(J-368) Tumor-on-Chip Utilizes Porous Silicon Nanoparticles for Uveal Melanoma Drug Study

Introduction:: Uveal melanoma is a rare tumor of the eye with disproportionately high risk of death due to lack of effective treatments. Low population incidence and high cost/failure of cancer drug discovery has realized minimal progress in uveal melanoma survivability over the last several decades. A lack of effective treatments and physiologically relevant disease models inspired our group to approach this problem with a novel tumor-on-chip platform for investigation of porous silicon nanoparticles (pSiNp) as a drug delivery system. To better replicate in-vivo tumor microenvironment (TME) conditions compared to standard cell culture models we developed our tumor-on-chip called, Apparatus to Simulate Tumor and Reproduce Organs in an Interactive and Dynamic System (ASTEROIDS) (fig. 1), which employs multiple distinct cellular chambers, mechanical force fluid perfusion, and hydrogel embedded uveal melanoma spheroids. To increase cancer drug specific targeting while decreasing capillary diffusion washout pSiNp’s provide an attractive delivery model. These nanoparticles are biologically non-toxic, photo-activatable, capable of loading with drugs, and have surface chemistries capable of functioning with molecules such as porphyrin and antibodies (fig. 2).


Materials and Methods:: Uveal melanoma spheroids are formed using ultra-low attachment culture plates. Two fluidic culture chambers of ASTEROIDS are seeded separately with TME associated cells on porous polycarbonate flexible membranes. Liquid diffusion occurs between these two fluidic culture side chambers through the spheroid embedded region of the device. A dextran assay was used to measure endothelial side chamber barrier permeability. Immunofluorescence staining (IF) was performed with a primary antigen antibody, and a secondary conjugate reporter antibody. Imaging was performed with florescent confocal microscopy. The pSiNp were produced by alternating current under hydrofluoric acid mediated electrophoresis of p++ silicon wafers, followed by roll milling sonication. Porphyrin functionalization was performed by allylamine chemistries. Complexion of anionic charged siRNA took advantage of the porphyrin cationic charged interaction.


Results, Conclusions, and Discussions:: Several Uveal melanoma cell lines were characterized, but only 92.1 cell line was used for further studies based on spheroid growth rate and shape experimental requirements. Uveal melanoma spheroids showed significant growth and markers of proliferation within our tumor-on-chip under physiologically relevant flow rates. Significantly less dextran diffusion out of ASTEROIDS fluidic culture chambers was observed when endothelial cells were present compared to no cells. Endothelial cell-to-cell junction barrier formation was further suggested by Ve-Cadherin peripheral cell membrane signaling by IF staining. A positive zeta potential shift of our nanoparticles is observed along with appearance of a UV-vis absorption peak between 400-500nm suggesting successful cationic porphyrin-functionalization. Endocytosis of these nanoparticles within uveal melanoma spheroid cells was observed by 2-photon fluorescent microscopy. Successful siRNA-nanoparticle complexion was indicated by an agarose gel retardation assay. We have characterized this tumor-on-chip disease model, optimized nanoparticle production and porphyrin-functionalization, studied cellular interaction with these nanoparticles, and produced siRNA-nanoparticle complexes. Uveal melanoma spheroid growth within ASTEROIDS provides a measurable target for future cancer drug efficacy investigation. The combination of fluidic forces, multicellular compartments, and cancer spheroid 3-dimensional culturing provides a more physiologically relevant disease model than previously realized in standard cell culture. Our porous silicon nanoparticle production and chemistries have been optimized and are ready for the next phase of exploring different drug efficacies in our tumor-on-chip conditions. We believe these steps firmly position our team to study novel uveal melanoma cancer drug therapies utilizing porous silicon nanoparticle and tumor-on-chip technologies in a more physiologically relevant manner.


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