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Improved Nanoparticle Preparation and Delivery Technology for DOTAP and Oligonucleotide Based Lipoplexes

Terp, Megan Cavanaugh

Abstract Details

2012, Doctor of Philosophy, Ohio State University, Chemical and Biomolecular Engineering.

The field of nucleic acid based therapeutics offers treatments for diseases at the most basic level of cellular biochemistry and its potential is boundless. Oligonucleotide therapeutics is also an invaluable tool in studying cellular processes and the precise effect certain genes have on disease models. Since passive cellular uptake of these highly charged molecules is low, an effective delivery method is imperative in order to achieve an observable effect. Lipid based nanoparticles with their numerous, well-documented advantages are often used for delivery and in this work, both pharmaceutical and engineering based approaches were explored to construct lipoplex nanoparticles. From the pharmaceutical side, the effect of lipid chirality on transfection efficiency was investigated. From an engineering perspective, even though liposomal nanoparticles represent an especially promising class of drug delivery vectors, conventional bulk mixing preparation methods have room for improvement with regards to yield and consistent control over structure and composition. Therefore, a device-based approach using arrays of microwells was studied to not only deliver particles directly to cells in a more controlled way but also to introduce control over the typically uncontrolled complexation process of lipids and nucleic acids.

DOTAP, as a racemic mixture, is a cationic lipid and a widely used transfection reagent. In this study, racemic and enantiomerically pure DOTAP were used in lipoplex formulations to deliver siRNA to MCF-7 cells, targeting the aromatase enzyme. The R enantiomer of DOTAP was found to be more efficacious than the S enantiomer or the racemate when used in combination with cholesterol. Specifically, the aromatase activity of cells treated with R-DOTAP lipoplexes was 50% lower than those treated with S-DOTAP or racemic lipoplexes at a 10 nM siRNA concentration. In other words, R-DOTAP lipoplexes were twice as effective at the low concentration. Amongst the DOTAP enantiomers, differences in lipid packing were found using membrane simulations and a differential cellular uptake pathway was found using confocal microscopy. These findings suggest an important role of lipid chirality in future development of lipid based drug delivery systems.

Directed delivery using microwells is a novel and simple way to use at least 10x less material for a successful transfection relative to bulk mixing, thereby improving yield. Microwell directed delivery of DOTAP-based lipoplex formulations was successful in delivering ODN in a linear, dose dependent manner to A549 lung cancer cells and MCF-7 breast cancer cells that was superior to traditional bulk mixing. Directed delivery of ODN was found to be up to 18x more effective than bulk mixing at equivalent doses. Microwell delivery of miR29-b to A549 cells resulted in mature miR29-b expression that 550x times more than untreated or bulk mixing using only 22 ng of material, compared to the μg quantities typically required. These promising results can be applied to many systems to improve performance and decrease the required amount of expensive, nucleic acid based biomolecules.

Directed assembly using microwells offers a novel way to achieve much tighter control on the particle structure and composition and it was found to better control the particle complexation process by allowing the components to interact in defined mini-reactors. Polymeric microwells were filled with pre-defined amounts of lipids and siRNA, thereby exactly controlling the particle composition and directing their assembly into particles. Fluorescence microscopy showed lipoplexes were formed in each well, and cryoTEM and AFM images suggested that siRNA molecules were not packed away into inaccessible interior layers. These simple and effective device based technologies could easily be expanded to many different nucleic acid and liposome formulations, and consequently lead to widespread clinical applications.

L. James Lee (Advisor)
Robert J. Lee (Advisor)
Jeffrey J. Chalmers (Committee Member)
Zhonga Liu (Committee Member)
200 p.

Recommended Citations

Citations

  • Terp, M. C. (2012). Improved Nanoparticle Preparation and Delivery Technology for DOTAP and Oligonucleotide Based Lipoplexes [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338301430

    APA Style (7th edition)

  • Terp, Megan. Improved Nanoparticle Preparation and Delivery Technology for DOTAP and Oligonucleotide Based Lipoplexes. 2012. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1338301430.

    MLA Style (8th edition)

  • Terp, Megan. "Improved Nanoparticle Preparation and Delivery Technology for DOTAP and Oligonucleotide Based Lipoplexes." Doctoral dissertation, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338301430

    Chicago Manual of Style (17th edition)