Cutaneous squamous cell carcinoma (cSCC) is a non-melanoma skin cancer and can be difficult to manage in locally advanced or recurrent disease, where wide excision may compromise function and aesthetics and systemic regimens are constrained by toxicity and limited selectivity. Gold(I) metallodrugs such as AuMTZ are attractive candidates because redox disruption can promote oxidative stress and regulated cell death in tumor cells; however, poor dispersion and chemical instability may limit translation. Nanostructured lipid carriers (NLCs) can protect labile compounds, modulate exposure, and support topical-oriented development. This study evaluated AuMTZ and tested whether nanoencapsulation (AuNLC) improves the antitumor/selectivity profile in cSCC-related models.

AuMTZ was synthesized and characterized following original chemical standards, and the nanoencapsulated formulation was denominated AuNLC. AuNLC was physicochemically characterized and shelf stability was monitored for 270 days. Antitumor response was assessed in FaDu, SCC-4, SCC-9, SCC-25 and non-tumoral keratinocytes (HaCaT). MTT (viability) and SRB (antiproliferative) dose–response assays were performed from 0.39–100 µM. Intracellular ROS (DCFDA), apoptosis by flow cytometry (Annexin V/7-AAD), migration (wound healing), invasion (Transwell-Matrigel), and 3D validation in spheroids generated in molds (n=260) by Live/Dead microscopy and morphometry.

AuNLC showed long-term physicochemical stability over 270 days (size ∼215–300 nm; PDI ∼0.16–0.30; zeta potential ∼ -29 to -13 mV), with no macroscopic aggregation. In MTT (0.39–100 µM), tumor lines showed concentration-dependent reductions, while HaCaT remained the most resistant within the same range. In SRB (TGI = 0% growth), both gold-based conditions were highly active in sensitive models: FaDu (AuMTZ =0.781 µM; AuNLC =0.781 µM) and SCC-9 (AuMTZ =0.781 µM; AuNLC =0.781 µM). In SCC-25, thresholds differed (AuMTZ =1.562 µM; AuNLC =12.5 µM), indicating cell line–dependent formulation impact. In HaCaT, both remained TGI >100 µM, supporting a favorable selectivity window. At fixed doses (3 and 25 µM), ROS increased (DCFDA) and flow cytometry confirmed apoptotic progression, with late apoptosis ranging 35–59% at 3 µM across the evaluated lines. Migration and invasion were reduced at both concentrations, with inhibition observed independent of matrix context. In 3D validation (n = 260), Live/Dead imaging showed treatment-dependent reductions in viability in tumor-derived spheroids, more pronounced under AuNLC, while HaCaT spheroids maintained high viability; global morphology remained preserved (solidity ∼1; Feret diameters: FaDu 425±39 µm, SCC-4 663±38 µm, SCC-9 678±33 µm, SCC-25 515±39 µm, HaCaT 609±27 µm).

AuMTZ displays strong antitumor activity in SCC models, and NLC nanoencapsulation provides a stable formulation with preserved tumor-directed effects and minimal impact on non-tumoral keratinocytes. The convergence of dose–response potency, fixed-dose ROS/apoptosis induction, suppression of migration/invasion, and consistent 3D efficacy supports AuNLC as a promising platform for topical-oriented translational development in cSCC.