Objective The use of lipid-based pharmaceutical nanocarriers, such as lipid-core polymeric micelles and liposomes have improved the pharmacokinetic and pharmacodynamic properties of many pharmaceuticals, especially in chemotherapy. These delivery systems have not only helped in increasing the solubility of poorly soluble chemotherapeutic drugs but have also acted as reservoir of large molecular weight active pharmaceutical ingredients (API) like proteins. The objective of the thesis was to explore surface modification options to further enhance the delivery of pro-apoptotic small molecules for cell-specific delivery and proteins for intracellular delivery. The thesis has been divided into two sections.
Section a The purpose of this study was to enhance the delivery of new pro-apoptotic small molecule, N-{[(2-hydroxy-5-nitrophenyl)amino]carbonothioyl}-3,5-dimethylbenzamide (DM-PIT-1), which is the non-lipid antagonist of phopshatidylinositol-3.4.5-triphopshate and inhibitor of the PI3-kinase pathway. Micelle-forming PEG2000-PE was used to solubilize DM-PIT-1, which has an aqueous solubility of 30 uM. To further improve the delivery of the micellar DM-PIT-1, cancer-targeting anti-nucleosomal mAb2C5 antibodies and/or Tumor necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) were attached to the surface of polymeric micelles.
Methods Plain micelles and surface-modified micelles (mAb2C5 and TRAIL) loaded with DMPIT-1 were prepared and characterized by dynamic light scattering. Drug loading efficiency was determined by HPLC. Cell viablity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) method.
Results DM-PIT-1 was effectively incorporated (> 70%) into 14–16 nm micelles, which had a negative surface zeta potential of 4–5 mV. Micellar DM-PIT-1 demonstrated high in vitro cytotoxicity against various cancer cells. An improved potency of the dual-activity DM-PIT-1/TRAIL combination nanoparticles in inducing death of TRAIL-resistant cancer cells was noticed. Similar effect was found mAb 2C5 conjugated DM-PIT-1 loaded micelles. The efficacy of the TRAIL therapy was enhanced by combining it with the mAb 2C5 DM-PIT-1 loaded micelles.
Section b Part 1 The purpose of this study was to develop octadecyl-rhodamine B (Rh)-modified liposomes loaded with fluorophore, FITC-dextran (FD), to achieve efficient lysosomal targeting and delivery.
Methods Plain or Rh-modified liposomes (Rh mol 1% and 3 mol %) were prepared from a mixture of phosphatidylcholine/cholesterol (7:3 molar ratio) by hydration of lipid films in PBS supplemented with FD. Alternatively, plain and Lip-Rh were loaded with 5-dodecanoylamino fluorescein di-b-D-galactopyranoside (C12FDG), a specific lipophilic substrate for the lysosomal enzyme β-galactosidase. HeLa cells were incubated with plain or Rh-modified liposomes. Delivery to lysosomes was investigated by confocal microscopy, flow cytometry and subcellular fractionation methods.
Results Incubation of HeLa cells with Lip-FD-Rh led to the accumulation of FD primarily in the lysosomes, which was evident from high rates of co-localization with specific lysosomal markers (Lysotracker Red and Lamp-2 antibody). The cells treated with the same concentration of plain Lip-FD showed low localization of FD in the lysosomes. Comparison of the fluorescence intensity of lysosome-enriched fractions showed that the efficiency of lysosomal delivery of FD by Rh-modified liposomes was 2-fold higher compared to plain liposomes. These results were confirmed by the flow cytometric analysis of the live intact cells treated with C12FDG-loaded liposomes, which showed increased lysosomal targeting by Rh-modified liposomes.
Section b Part 2 The purpose of this study was to develop octadecyl-rhodamine B (Rh) modified liposomes loaded with VPRIV® (velaglucerase alfa for injection), a lysosomal glucocerebroside-specific enzyme indicated for long-term enzyme replacement therapy (ERT), and determine the lysosomal targeting efficiency of the delivery system.
Methods Plain or Rh-modified liposomes (Rh mol 1%) were prepared from a mixture of phosphatidylcholine/cholesterol (7:3 molar ratio) by hydration of lipid films in PBS supplemented with VPRIV®. Alternatively, plain and Rh1%-lip were loaded with FD40K. Gaucher's fibroblasts and monocyte derived macrophages (MDMs) were incubated with plain or Rh-modified liposomes. Delivery to lysosomes was investigated by confocal microscopy and flow cytometry methods.
Results Incubation of Gaucher's fibroblasts and MDMs with Rh1%-liposomes led to accumulation of VPRIV primarily in the lysosomes which was evident from the resultant fluorescence intensity when the treated cells were incubated with a lysosome-specific substrate. The cells treated with the same concentration of plain liposomes showed lower targeting. Comparison of the fluorescence intensity showed that the efficiency of lysosomal delivery of enzymes by Rh-modified liposomes was 1.5-fold higher compared to plain liposomes.
Conclusions The lipid-core polymeric micelles form increase the solubility of poorly soluble DM-PIT-1. Their surface modification with mAb2C5 or TRAIL enhances the cell killing effect of this novel chemotherapeutic agent. This system can be used for targeted combination therapy against TRAIL-resistant cancers.
The modification of the liposomal surface with octadecyl-rhodamine B significantly increases the delivery of liposome-loaded FITC-dextran to lysosomes. Therefore, the delivery system was applied to Gaucher's fibroblasts and MDMs, which is a Gaucher's cell model. The octadecyl-rhodamine-modified liposomes significantly increased the delivery of liposome-loaded VPRIV into the lysosome.
These in vitro studies open up avenues for better targeting of therapeutic proteins like VPRIV (known for developing antibodies when administered in free form), encapsulated in reservoir-type nanocarrier systems.
