ASSESSMENT OF THE BEHAVIOR OF GEMINI SURFACTANT NANOPARTICLES WITHIN BIOLOGICAL SYSTEMS USING MASS SPECTROMETRY
Date
2020-01-15
Authors
Journal Title
Journal ISSN
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Publisher
ORCID
Type
Thesis
Degree Level
Doctoral
Abstract
Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as non-viral gene delivery vectors. However, the biological behavior of gemini surfactant nanoparticles has not been well understood and little is known about their cellular uptake, distribution, and metabolism after entering a biological system. Such knowledge is of great importance as it could explain their varying efficiencies and toxicities, and ultimately contribute to the development of novel gemini surfactants with enhanced efficiency and reduced toxicity. Therefore, my Ph.D. research investigated the biological behavior of gemini surfactant nanoparticles using mass spectrometry with a focus on their quantitative determination in the cellular matrix and identification of their potential metabolites.
To determine the cellular uptake and distribution of gemini surfactants, a simple flow injection analysis-tandem mass spectrometry (FIA-MS/MS) method was developed and validated for the quantification of three model gemini surfactants, unsubstituted (16-3-16), with pyridinium head groups (16(Py)-S-2-S-16(Py)), and substituted with a glycyl-lysine di-peptide (16-7N(GK)-16), in the cellular matrix. To our knowledge, this is the first FIA-MS/MS method that was developed for the determination of three gemini surfactants belonging to different structural families. The method is superior to previous liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, developed in our laboratory, with respect to sensitivity and time of analysis. The application of the method allowed for a time-course monitoring of the cellular uptake and subcellular distribution of gemini surfactants.
Differential cellular uptake and distribution were observed among three gemini surfactants, which explained their varying efficiencies and toxicities. In general, high cellular uptake of gemini surfactant corresponds to high transfection efficiency, while non-preferential accumulation in the nucleus may have contributed to the observed toxicity. The gemini surfactant 16-7N(GK)-16 displayed the highest cellular uptake among three compounds, consistent with its high efficiency in gene transfection; whereas 16(Py)-S-2-S-16(Py) showed the highest distribution in the nucleus, corresponding to its high toxicity. In addition, the DNA binding capability and the shape of aggregates of the gemini surfactants explained their different behaviors in biological systems. The gemini surfactant 16-7N(GK)-16 has a balanced DNA binding property and tends to form aggregates with flexible bilayer structures, resulting in its high efficiency and low toxicity. At the same time, 16(Py)-S-2-S-16(Py) and 16-3-16 display relatively strong DNA binding properties and have cylindrical micelle aggregates, leading to their low efficiency and high toxicity.
Different metabolic pathways for the three gemini surfactants were also determined for the first time. The gemini surfactant 16-3-16 was not metabolized in PAM 212 cells, which suggests it most likely remains intact during cellular detoxification and elimination. On the other hand, 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I biotransformations, including oxidation and dealkylation. Finally, the gemini surfactant 16-7N(GK)-16 was metabolized mainly via phase II biotransformations, such as methylation, acetylation, and conjugations (glucose, palmitoyl and stearyl conjugations). The metabolism studies of gemini surfactants provide insight for future directions in the design and development of novel compounds with low toxicity for gene delivery.
Together, the work accomplished in this Ph.D. study lays the foundation for investigating the behavior of gemini surfactant nanoparticles in biological systems and feeds into the rationale for designing novel gene delivery systems. Ultimately, the results would contribute to the development of more effective and less toxic novel gene delivery systems.
Description
Keywords
Gemini surfactants, Gene delivery, Subcellular distribution, Efficiency and toxicity, Metabolites, Mass spectrometry
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Pharmacy and Nutrition
Program
Pharmacy