Track: Formulation and Delivery - Chemical - Drug Delivery - Nanoparticles
Category: Poster Abstract
Development and Evaluation of Transferrin Conjugated Amodiaquine Nanoparticles - Non-Small Cell Lung Cancer (NSCLC) Targeted Therapy
Purpose: Non-small cell lung cancer (NSCLC) is a malignant tumor type leading to very high mortality. Transferrin receptors are overexpressed in many NSCLC, playing a crucial role in cellular proliferation. Targeting these receptors through specific targeting ligands (Transferrin: Tf) is advocated to achieve superior anti-cancer efficacy. As current treatment is limited by presence of increased resistance towards chemotherapeutic agents, amodiaquine (AQ; FDA approved antimalarial) has been chosen due to its potent autophagy inhibition, anti-cancer efficacy in melanoma as reported in the literature and its feasibility in repurposing. We aim to develop transferrin conjugated AQ-loaded nanoparticles (Tf-AQ NPs) for targeted and efficient NSCLC treatment regimen, using an efficient process of high-pressure homogenization (HPH). Methods: Unconjugated and Tf conjugated AQ loaded nanoparticles were formulated by HPH at optimized conditions. Tf-PLGA was prepared by activation of PLGA through NHS/EDC reaction for 4 hours at room temperature, then incubated activated PLGA with Tf (100µg/ml) followed by collection of Tf-PLGA through addition of water on next day (1). Schematic representation of conjugation of Tf with PLGA can be seen in Fig. 1. Briefly, a pre-emulsion was formed from 50:50 PLGA/50:50 PLGA with Tf-PLGA and AQ with 1% polyvinyl alcohol solution using a probe homogenizer at 25,000 rpm, which was further processed through high pressure homogenizer (Nano DeBee) for 7 cycles at 30,000psi (2). Product was subjected for overnight stirring and was subsequently washed. Nanoparticles were evaluated for physicochemical properties, targeting capability (cellular uptake), in-vitro lung deposition behavior and therapeutic potential against NSCLC in-vitro and ex-vivo. Anti-cancer activity was determined through colony formation and apoptosis assays. Ex-vivo studies to establish superior efficacy of Tf-AQ NP were performed using 3D spheroid cell culture studies with established protocols (3). Results: Physical characterization revealed that AQ-loaded nanoparticles without (AQ NP) and with Tf (Tf-AQ NP) were found to have uniform size distribution with mean particle size: 223.3±41.8nm, 244.1±10.5nm; zeta potential: -11.4 ±5.6 mV, 8.2±1.3 mV; % drug entrapment: 31.8±1.5%, 41.0±9.8% respectively. In-vitro cellular uptake studies (Figs. 2A&B) revealed enhanced internalization of Tf-AQ NPs compared to unconjugated NPs. As observed in cytotoxicity studies, transferrin conjugated AQ nanoparticles showed enhanced cytotoxic efficacy of AQ in both A549 and H1299 cell lines with a prominent reduction in IC50 values. IC50 values for plain AQ, AQ NP and Tf-AQ NP were found to be 25.3±11.0µM, 3.0±1.7µM and 3.0±2.1µM: A549; 42.2±3.0 µM, and 37.1±11.5µM and 18.0±5.0µM: H1299 respectively. Tf-AQ NP exhibited excellent aerosolization (Fig. 2C), colony inhibition (Fig. 2D) and apoptosis induction properties demonstrated from the induced caspase levels (Fig. 3C). From 3D spheroid cell culture studies, it was found that spheroid volumes (day 12) were 11.1±4.2 mm3: control, 11.9±3.2 mm3: AQ, 13.6±2.7 mm3: AQ NP and 1.9±3.3 mm3: Tf-AQ NP at 10µM (Figs. 3A&B); Tf-AQ NP demonstrating ~5.8-7-fold reduction in spheroid volume compared to control and AQ NP. Also, Tf-AQ NP were found to inhibit autophagy, thus aid in hindering cancer cell proliferation. Hence, it was understood that Tf-AQ NP are capable of exhibiting superior anti-cancer efficacy in NSCLC due to their specific and augmented interaction with overexpressed transferrin receptors in NSCLC. Conclusion: It can be concluded that transferrin conjugated amodiaquine nanoparticles can be a potential inhalable treatment strategy for targeting NSCLC. While the in-vitro and ex-vivo results are promising, in-vivo studies are required to encompass the full spectrum of this approach’s feasibility.
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