Dual cell-penetrating peptide-conjugated polymeric nanocarriers for miRNA-205–5p delivery in gene therapy of cutaneous squamous cell carcinoma

Despite the potential of microRNAs (miRNAs) in suppressing tumorigenesis, the main challenges are achieving tumor-specific selectivity and efficient delivery into cancer cells. In this study, miR-205–5p-loaded polymeric nanoparticles conjugated with dual cell-penetrating peptides (CPPs) were designed for targeting and treating cutaneous squamous cell carcinoma (cSCC). The CPPs, R9, and p28, demonstrated high cell-penetrating/targeting abilities and antitumor activity. The anti-cSCC effect of the nanocarriers was examined using in vitro cellular 2D and 3D models and in vivo spheroid-xenografted murine models. The average size of the dual CPP-conjugated nanocarriers was 193 nm with a zeta potential of 5.7 mV. These nanocarriers were readily internalized by A431 cells, resulting in decreased proliferation compared to naked agomiR and nanoparticles with a single CPP. The nanocarriers induced cell cycle arrest in the G0/G1 stage. By loading the miR-205–5p mimic, the dual CPP-conjugated nanoparticles enhanced cell apoptosis threefold compared to the control, activating caspases and poly(ADP-ribose) polymerase (PARP). The wound healing assay demonstrated that the nanocarriers significantly inhibited the migration and invasion of cSCC cells. Additionally, the CPP-conjugated nanocarriers penetrated cSCC 3D spheroids, reducing spheroidal size and proliferation. In vivo studies demonstrated that the intratumoral CPP-conjugated nanocarriers achieved a 30 % reduction in tumor volume than the PBS control. The number of Ki67-positive cells in the nanocarrier-treated tumor decreased fivefold than the untreated tumors. The nanoparticulate agomiR (1 μM) exhibited no cytotoxicity towards normal keratinocytes. No significant toxicity was observed in the skin and peripheral organs following subcutaneous administration of the nanoparticles in healthy mice. These findings demonstrate that miR-205–5p mimic delivery via dual CPP-conjugated nanocarriers can promote efficient and safe cSCC regression.

Statement of significance

Cutaneous squamous cell carcinoma (cSCC) is a highly invasive skin malignancy with limited treatment options. This study introduces dual cell-penetrating peptide (CPP)-conjugated polymeric nanoparticles for delivering miR-205–5p, a tumor-suppressor microRNA, to cSCC cells. The nanosystem enhances cellular uptake, inhibits cell proliferation, and promotes apoptosis in both 2D and 3D tumor models. In vivo, the nanocarriers demonstrate significant antitumor efficacy with minimal toxicity, highlighting their potential as a targeted, non-invasive therapy. This research represents a promising advance in gene therapy for cSCC by combining nanotechnology and CPPs to address challenges in miRNA delivery and tumor targeting.