Category: Formulation and Quality
Purpose: Sorafenib (SF), a multi-kinase inhibitor, has been approved for treatment of advanced renal cell carcinoma, hepatocellular carcinoma and metastatically differentiated thyroid cancer by FDA in the last decade. Owing to its wide range of mechanism on cancer, it has also been explored for non-small cell lung cancer (NSCLC). While sorafenib has shown the potential to be effective against NSCLC, its use has been limited due to reasons such as poor solubility, low bioavailability and limited routes of administration. In addition, sorafenib is highly protein bound (~99.5%) resulting into a narrow therapeutic window drug and hence requires increased dose for effective therapeutic activity resulting into severe toxicity. In this project, we pursue to load sorafenib in cationically modified nanoparticles as inhalable carriers for treatment of NSCLC resulting into enhanced bioavailability, localized drug delivery, improved therapeutic activity and establishing safety of sorafenib via reduced toxicity and side effects.
Methods: Materials: Sorafenib was obtained from LC Laboratories; Resomer® RG 502 H, Poly (D, L-lactide-co-glycolide) (PLGA), Poly-L-arginine (PLA) and Polyethyleneimine (PEI) was procured from Sigma-Aldrich.
Sorafenib-loaded nanoparticles were prepared by single o/w solvent evaporation method. Briefly PLGA (60 mg) and sorafenib (0.5 mg) were dissolved in organic solvent mixture (DCM: DMSO) and emulsified along with 1% PVA solution using probe sonicator. The emulsion was stirred overnight for evaporation of organic solvent followed by centrifugation and washing with water to remove free drug. The PEI and PLA nanoparticles were prepared by adding 1% or 2% of PEI; or 1mg PLA to 1% PVA during emulsification process. The formulations were evaluated for particle size, poly dispersity index (PDI) and zeta potential using Malvern® Zetasizer. % Drug entrapment and % drug loading was analyzed by lysing nanoparticles and quantifying amount of drug encapsulated using UPLC (Waters, Inc). Cytotoxicity studies were performed in five NSCLC cell lines; A549, H4006, H157, H358 and H460. Cell viability was assessed after treatment period by using MTT assay. The therapeutic effect of nanoparticles was also assessed using cellular uptake studies, scratch assay and clonogenic assay. The in-vitro tumor model simulation was performed using 3D spheroid with supporting evidence from caspase assay.
Results: As shown in Table 1, all the formulations had average particle size < 200 nm and narrow particle size distribution indicating uniformity of particle size. There was reduction in zeta potential with introduction of PLA and PEI (varied concentration) (Table 1). The % drug entrapment ranged from 19.4% to 42.4% during the development and optimization of nanoparticles. Cytotoxic studies data (Fig. 1) reveal significantly superior efficacy of PLA coated nanoparticles in enhancing cytotoxicity of sorafenib owing to better interaction with cell membrane. The IC50 reduction; sorafenib (~5 µM) to < 2.5 µM via uncoated nanoparticles and < 0.9 µM using PLA coated nanoparticles. Significantly lower IC50 values observed with PLA nanoparticles indicate that reduced surface energy of nanoparticles cause increased intracellular accumulation of particles. This therapeutic activity was also in accordance to the results obtained from various other in-vitro assays, thus indicating the efficacy of developed nanoparticles in enhancing therapeutic activity of sorafenib.
Conclusion: The results obtained demonstrate the effectiveness of sorafenib loaded nanoparticles as effective delivery carrier for treatment against NSCLC. This is one of first reports to explore sorafenib loaded nanoparticles for NSCLC treatment and hence substantial in-vitro and in-vivo or simulated in-vitro model studies will be implemented to support the development and applicability of the proposed delivery system.