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Formulation and Delivery - Chemical

(T0930-07-41) Pulmonary Delivery of Trametinib-Encapsulated Nanostructured Lipid Carriers for Non-Small Cell Lung Cancer

Tuesday, October 22, 2024
9:30 AM – 10:30 AM MT
Purpose: Lung cancer is a leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for over 80% of cases. Recent advances in targeted therapies have shown promise in improving outcomes for NSCLC patients, particularly those focusing on the mitogen-activated protein kinase (MAPK) pathway. The MAPK pathway regulates cell proliferation and survival and is constitutionally overactive in 30% of cancers. Two enzymes in this pathway, v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) and mitogen-activated extracellular signal-regulated kinase (MEK), are target kinases which play crucial roles in the cell cycle. The first therapies to target this pathway were BRAF inhibitors, but intrinsic and acquired tumor resistance quickly led to treatment failure. Trametinib is a reversible and highly allosteric MEK inhibitor which shows anticancer activity against BRAFV600E mutation. In this study, we have prepared a lipid-based nanoparticle system (nanostructured lipid carriers) that can be delivered via nebulization and leads to increased drug concentration at the tumor site.  

Methods: The nanostructured lipid carriers (NLCs) were prepared by high shear ultrasonication method. Briefly, the lipid phase, comprising oleic acid, glyceryl dibehenate, and the drug was combined with the aqueous phase containing Kolliphor® P 188 and Span™ 80. This mixture was heated and subsequently homogenized using a probe sonicator to form Trametinib-loaded NLCs (TRB-NLCs). The prepared formulation was 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) and Powder X-ray Diffraction (PXRD). The aerosolization properties of TRB-NLCs was tested using Next Generation Impactor™ (NGI) and PARI LC Plus® nebulizer cup. The in vitro cytotoxicity of TRB-NLCs was evaluated on adenocarcinoma cell line, HCC364 using MTT assay. The drug release from the TRB-NLC was determined after suspending a known amount of sample in PBS supplemented with 0.2% SLS and drug released at each time point was quantified. The stability studies for the TRB-NLC were performed upon storage at refrigerated (4 °C) and room temperature (25 °C) conditions. 

Results: The NLCs exhibited a particle size of around 102.09 ± 1.53 nm with a polydispersity index of 0.29 ± 0.05 and a zeta potential of -24.78 ± 1.05 mV (Fig. 1). Additionally, the entrapment efficiency and drug loading were calculated to be 66.50 ± 4.45% w/w and 21.94 ± 2.03 μg/mg of lipid, respectively (Fig. 1). The disappearance of intense peaks in the XRD diffractogram for the TRB-NLCs compared to the free drug indicated successful encapsulation of the drug into NLCs (Fig. 2A). The absence of the melting peaks in the DSC thermograms for the formulation further ascertains the entrapment of the drug within the NLCs (Fig. 2B). The potential of the TRB-NLCs 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. 2C). The mass median aerodynamic diameter (MMAD) depicts the median aerodynamic particle size distribution by mass and was found to be 4.78 μm. The fine particle fraction (FPF) was greater than 75% showing excellent aerosolization performance of the TRB-NLCs. The trametinib-loaded NLCs resulted in a 2.6-fold reduction in the inhibitory concentration (IC50) value as compared to the free drug, thereby demonstrating improved cytotoxicity of the formulation (Fig. 3A). The in vitro drug release profile of TRB-NLCs showed a sustained release (~15%) of the drug in 24 h. Further stability studies on TRB-NLC showed stability when stored at 4˚C and 25 ˚C (room temperature) for 4 weeks.  

Conclusion: Overall, this study demonstrates the successful development of inhalable trametinib-loaded nanostructured lipid carriers. This can be a promising approach to minimize the acquired drug resistance to BRAF inhibitors for non-small cell lung cancer therapy. 



 Figure 1. Formulation Development. Physiochemical characterization of Trametinib-loaded Nanostructured Lipid Carriers (TRB-NLCs). Data represents mean ± SD (n = 5).

Figure 2. Formulation Characterization. A. The XRD diffractogram of TRB, physical mixture, and TRB-NLCs. B. The DSC thermogram of TRB, physical mixture, and TRB-NLCs. C. In vitro aerosol deposition profile represented as percent of drug deposited on each stage of Next Generation Impactor (NGI). Data represents mean ± SD (n = 3)

Figure 3. Cytotoxicity Assay and Stability Studies. A. Cytotoxicity studies after 72 h treatment with TRB, and TRB-NLCs determined using MTT assay in HCC364 cell line. Data represents mean ± SD (n = 3) B. Stability studies for TRB-NLCs upon storage at room temperature (25 ˚C) and refrigerated (4 ˚C) conditions.