Category: Formulation and Quality
Purpose: Bortezomib (BTZ) is a proteosome inhibitor, approved by U.S FDA in 2003, which was also effective against solid tumors. The present study was intended to evaluate biotinylated HPMA (N-2-hydroxypropylmethacrylamide) and PLA (poly-l-lactic acid) polymeric micelles (PMC) to deliver the anticancer drug. Biotin, a targeting ligand, is a brilliant guide to deliver therapeutic agent specifically to the tumors. The in vitro studies included drug loading capacity (DLC), drug entrapment efficacy (DEE), release, hemolytic toxicity, cell line studies, cellular uptake studies, and pharmacokinetic studies.
Methods: HPMA-biotin, HPMA-PLA (1) and HPMA-PLA-biotin (HPLA-BT) conjugates were synthesized and BTZ was encapsulated to fabricate nano-assemblies, PMC. HPMA-PLA was conjugated using DCC, DMAP and to this copolymer, biotin was conjugated via thionyl chloride in two steps. The synthesized conjugates were characterized by 1H-NMR, 13C-NMR, and FT-IR. The DLC, DEE and in vitro release studies were determined using dialysis method in phosphate buffer saline (pH 7.4). Hemolytic toxicity study was performed in human RBCs and cytotoxicity studies were performed against cancer cell lines (2).
Results: In FT-IR spectrum of HPMA-biotin, the peaks at 2925.6 cm-1 (CH3) 2861.8 cm-1 (-NH), 1630.6 cm-1 (-CONH), 3443.54 cm-1 and 1382.7 cm-1 peaks were observed. The conjugation of HPLA-BT was ensured by 1H and 13C NMR results. 1H NMR (500 MHz, DMSO) δ ppm: 9.64 [-NH, BT (a)], 4.43 [m -CH (b)], 1.13 [m -CH (e)], 1.53 [s CH3 (f)], 3.26 [d CH (d)], 2.94 [CH2 (c)]. 13C NMR δ ppm: 162.99 [CO (a)], 22.99 [CH3 (b)], 14.13 [CH3 (c)], 23.34 [CH2 (d)], 53.32 [CH2 (e)], 77.85 [m CH (f)] characterstics peak were noticed. The mean size of HPLA-BT was found to be 199.7±0.02 nm with 0.196±0.11 polydispersity index (PDI). The DLC and DEE of prepared PMC were found to be 19.73±0.03 and 82.20±0.14, respectively. In vitro release data of PMC showed sustained release pattern of encapsulated drug (Figure 1). PMC exerted less hemolysis than pure drug and was highly effective against MCF-7 breast cancer cell lines in the cytotoxicity studies (Figure 2).
Conclusion: HPMA-PLA-biotin was successfully conjugated, and PMC were prepared. In vitro release studies, cytotoxicity studies resulted into favorable delivery of BTZ using PMC approach. A sharp reduction in the IC50 value of the drug was encouraging for BTZ-HPLA-BT PMC than the pure drug. Further treatment of MCF-7 breast cancer cells with the prepared formulation in cellular uptake study (Figure 3) revealed that the higher uptake of formulations may be due to the biotin tethering and surface modification characteristics that enhanced the selectivity and targeting of formulations efficiently than the non-tethered biotin formulation such as HPLA PNPs. The in vivo kinetics results inferred the increase in bioavailability, half-life, and decrease in clearance rate confirmed the increment in the retention time of the formulations.
Umesh Gupta– Ajmer, Rajasthan, India