DNA/RNA Hybrid Hairpin Gold Nanobeacons Targeting miR-31 Reprogram Invasion in Lung Cancer and Remodel Tumor Histo-Architecture <i>In Vivo</i>

Readout-enabled delivery of anti-microRNA (miRNA) therapeutics remains a bottleneck for translating RNA nanomedicine against invasion and metastasis. We report ∼60 nm PEG-gold nanobeacons bearing a Cy3-labeled anti-miR-31-5p hairpin and establish a chemistry-to-phenotype pipeline spanning physicochemical control, cellular trafficking, functional phenotyping, miR-31 kinetics, transcriptomics, and histology-only in vivo analysis. The formulation exhibits invariant plasmonics, stepwise growth in hydrodynamic diameter with low dispersity, and moderated ζ-potential, confirming a stable, readout-enabled architecture. In lung cancer cells with high endogenous miR-31, nanobeacons are efficiently internalized, traffic predominantly to lysosomes, and selectively suppress 3D invasion at 10–20 nM while leaving 2D migration and viability unchanged. A low-miR-31 cell line shows minimal response, supporting target dependence. Time-course qRT-PCR reveals a delayed decrease in mature miR-31 (120–144 h) consistent with a two-phase mechanism: early functional sequestration by a minority cytosolic fraction followed by transcriptional down-tuning. Bulk RNA-seq at 72 h captures early remodeling of adhesion/ECM, integrin/TGF-β/WNT, and cytokine programs with directionality consistent with derepression of the miR-31 regulon. In xenografts locally exposed to nanobeacons, histology shows intratumoral Cy3-positive clusters, innate-immune-like infiltrates, focal reductions in Ki-67, fibrotic stroma, increased necrosis and inflammation, spatial evidence of local microenvironment remodeling without invoking tumor-size end points. Together, these results link precise materials design to a target-engaged, noncytotoxic anti-invasive outcome and define actionable levers, especially enhanced endosomal escape and pathway-level validation, to advance readout-enabled miRNA nanotherapeutics toward translation.