Ultrasound-Enhanced CRISPR-Curcumin Nanoparticles for Gene-Modulating Therapy in Metastatic Pulmonary Lesions.

[BACKGROUND] Curcumin exhibits strong gene-regulatory, epigenetic-modulating, and oncogene-suppressive properties, but its poor aqueous solubility and low intracellular bioavailability limit therapeutic application. Gene-targeted nanodelivery platforms combined with ultrasound-mediated permeabilization provide a promising approach to enhance the precision and potency of curcumin-based gene therapy. This study evaluates CRISPR-supported, curcumin-loaded polymeric nanoparticles (CRISPR-CuNPs) designed to silence the KRAS-G12D oncogene while enhancing tumor-suppressor gene expression (TP53, PTEN) in metastatic pulmonary cancer cells, using experimentally optimized ultrasound conditions validated for delivery efficiency and cellular safety.

[METHODS] Curcumin-loaded polymeric nanoparticles were co-formulated with plasmid DNA encoding CRISPR/Cas9 elements targeting KRAS-G12D and delivered to cancer cells with or without experimentally optimized low-intensity pulsed ultrasound (1 MHz, 0.5 W/cm). Nanoparticle characterization included size, zeta potential, polydispersity index, and encapsulation effectiveness. Nanoparticle stability, morphology, and plasmid retention were evaluated under physiological temperature and serum conditions before and after ultrasound exposure. Intracellular uptake, endosomal escape, and nuclear localization were quantified using luminescence microscopy and flow cytometry. Gene silencing and gene improvement were analyzed using qPCR, Western blotting, and chromatin immunoprecipitation assays for epigenetic profiling. The MTT, Annexin V, and three-dimensional tumor models were used to assess cytotoxicity, apoptosis, and spheroid loss, respectively.

[RESULTS] After ultrasound exposure, CRISPR-CuNPs showed a 2.7-fold increase in nuclear-associated nanoparticle fluorescence, suggesting improved endosomal escape and nuclear entry for effective gene delivery and modulation. A low polydispersity index (0.18 ± 0.02) determined by dynamic light scattering minimized variability in uptake across replicate organs. KRAS mRNA and protein levels were reduced by 72 ± 4%, with greater suppression observed in CRISPR-CuNP-treated cells compared with curcumin-only controls, confirming CRISPR-mediated gene disruption. Curcumin-mediated chromatin remodeling was evidenced by enrichment of H3K27ac at TP53 and PTEN promoters, resulting in upregulation of TP53 (3.5-fold) and PTEN (2.8-fold). Ultrasound-enhanced CRISPR-CuNPs further increased KRAS protein repression to 90 and elevated PTEN expression 46-fold. Apoptosis induction reached 87 ± 3, synergistic interaction between KRAS silencing and epigenetic activation of tumor-suppressor pathways, while metastatic lung cancer spheroids showed an 80% reduction in viable tumor volume. Minimal γ-H2AX induction indicated low acute DNA damage, supporting short-term genomic safety.

[CONCLUSIONS] Ultrasound-stimulated CRISPR-CuNPs provide a potent, non-radiotoxic platform for targeted gene therapy. By integrating experimentally validated ultrasound parameters with KRAS gene silencing and curcumin-driven epigenetic activation of TP53 and PTEN, this approach achieves synergistic suppression of metastatic pulmonary cancer phenotypes. While sequencing-based genome-editing confirmation and long-term genomic stability studies remain necessary, this system holds significant promise for gene-enhancing, minimally invasive therapeutic strategies for the unborn.