D. Dhamecha, J.U. Menon
University of Rhode Island,
Keywords: porous microparticles, 3D culture, lung tumor, in vitro models, drug screening
Summary:Conventionally used in vitro cancer models involving cells cultured as monolayers in a petridish, are not representative of the in vivo environment. Systematic generation of spheroids in biologically simulated polymeric scaffolds represent cell-cell or cell-extracellular matrix interactions features of solid tumors formed in humans and hence can be used for accurate early screening of promising drugs. The present research was focused on the formulation of porous PLGA (poly (lactic-co-glycolic acid)) microspheres (PPMS) using a novel alginate microspheres (AMS) porogen which can subsequently be used to develop three-dimensional (3D) cancer spheroids with uniform size and morphology. The PPMS was developed by double emulsion solvent evaporation technique where AMS solution was incorporated in the first water phase. The AMS porogen was developed by water-in-oil emulsion technique as described by Zhu et al. with minor modification. Formulated microspheres were then treated with ethylenediaminetetraacetic acid (EDTA) to digest AMS to get porous PPMS. The AMS had a diameter of 10.3 ± 4 µm whereas PLGAMS and PPMS had diameters of 79 ± 22 and 103 ± 30 µm respectively. Scanning electron microscopy (SEM) images showed that PPMS and PLGAMS were spherical, and cross-sections of the particles showed the presence of interconnected pores for PPMS and non-porous, smooth internal structure for PLGAMS. Degradation studies demonstrated that the PPMS degraded faster than PLGAMS at 37oC. 24% weight of PPMS was remaining at day 28 compared to 40 % weight remaining for PLGAMS. Preliminary in vitro data using A549 lung adenocarcinoma cells demonstrated that the cells were able to attach onto the surface of the PLGAMS and PPMS and proliferate for up to 28 days. The cells on the microparticles were viable, which was confirmed by confocal microscopy following live/dead staining. We are now investigating the responses of these microparticle-based lung tumor models to therapeutic agents used for first-line lung cancer therapy, and comparing the results with conventionally used 2D monolayer cultures. In conclusion, PPMS formulated in the present investigation demonstrated a clear porous nature. Although porous microparticles have been used in various research applications, we report a novel method of safe and controlled pore formation using AMS and EDTA. The PPMS thus formed had a robust and favorable architecture for cell attachment and proliferation to develop an in vitro lung tumor model.