Category: Formulation and Quality
Purpose: Afatinib (AFA) is a novel, potent, aniline–quinazoline derivative, which was approved by Food and Drug Administration (FDA) as an oral once-daily tablet for first-line treatment of metastatic Non-small-cell lung cancer (NSCLC). However, it has limitations as poor solubility, systemic toxicity and low bioavailability, thus limiting its clinical applications. Polymeric nanoparticles (PNPs) have attracted considerable interest over the last few years due to their unique properties and behaviors resulting from their small size. Poly (lactide-coglycolide (PLGA), an FDA approved polymer, has been proven to be safe in various clinical applications and being a biodegradable polymer, it has been known to be biocompatible and can be easily absorbed in natural conditions. Formulation of AFA loaded PLGA nanoparticles is postulated to enhance the efficiency for drug delivery and will also aid in extending release, minimizing degradation, increasing bioavailability and reducing drug toxicity. One of the limitations for PLGA is that PLGA-NP will be negatively charged which will hinder its interaction and consequently adhesion and penetration through negatively charged cellular membrane. Poly-l-arginine (PLA), polycationic biopolymer, therefore, will be investigated for coating to promote adhesion. In this project, we aim to develop biodegradable PLGA nanoparticles of AFA using double emulsion-solvent evaporation method, both uncoated and coated with PLA. We hypothesize that loading of AFA in biodegradable nanoparticles will be instrumental in improving therapeutic outcomes in NSCLC patients.
Methods: AFA loaded PLGA nanoparticles were formulated using double emulsion-solvent evaporation method. Briefly, AFA (1 mg) was first dissolved in organic solution (dichloromethane) of PLGA (Resomer RG 502 H, 60mg PLGA /3ml of DCM). Ultra-probe sonication was done for 2 min (40% amplitude, 10 sec on-off cycle). This primary emulsion was probe sonicated for 4 min (10 sec on-off cycle) in 1% polyvinyl alcohol (PVA) stabilizer solution, with or without PLA (1 mg/ml) and the secondary emulsion was obtained. The nanoparticles were then allowed to stir overnight for removal of organic solvent and were subsequently washed to remove excess PVA. The final formulation was analyzed for particle size, zeta potential and entrapment. Physical and chemical compatibility testing were confirmed for both coated and uncoated using differential scanning colorimetry, XRD and FT-IR. In-vitro drug release study was performed 0.1% (w/v) Tween 80 PBS (pH 7.4) at 37°C. Samples were withdrawn at 0.5,1, 2, 4, 6, 12, 24 and 48 hr. Cytotoxicity potential against NSCLC was tested in 2 NSCLC cell lines; A549, and H358 by testing for cell viability following 48 hour drug incubation with MTT assay. These cell lines were known to exhibit KRAS mutations where afatinib was proven to impair K-RAS-driven lung tumorigenesis. A 4-week stability study was also performed for both coated and uncoated nanoparticles at 4°C, 25 and 37 °C.
Results: Both coated and uncoated formulated AFA nanoparticles were found to have uniformly sized particles with a narrow polydispersity index and significant % entrapment efficiency. For coated nanoparticles size, polydispersity index, zeta potential and % entrapment efficiency were measured to be 221.7 nm, 0.253, -12.1 mV and 39.20%, While in case of uncoated nanoparticles was measured to be 191.2 nm, 0.082, -23.1 mV, and 30.59%. With respect to in-vitro drug release, cumulative drug release percentage after 24 hours was (61.1+11.1%, and 39.3+2.3%) for coated and uncoated respectively. Both coated and uncoated nanoparticles showed sustained release profile with significant difference (p< 0.01) when comparing the release of both. As observed in cytotoxic studies (Fig. 1), AFA encapsulation in PLGA nanoparticles significantly enhanced its cytotoxic potential in both NSCLC cell lines (A549 and H358) with an IC50 reduction of at least 3-8 folds as compared to plain afatinib. The IC50 values for afatinib decreased significantly with nanoparticles in A549 (3.125 µM vs 0.78 µM and 1.56 µM) in case of drug vs coated and uncoated nanoparticles; respectively and H358 (0.78 µM vs 0.095 µM) in case of drug vs both coated and uncoated nanoparticles; respectively. These results reveal the significant increase in intracellular accumulation of drug-loaded nanoparticles in cancer cells as the developed nanoparticles delivery system can deliver the drug into cells effectively due a smaller particle size in case of both coated and uncoated and a positive charge on their surface in case of coated. Both coated and uncoated PLGA nanoparticles were found to be physically and chemically stable for 4 weeks in the stability study at three storage conditions. Further clonogenic assay, cellular uptake study are required to ensure the significance of this approach and evaluate the superiority of cellular uptake of PLA coated versus uncoated AFA-PLGA nanoparticles.
Conclusion: From the results, it can be concluded that AFA-PLGA nanoparticles formulated through double emulsion-solvent evaporation method could be a promising approach for efficient treatment of NSCLC with reduced systemic toxicity.