact of polyanions on disulfide bond formation: implications for gene transfer vectors.

Compacting plasmid DNA (pDNA) into a small size is a fundamental necessity for the efficient in vivo transfer of nucleic acids to somatic cells. An approach for accomplishing this is to condense pDNA using cationic detergents with sulfhydryl groups, near their critical micelle concentration. In this study, a model surfactant was used to study how the rate of disulfide bond formation relates to environmental factors. It was shown that the thiol detergent had the ability to form a disulfide bond when oxidized and the presence of polyanions was significantly increased. The addition of a reducing agent disrupted the disulfide bonds initially, but this was followed by disulfide bond reformation in a short time period.

A gene delivery approach for antimicrobials: Expression of defensins

Introduction: Peptide antibiotics as new therapeutic agents are becoming a popular option to investigate due to their broad bacterial target selectivity and limited resistance problems. Although attractive, these new drug candidates have several limitations including low potency and delivery issues which face all peptides/proteins. Methods: In this study, we designed a plasmid expression system for human beta defensin 3. This sequence was cloned from a human epithelial lung cell into a CMV driven expression cassette. This expression plasmid was then evaluated for its ability to produce human-beta defensin 3 with the use of the non-viral transfection agent, polyethylenimine (PEI). Results: The results indicate the expression cassette was transcriptionally active in HEK 293 cells, as measured by RT-PCR and that a beta defensin peptide was produced by the cells as confirmed by Western blot. The biological activity of the peptide was confirmed against both gram negative E. coli and gram positive Bacillus species using in vitro screening. Conclusion: Both the cultured media as well as the transfected cell lysate demonstrated biological activity demonstrating the peptide is also secreted.

Nucleic Acid Cellular Delivery

The potential of using nucleic acids to correct a malfunctioning, nonexpressed gene or to supplement the production of a natural occurring protein has generated much enthusiasm from researchers and the lay public. Even though the potential for medical advances exists, there are still obstacles that must be overcome before this gene-therapy paradigm becomes a viable option. One of the more substantial obstacles is the delivery vector for the nucleic acids (e.g., plasmid DNA, oligonucleotides, siRNA, ribozymes, etc.). As a whole, nucleic acids offer unique challenges in the design and development of drug-delivery systems because of their poly anionic nature and their lack of resistance to metabolic degradation. Historically, the major theme in the design of modern nucleic acid delivery systems is to mimic the viral method of nucleic acid transfer.