Development of peptide conjugated nanoparticles as drug delivery system for specific targeting to Leukemia cell lines

Abstract

Leukemia is the malignant disease of blood-forming in bone marrow characterized by an abnormal increasing of immature blood cells. The advance therapeutics of leukemia are endeavoring for improved the efficacy and specificity of treatments. Active targeting carrier is becoming such one candidate for leukemia therapeutic by targeting molecular markers differentially expressed on cancerous cells. Thus, the targeting marker present as an important factor for the development of active targeting carriers. The aim of this thesis is to develop the specific drug deliver system for leukemic cells. Here, lymphocyte function-associated antigen-1 (LFA-1) was used as the targeting receptor for this system due to the differential expression on various types of leukemia cells. The role and expression of LFA-1 on leukemic cells and the possibility of using this integrin as a target included where cIBR, a cyclic peptide derived from a binding site of LFA-1, was conjugated to the surface of polymeric nanoparticles and used as a targeting ligand. PLGA nanoparticles were prepared by solvent displacement method. The method of conjugation was provided by carbodiimide reaction. Four different types of leukemic cell lines (Molt3, Molt-4, HL-60, and U937) were investigated the expression of LFA-1 and the degree of cellular uptake. Molt-3 cells showed the highest level of LFA-1 on their membranes when compare to the other cell lines. In comparison of the cellular uptake of untargeted naopaticles (NPs) and cIBR-conjugated nanoparticles (cIBR-NPs), the results presented that cIBR-NPs showed the greater degree of uptake when compared to untargeted NPs, indicated the targeting properties of cIBR-NPS. The cellular uptakes of cIBR-NPs by Molt-3 cells were also showed the highest degree of uptake, significantly. These studies revealed a correlation of LFA-1 expression level on leukemic cell lines and binding and internalization of cIBR-NPs suggesting a differential binding and internalization of cIBRNPs to leukemic cells overexpressing LFA-1. Due to the expression of LFA-1 on cancer cell diverges from the physiological condition, thus it is the attractive point to investigate the cellular binding of cIBR-NPs to LFA-1 on the leukemic cell line and PBMC, a normal leukocyte, to prove the selectivity of this system in the condition that consist of normal leukocyte and leukmic cell line lead to the safety profile of cIBR-NPs. LFA-1 on the PBMCs, Mixed PBMC-Molt-3 cells and Molt-3 were determined the expression and were used as the targeted receptor. The expressions of LFA-1 on Molt-3, from flow cytometry and Western blot, possessed the highest level whereas PBMCs showed the lowest level. The kinetic binding profiles of cIBR-NPs were obtained by flow cytometry. The degree of cellular uptake presented a similar trend with the level of LFA-1 indicating a correlation with the level of expression and degree of uptake via LFA-1. the co9nformation change of LFA-1 had a slight effect on the function of cellular binding of cIBR-NPs. Take the advantage from LFA-1/ICAM-1 interaction, cIBR peptide derived from the ICAM-1 was also utilized as the targeting moiety for paclitaxel loaded nanoparticles for direct targeting to LFA-1 expressing cancer cells. The two different methods of preparation, solvent displacement and emulsification evaporation, were employed in this thesis to provide the optimal method of paclitaxel-loaded nanoparticles’ preparation. The three different sequences of conjugation were employed to compare the effectiveness of each method in the terms of physicochemical properties, the density of peptides, drug entrapment effiency, cellular uptake and cytotoxicity. The developed formulation and conjugation techniques given small particle size with narrow size distribution, negatively charge and high degree of uptake were discussed. Overall the results demonstrated that cIBR-NPs enhanced cellular uptake and improved the selectivity of drug carriers to LFA-1 on the leukemia cells, which related with the order of LFA-1 expression. Nanoparticles conjugated with a cyclic peptide against an accessible molecular marker of disease hold promise as a selective drug delivery system for leukemia treatment.