Application of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) for Lipid-based Nanomedicine Development

Abstract

Secretory phospholipase A2 (sPLA2) are increased in various cancers. The lipid specificity and reactivity of sPLA2 and its ability to interact with PLA2 receptors (PLA2R) are potential targets for development of liposome drug delivery systems. However, many phospholipids used to prepare liposomal formulations can be found endogenously and have biological isomers that complicate quantitative analysis. Thus, the challenges associated with the development and optimization of liposome nanoparticles are the difficulty to extract them from biological milieu and to distinguish liposome formulations and their metabolites from endogenous phospholipids. The purpose of this work is to establish a quantitative method that is capable to distinguish and quantify liposomes and their metabolites in biological samples. To achieve this purpose, we incorporated deuterated lipids in our sPLA2 responsive liposomes (SPRL) and developed an acidified Bligh-Dyer extraction method in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to evaluate their intracellular uptake and degradation in prostate cancer. Chapter 1 is an overview of the technical challenges to evaluate intracellular uptake and disposition of lipid-based Nano-carriers. The challenges are stated in two aspects of developing a quantitative method: extraction and differentiation. The current available methods that researchers use to overcome these challenges are described as well. Chapter 2 addresses the extraction challenge. We tested the extraction efficiency of 6 commonly used phospholipids for lipid-based nanomedicine using Bligh-Dyer (BD) extraction method, and proposed two different acidification procedures for certain anionic phospholipids with a poor extraction efficiency using conventional BD extraction: DSPA, one-step acidification in aqueous solution before extraction; DSPE, two step-acidification before and after extraction. Chapter 3 introduces the development of LC-MS/MS method for differentiation and quantification of liposome. Deuterated (d70)-1,2-distearoyl-sn-glycero-3-phosphocholine (d70-DSPC) was used as a substitute for DSPC to increase the uniqueness of SPRL formulations, which has no changes on the physiochemical or biochemical properties of the formulations. Both deuterated parent phospholipid (d70-DSPC) and one of its metabolites (d35-LysoPC) were able to be distinguished and quantified. The method was validated for its specificity, linearity, sensitivity (LOD and LLOQ), accuracy and precision. Chapter 4 is the application of the newly developed LC-MS/MS method for in vitro and in vivo evaluation of intracellular uptake and disposition of SPRL. The experiments were carried out on human prostate adenocarcinoma (PC-3) cells and mouse xenograft model of PC-3 cells implanted subcutaneously in athymic NCr (nu/nu) mice. The method provided detailed uptake and disposition information that pre-existing methods weren’t able to, which is critical for understanding uptake mechanisms and further optimizing the formulation. The establishment of this new LC-MS/MS method fills in the technical gap for evaluating in vivo performance of lipid-based nanomedicine. This method strengthens the ability to evaluate and optimize lipid-based drug carriers such as liposomes. Such tools are critical to gaining mechanistic insights into the distribution and intracellular fate of nanomedicines.