Delivery of cytotoxic agents using low density lipoprotein (LDL) - physico-chemical and biological evaluation of LDL-drug conjugates

Low density lipoprotein (LDL) particles appear very promising for delivering anticancer drugs specifically to tumor or macrophage cells by exploiting the LDL or the scavenger receptor pathway. I have chosen doxorubicin [Dox] to investigate the feasibility of this approach in cancer chemotherapy. Dox could be directly incorporated into LDL particles. A lipophilic cholesteryl ester [CE] analogue, cholesteryl iopanoate [CI] was proposed as a radiotracer to study the in vivo fate of LDL. CI was radioiodinated with ¹²⁵I by a pivalic acid exchange reaction and the radiochemical purity was determined by HPLC in conjunction with γ-counting and was found to be more than 95% pure. -- A new reverse phase HPLC procedure with UV detection was developed for the quantitation of CI. The regression line, and intra- and interday variations for a set of standards were determined and were found to be statistically valid. The minimum detection range was less than 10 ng for the compound. The % recovery from LDL was found to be more than 95%. Plasma protein binding of Dox was studied ex-vivo. Dox was found to be more than 30% lipoprotein bound. The plasma distribution of Dox was refashioned by preincubating plasma with oleic acid. With oleic acid, Dox association with lipoproteins increased from less than 30% to approximately 70%. -- Mainly the contact method or the direct addition method was adapted to incorporate drugs into LDL particles. The loading techniques were optimized in terms of incubation time, temperature, and stoichiometry of LDL-drug conjugates. A four to six hour protocol and 37° incubation temperature were chosen with a drug to protein ratios more than 1. The effect of various wetting agents, such as Tween 20,40, 60 and 80, Span 60, Triton-X, and Celite 545, and ethanol was investigated for LDL-Dox conjugates in the contact method. Tween 20 was chosen for its favorable loading efficiency (more than 5-fold compared to the dry film method). Liposomal Dox was prepared by an extrusion technique with 24% loading efficiency. The liposomal Dox preparation was found to be the most suitable (45 molecules of Dox/LDL particle) compared to other methods such as the dry film method, the contact method with Tween 20, and the direct addition method. -- All these incorporation methods were found to be suitable for generating LDL-drug conjugates without disrupting the native integrity of LDL particles when characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE], electron microscopy [EM], and differential scanning calorimetry [DSC]. -- An insect lipid transfer catalyst [LTP] was studied and found to enhance drug loading into LDL particles by at least 2 to 5-fold, depending on the drug molecules and the incubation conditions. The LDL-drug conjugates generated by this transfer particle were characterized by SDS-PAGE and EM and found to be similar to native LDL. The site of drug location in LDL was studied by DSC and UV-visible scanning. The drug was found to be located both in the core and the outer monolayer of LDL for Dox. This kind of enhancement was not observed with human cholesterol ester transfer protein [CETP]. -- Dox interference in bicinchoninic acid [BCA] protein assay method was examined and it was found that Dox interfered [33-fold more sensitive compared to protein] with the protein assay method. A solution to overcome this interference was also suggested using the Bradford method. -- To target macrophages [Mϕ], native LDL was modified by acetylation [acetylated LDL, AcLDL] and Dox was loaded in AcLDL and physico-chemically characterized by SDS-PAGE and EM. The loading efficiency of AcLDL-Dox conjugates was comparable to that of LDL-Dox conjugates. -- Different formulations of Dox were evaluated in cell culture studies using a human tumor cervical cell line, HeLa, and a mouse Mϕ cell line, J774.A1. LDL-Dox conjugates were found to be greater than 18-fold more cytotoxic than the corresponding free drug Dox using the [3-[4,5-dimethylthiazoyl-2-yl]-2,5-diphenyl-tetrazolium bromide], [MTT] assay in HeLa cells. When the cytotoxicity of AcLDL-Dox conjugates was examined in J774.A1 cells, a more than 7-fold increase in cytotoxic effects was observed in comparison to its free drug counterpart, Dox