(W1030-03-17) Fabricating Inhalable PLGA Nanoparticles for Targeted Therapy in Non-small Cell Lung Cancer

Purpose: Lung cancer is among the leading causes of cancer-related deaths globally, with non-small cell lung cancer (NSCLC) accounting for majority ( >80%) of those cases. With the advent in targeted therapies, there has been a major improvement in the outcomes for NSCLC patients. Targeted therapy involving the use of epidermal growth factor receptor (EGFR) inhibitors have been very successful for NSCLC patients with EGFR overexpression. However, continuous employment of these EGFR inhibitors leads to drug resistance and further induces the activation of other signalling pathways. Recently, an investigational drug, INA, has shown efficacy as a targeted therapeutic agent in various cancer cells. In this study, INA was encapsulated into polymer-based nanoparticle system and further spray-dried into a dry powder. Pulmonary administration of the dry powder was utilized for safe and efficacious delivery of the drug to the target site i.e. the lungs, limiting systemic toxicity.

Methods: The PLGA nanoparticles were prepared by the single emulsion solvent evaporation technique. Briefly, PLGA and the drug were first dissolved in dichloromethane. This mixture was then added to a 2.5% PVA (polyvinyl alcohol) solution, and the resulting dispersion was sonicated. Further, the dispersion was slowly added under continuous stirring to a 0.75% PVA solution. This solution was kept under stirring for 4 h to remove organic solvent. Furthermore, the resulting PLGA nanoparticles dispersion (INA PLGA NPs) was spray dried with an excipient (L-Leucine) using a Buchi B-290 to obtain a dry powder (SD-INA).  The prepared formulations (INA PLGA NPs and SD-INA) were further characterized for particle size, polydispersity index, zeta potential, entrapment efficiency, and drug loading. Furthermore, solid-state characterization of the formulation was performed using Differential Scanning Calorimetry (DSC). The aerosolization properties of SD-INA was tested using Next Generation Impactor™ (NGI) and RS01^® dry powder inhaler. The in vitro cytotoxicity of INA was evaluated on adenocarcinoma cell line A549 using MTT assay.

Results: The PLGA nanoparticles exhibited a particle size of 246.03 ± 0.57 nm with a polydispersity index of 0.24 ± 0.02 and a zeta potential of –15.13 ± 3.45 mV (Fig. 1). Additionally, the entrapment efficiency and drug loading were calculated to be 81.49 ± 7.91 % w/w and 36.01 ± 0.41 μg/mg of polymer, respectively (Fig. 1). The spray-dried powder (SD-INA) exhibited a resuspendibility index of 1.9 with a polydispersity index of 0.28 ± 0.02, yield of 51 % w/w, and a drug loading of 32.85 μg/mg of polymer. The DSC thermograph of pure drug INA revealed a sharp endotherm at 232 °C, representing its melting point and confirming the crystalline nature of the drug (Fig. 2). The formulation (SD-INA) also demonstrated an endothermic transition around 220 °C (Fig. 2). This behaviour can be attributed to the degradation of the excipient, L-leucine at that temperature which has been previously studied by our group. The potential of SD-INA to be deposited in the respiratory airways was investigated using NGI. Majority of the particles were deposited between Stages 3 – 7 representing the lower respiratory tract (Fig. 2). The mass median aerodynamic diameter (MMAD) depicts the median aerodynamic particle size distribution by mass and was found to be 2.29 ± 0.16 μm. The fine particle fraction (FPF) was 81.32 ± 2.89 % w/w, showing excellent aerosolization performance of the nanoparticles. The investigational drug demonstrated a IC50 value of 0.44 ± 0.01 µM in lung adenocarcinoma cells (A549), thereby indicating an effective anti-cancer activity in NSCLC cells (Fig. 3).

Conclusion: Overall, this study demonstrates the successful development of inhalable INA loaded PLGA nanoparticles, which can be a promising approach for targeted therapy for non-small cell lung cancer therapy.

Figure 1. Formulation development. Physicochemical characterization of PLGA nanoparticles.

Figure 2. Formulation characterization. A. The DSC thermogram of free drug, polymer, physical mixture, and spray-dried INA PLGA NPs B. In vitro aerosol deposition profile represented as the percentage of drug deposited on each stage of Next Generation Impactor (NGI).

Figure 3. Cytotoxicity Assay. Cytotoxicity studies after 72 h treatment with the free drug determined using MTT assay in A549 cell line.