J6 - Functionalized porous silicon nanoparticles for the vectorization and photoactivated siRNA delivery against uveal melanoma

Introduction: : Uveal melanoma (UM), a rare but lethal ocular malignancy, poses significant therapeutic challenges due to its propensity for metastatic spread and limited treatment options. The primary standard of care for UM is radiotherapy and surgical intervention which both carry a high risk of permanent vision loss. Alternatives like photodynamic therapy (PDT) for thin uveal melanomas ( < 4 mm) reduces radiation-induced vision loss but have high recurrence rates. Gene therapy, using targeted nucleic acid delivery like siRNAs, is a promising option but often relies on viral vectors, which carry inherent risks of immune activation and tumorigenesis. Here, we will report advances in developing a non-viral nucleic acid delivery system using biodegradable porous silicon nanoparticles (pSiNPs), which offer low toxicity and intrinsic photoluminescence properties. Porphyrin-grafted pSiNPs enable light-triggered gene delivery via near-infrared excitation, while supporting photochemical internalization under one- and two-photon excitation, compatible with the eye environment for a targeted and safe release.

Materials and

Methods: : The pSiNPs were synthesized using the etching method “electrochemical perforation etching” on a boron-ultradoped crystalline silicon wafer with a current density of 100 mA.cm-2. Once synthesized and characterized by dynamic light scattering and nanoparticle tracking analysis, the pSiNPs were functionalized by hydrosilylation with allylamine to graft the cationic porphyrin. 92.1 cell line was used as UM model and cultured in RPMI 1640 + 2mM Glutamine + 10% Fetal Bovine Serum, either as a monolayer or spheroids in an ultra-low attachment round bottom plate supplemented with 5% Matrigel. siRNA was grafted to the porphyrin-functionalized pSiNPs through passive incubation using a ratio of 20:1 (w/w) at 37°C for 90 min. siRNA-conjugated pSiNPs were incubated with both UM monolayer and spheroids for 24h and release efficiency was confirmed by PCR and western blot. PDT treatment (650 nm, 4mW, for 20 min at working distance of 7 cm) was also performed on UM cells to assess phototoxicity and effect on siRNA release.

Results, Conclusions, and Discussions:: The average size of the pSiNPs was 267.2 ± 22.3 nm with a zeta potential of -25 mV and +30.49 ± 0.53 mV when functionalized with porphyrin. siRNAs against different targets, including inositol polyphosphate-5-phosphatase A (INPP5A) and inhibitor of apoptosis (IAP) proteins, were successfully conjugated as demonstrated by gel retardation. Our biological characterization demonstrated that the pSiNPs are biocompatible at physiologically relevant concentrations (up to 25 µg/mL) and can be internalized by UM cells. Current investigations are elucidating the endocytic mechanisms governing pSiNP internalization to delineate nanoparticle-cell interactions and enhance intracellular delivery efficiency. Interestingly, siRNA-conjugated pSiNPs mediated significant downregulation of RNA and protein expression in UM cells within 24 hours, achieving gene silencing efficacy comparable to conventional cationic lipid transfection systems. PDT demonstrated high efficacy in reducing UM cell survival and proliferation in both monolayer and spheroid models. On-going experiments aim to elucidate whether PDT enhances siRNA delivery efficacy and if combinatorial treatments (siRNA with PDT or X-ray radiation) induce synergistic cytotoxicity to further suppress tumor cell viability and offer approaches for treatment de-escalation to preserve vision. To enhance biological characterization, UM tumor microenvironment is currently modeled using a novel tumor-on-chip (named ASTEROIDS) that integrates 3D cell culture with independently perfusable vascular compartments, recapitulating critical tumor-stroma interactions and drug transport dynamics. Preliminary data showed that UM spheroids can grow in the ASTEROIDS and can internalized pSiNP when injected either directly in the spheroids (mimicking intratumoral injection) or in the vascular chamber (intravenous injection). Additional investigations are currently developing PDT protocols for ASTEROIDS and are assessing pSiNP stability and efficiency in a physiologically relevant (i.e., dynamic, multicellular) environment. Collectively, these results demonstrated that pSiNPs exhibited biocompatibility, efficient UM cellular internalization, and functionalization capacity for therapeutic siRNA delivery. This platform presents a novel UM treatment strategy that, when combined with existing PDT or radiotherapy approaches, may enable safer and more effective therapeutic outcomes.

Acknowledgements and/or References (Optional):: We thank the UArizona-CNRS PhD Joint Program for financial support.