Phospholipases hydrolyze the phosphodiester bonds of phospholipid substrates releasing fatty acids and lysophospholipids. Phospholipases can be classified into five superfamily enzymes based on their differences in hydrolyzing the substrate. One of them, phospholipase A2 (PLA2) cleaves the ester bond at the sn-2 position of the phospholipids. Secreted phospholipases A2 (sPLA2) are the cell-secreted PLA2s ubiquitously present throughout the body. The sPLA2s are smaller in size and structurally sturdier than their PLA2 congeners due to the presence of several disulfide bonds in their tertiary structure. These enzymes dock on the bilayer membrane and work at the water/oil interface of lipid supramolecular assemblies, unlike other hydrolase enzymes, which process their substrates individually. Different subclasses of sPLA2 have preferences for different phospholipids, withPLA2-GIB hydrolyzing PCs more than PLA2-GIIA as a typical example. Drug-loaded liposomal membranes could be impaired by sPLA2, triggering drug release at the site of sPLA2 expression. This class of enzymes is therefore an interesting target for developing drug delivery systems for diseases where PLA2s are overexpressed, such as inflammation, rheumatoid arthritis, and various cancers. In order to develop a delivery system targeting PLA2, one needs an assay system for PLA2s based on a natural substrate (phospholipid-based). A limited number of studies were found in which PLA2 kinetics were assessed using a natural phospholipid-based substrate, with none recent. Thus, in Chapter 2, we developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye, bromothymol blue (BTB), in which different POPC self-assemblies (liposomes or mixed micelles with Triton X-100) were used to assess the enzyme activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies, and we determined the kinetic parameters of sPLA2 isozymes IB and IIA for each supramolecular POPC assembly. We also validated the assay using the standard sPLA2 inhibitor varespladib. In Chapter 3, we studied the interaction of PLA2 with bilayer vesicles (lipid/polymer-based) and we found that this interaction varies depending on the distribution of PEG corona on the bilayer surface. Within this framework, we have investigated the action of phospholipases PLA1 and PLA2 towards destabilizing liposomes made of natural glycerophospholipids. We studied the impact of co-lipids such as cholesterol and poly-ethylene-glycol (PEG)-conjugated lipids such as DSPE-PEG2000 on the stability of these liposomal delivery systems against PLA1 and PLA2-mediated hydrolysis, using calcein as a model encapsulated drug. Continuing the studies from Chapter 3, in Chapter 4, we developed a liposomal PEGylated (stealth) doxorubicin formulation that is metastable – relatively stable in circulation (but with a t1/2 less than DOXIL®), aimed to reduce the PPE (Palmar-Plantar Erythrodysesthesia) side effect of DOXIL® but vulnerable to sPLA2 enzymes expressed in certain breast cancers. We loaded the liposomes with doxorubicin, purified and characterized them, and subsequently subjected them to sPLA2 action to assess the impact of sPLA2 on their stability and DOX release effect on MDA-MB-231 as a triple-negative cancer cell model expressing sPLA2s. In Chapter 5, we optimized a delivery system with a slower drug delivery profile, based on biocompatible/biodegradable PLGA ester copolymer. PLGA polymer-based nanoparticles were developed and optimized for loading with a hydrophobic model drug CAI-29. PLGA nanoparticle formulation was optimized using different stabilizers to maximize the encapsulation efficiency of the highly hydrophobic drug. The optimized drug-loaded formulation was also scaled up and tested on the same breast cancer cell model, MDA MB 231, for its ability to suppress the growth of this carcinoma.
Shibbir Ahmed Khan (Thu,) studied this question.