Author ORCID Identifier

https://orcid.org/ 0000-0003-3009-7686

Date Available

8-5-2022

Year of Publication

2022

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

Advisor

Vincent J. Venditto

Co-Director of Graduate Studies

David J. Feola

Abstract

Lipid based nanoparticles (LBNs) are used in myriad applications in medicine from small molecule drug delivery to mRNA vaccines. A major contributing factor to the development of the field has been the ongoing development of novel compounds that retain the functionality of natural lipids but expand upon them through inclusion of functional moieties that can be applied to specific scientific and biomedical questions. In the body of this dissertation, an extensive overview of LBNs is provided, focusing primarily on their use in immune modulation. The research presented herein begins with the synthesis of a novel class of lipids based on the triazine (TZ) cyanuric chloride. Twelve compounds were synthesized and assessed for their biophysical behavior and ability to form LBNs. Of the 12 compounds, 10 were able to form nanoparticles and these were assessed for in vitro toxicity. The toxicity of the nanoparticles differs based on the nanoparticle charge and approximate that observed for similarly charged compounds. The cationic TZ lipids were then tested in vitro for their ability to deliver plasmid DNA into cells where they showed improved efficacy compared with the cationic lipid DOTMA, and similar toxicity. Finally, TZ lipids were used to lipidate peptides in a liposomal peptide vaccine where they induced similar anti-peptide titers as a CHEMS conjugate. Following these experiments, the in vivo toxicity and potential for plasmid delivery was evaluated for the cationic TZ lipids. TZ lipids led to toxicity similar to other cationic lipids. Of note, the PEG length in the nanoparticles was studied for its effect on transfection efficiency as was the effect of the helper lipid in the formulation. These experiments showed improved transfection efficiency with DOPE and with shorter length PEG chains on the nanoparticle surface. Evaluation of immune responses toward the transgene studied showed a similar titer response as the free protein. However, when the protein was delivered with a cationic lipid as control, titers increased significantly, particularly for the TZ lipid used, which increased titers 1000-fold. These data provide evidence for continued evaluation of TZ lipids as gene delivery vectors and as potential vaccine adjuvants. Finally, in continuing the evaluation of LBNs to improve gene therapy, an LBN based system was evaluated to deplete anti-AAV8 antibodies. As one of the most promising strategies to deliver transgenes since AAV provides an excellent platform that is unfortunately affected by the presence of anti-viral antibodies. This system, using doxorubicin liposomes coated with recombinant VP1 protein bound to DGS-NTA-Ni lipid or DSPE-PEG2000-Maleimide, failed to deplete circulating antibodies to AAV. However, the results of the experiments carried out shed light on how this strategy might be improved upon at a later time. Finally, in an attempt to better understand the immune targets on AAV, the antibody response toward AAV8 was tested in human samples from deidentified blood donors and compared with that of mice and monkeys treated with the virus. Serum from these species was scrutinized for its ability to neutralize the virus in vitro and evaluated using a peptide array for targets against the viral capsid protein VP1. Collectively, the studies presented in the body of this dissertation demonstrate the utility of LBNs in gene delivery, both as vectors and as aids for viral delivery.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.280

Funding Information

UK College of Pharmacy

Lyman T. Johnson Award

NIH 5TL1TR001997-04

NIH P20GM130456

NIH RO1HL152081

AHA 17SDG32670001

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