Poster S3 - Photo-activated Combinatorial microRNA Delivery Induces Apoptosis in Head and Neck Cancer

Introduction: Head and Neck Cancer (HNC) accounts for 3% of cancer incidences and approximately 54,000 annual diagnoses in the United States. GLOBOCAN estimates that by 2030 the incidence rate of HNC will increase by 30%, specifically in younger populations. Lifestyle changes such as the increased combination of tobacco and alcohol consumption or the higher prevalence of HPV related oropharyngeal cancer significantly contribute to this increase. Standard treatments regimens such as surgical resection, chemotherapy, and radiotherapy present significant drawbacks including permanent disabilities in speech, respiration, swallowing, and cosmesis due to tissue loss. With the significant side effects of current treatment methods and the increasing prevalence in younger generations, innovative technologies are necessary to preserve tissue and improve quality of life after diagnosis with HNC.

Recently, nucleic acid therapeutics emerged as an alternative strategy for the treatment of solid tumors as they can simultaneously modulate multiple genetic pathways essential to cancer progression including apoptosis, proliferation, and metastasis. The delivery of microRNA, a short non-coding RNA 18-22 nucleotides in length, works quickly to modulate protein expression in 4-24 hours. Our lab has developed a photo-activated, gold-silver-gold core-shell-shell nanoparticle (CSS NP) that enables the delivery of microRNA mimics with spatiotemporal control (Figure 1A). This study focuses on the combinatorial delivery of microRNA by the CSS NPs to increase the treatment’s efficacy and applicability in HNC. In vitro analysis shows that the delivery of multiple microRNAs increases the efficacy and breadth of HNCs that the delivery system can treat.

Materials and

Methods: Gold chloride and sodium citrate were refluxed to form the gold core of the CSS NPs. The gold seeds were reacted with ascorbic acid, silver nitrate, and sodium hydroxide to form the silver shell. This intermediate was reacted with gold chloride, hydroquinone, and sodium citrate to form the final gold shell. The CSS NPs were functionalized with a furan-based Diels-Alder (DA) linker and microRNA via N-Hydroxysuccinimide and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (NHS:EDC) bioconjugation.

Nanoparticles functionalized with this furan DA linker can undergo a retrograde reaction due to plasmonic activity when exposed to near-infrared light (NIR). This reverse DA reaction yields the initial biocompatible reactants and releases the nucleic acid therapeutic from the surface of the CSS NP. Therefore, the use of NIR light as an external stimulus is well suited for HNC as it can penetrate tissue and provide spaciotemporal control over delivery of microRNA, with minimal off-target effects. Following synthesis, nanoparticles were characterized via DLS and UV-VIS.

Various microRNA combinations were screened by delivering microRNA mimics to FaDu cells via Lipofectamine RNA iMAX. The miRNA mimics miR-34a-5p and miR-148b-3p were selected for combinatorial delivery. Qiagen IPA was used to perform bioinformatics on selected mimics to determine the relevant gene targets responsible for inducing apoptosis in FaDu cells (Figure 1B). CSS NPs were functionalized with singular miRNA mimics. Efficacy of combinatorial delivery was analyzed in vitro with representative viability via Live/Dead, cell count via PicoGreen, uptake via confocal microscopy/flow cytometry, and gene expression via RT-qPCR.

Results, Conclusions, and Discussions: Results and

Discussion:
Transfection of microRNA mimics via Lipofectamine RNA iMAX revealed that the combination of miR-34a-5p and miR-148b-3p induced the most death in FaDu cells. Bioinformatics analysis via Qiagen IPA suggested that these mimics induce apoptosis via targeting mRNA for BCL2 and BCL2L11, among other possible targets (Figure 1B). Confocal microscopy and flow cytometry results suggest that uptake of miRNA by cells via CSS NPs is significantly increased in comparison to uptake via Lipofectamine. The concentration of miRNA delivered was lowered from 100 nM to 25 nM accordingly. Live/Dead and PicoGreen analysis exhibited significantly increased knockdown of FaDu cells in the combinatorial treatment groups compared to minimal knockdown in the solo treatment groups (Figure 1C).

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Figure 1. (A) Graphical depiction of the CSS NP uptake, miRNA release triggered by NIR irradiation, and the resulting programmed cell death. (B) Bioinformatic evaluation of combinatorial miRNA targets in apoptosis including BCL2 and BCL2L11 analyzed via Qiagen IPA. (C) Representative Live/Dead images at 24, 48, and 72 hours following NIR irradiation showcasing reduced cell survival in the combinatorial treatment group. Scale bar = 200 um.

Conclusion:
Combinatorial delivery of miR-34a-5p and miR-148b-3p resulted in significant knockdown of FaDu cells while solo delivery was ineffective for this HNC cell line. Delivery via CSS NPs resulted in significantly improved uptake allowing for reduced concentration of the nucleic acid therapeutic. Ongoing work includes in vivo evaluation of the CSS NPs described in this study, testing the CSS NPs on the HPV+ HNC cell line UM-SCC-47, and designing CSS NPs which deliver siRNA targeting BCL2 and BCL2L11.

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