(T1130-08-53) Microfluidic-Assisted Preparation of ARV 825 and Osimertinib Loaded Liposomal Formulation as a Potential System for Colorectal Cancer Therapy

Purpose: Colorectal cancer (CRC) is the second most lethal and the third most common malignant tumor globally. With the over-escalating need for new therapeutics due to existing treatment drawbacks, another active field is the development of protein degraders, i.e., proteolysis targeting chimeras (PROTACs). Since the last 5 years, PROTACs have emerged as a breakthrough technology that targets overexpressed protein moieties by recruiting E3 ubiquitin ligase. BET proteins, mainly BRD4, is known to control numerous gene expression related to cell growth and cancer progression suggesting it to be an epigenetic protein that regulates the transcription of specific promoters like c-MYC and BCl2. ARV-825 (ARV), one of the cereblon-based PROTAC is responsible for BRD4 protein degradation. Also, overexpression of EGFR has been observed in 25–77% of CRCs, and EGFR can lead to the growth, proliferation, survival, and metastasis of neoplastic cells. In this context, we propose the use of a selective BRD4 protein degrader, ARV-825, and third-generation EGFR inhibitor Osimertinib (OSI) for CRC therapy. Our research objectives were: (a) to investigate the synergism between ARV and OSI in 2D and 3D colorectal cancer models, and (b) to develop and characterize ARV and OSI -loaded Liposomal formulation (AROSIB) using Microfluidics technology.

Methods: Cytotoxic activity of ARV and OSI was analyzed in HCT 116 and HT 29 colorectal cancer cell lines using MTT assay. ARV-OSI combination assay was carried out at different molar ratios to evaluate the synergistic interaction between both drugs in different cancer cell lines and the combination index was calculated. For the production of liposomes using microfluidics, two syringes placed on syringe pumps (Fusion 100 Syringe Pump) were linked to the microfluidic chip made of polypropylene that was 3D printed using FDM on an Ultimaker 3 printer (Ultimaker, The Netherlands). The material was selected because of its high chemical resistance with solvents and low interaction with molecules. The chips present a “zigzag” bas-relief or a split and recombine circular subchannels patterns that resulted in effective to achieve complete micromixing into the channels. The Organic phase consists of lipids and drugs dissolved in ethanol pumped against the aqueous water phase at an optimized flow rate, and the samples were collected from the outlet (Fig. 2). Different lipids concentrations, flow rate ratios, and total flow rates were screened to optimize the size and entrapment efficiency. Batches with DOPC, cholesterol, and DSPE-PEG lipids with ARV and OSI were prepared followed by physicochemical characterization for particle size, polydispersity index, zeta potential, and drugs entrapment efficiency. In vitro hemolysis assay was performed at different concentrations of ARV and OSI in AROSIB. In vitro assays including migration, clonogenic, and apoptosis, of AROSIB are currently under investigation. Furthermore, the effect of AROSIB within 3D multicellular tumor spheroids will be studied.

Results: The primary objective of rationalizing the combination of ARV and OSI was achieved by estimating the combination index of both molecules in colon cancer cells. IC₅₀ of ARV in combination was found to be 0.084 ± 0.007 µM and 0.111 ± 0.004 µM in both the cell lines, which was significantly lower than IC50 of ARV alone. Similarly, IC50 of OSI in combination was found to be 0.419 ± 0.043 µM and 0.555 ± 0.087 µM, which was significantly lower than IC50 OSI alone in both the cell lines (Fig. 1A-1B). ARV and OSI revealed strong synergism at a molar ratio of 1:5 in colorectal cancer with the lowest combination index value of 0.362 ± 0.001 and 0.253 ± 0.006 in HCT 116 and HT 29 cells (Fig. 1C). Acidified sterile water (pH 4) with higher flow rate (than organic phase) was key factors driving the entrapment efficiency and stability. Optimized AROSIB resulted in a particle size of 198.4 ± 0.215 nm, polydispersity index of 0.145 ± 0.01, and ζ-potential of +35.3 ± 0.042 mV (Fig. 3A-3B). Additionally, Cryo-TEM was used to verify the particle size and structure of AROSIB (Fig 3D). The encapsulation efficiency of ARV was found to be 73.59 ± 2.96 % and OSI 96.82 ± 2.34 % in AROSIB (Fig 3C). Positive zeta potential is clearly indicative of the orientation of both the weakly basic molecules in the outer leaflet of liposomes. Despite the positive charge, no significant hemolysis or non-specific cytotoxicity was observed. As shown in Fig. 3E, AROSIB showed negligible hemolysis ( < 1%) at different concentrations of ARV and OSI in contrast to the positive control (sodium dodecyl sulfate), which indicates 100% hemolysis. The fact that RBCs redispersed quickly and completely reveals that the AROSIB had not changed their surface properties.

Conclusion: Our results demonstrate that combining BRD4 PROTAC and EGFR inhibitor leads to synergistic antitumor effects against colorectal cancer. The development of nanoliposomes “AROSIB” further augments the efficacy of this treatment cocktail for its successful therapy in colorectal cancer. Microfluidic can be helpful in the continuous production of liposomes with desired drug ratios.