Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles

Purpose: siRNA has therapeutic potential, but in vivo delivery is limited by rapid clearance, nuclease degradation, and nonspecific distribution. A tumor-targeted, biocompatible delivery system was needed to increase intratumoral siRNA accumulation, reduce off-target effects, and enable silencing of growth-promoting genes in ovarian cancer.

Hypothesis: If siRNA is incorporated into cyclic RGD peptide-labeled chitosan nanoparticles, then the nanoparticles will bind αvβ3 integrin-expressing tumor cells and tumor vasculature, selectively deliver siRNA in vivo, silence target genes, and produce antitumor efficacy in orthotopic ovarian cancer models.

Aims: Develop and characterize RGD-conjugated chitosan nanoparticles (RGD-CH-NP) loaded with siRNA. - Confirm binding specificity for αvβ3 integrin in vitro. - Evaluate in vivo tumor-targeted delivery and gene silencing of POSTN, FAK, and PLXDC1. - Assess antitumor efficacy alone and in combination with docetaxel in multiple orthotopic ovarian cancer models.

Delivery system: Platform: Chitosan nanoparticles (CH-NP) prepared by ionic gelation with tripolyphosphate and siRNA. - Targeting ligand: Cyclic RGD peptide c[RGDfK(Ac-SCH2CO)] conjugated to chitosan via thiolation using SPDP. - Payload: siRNA targeting POSTN, FAK, PLXDC1, or control siRNA; Alexa555-labeled siRNA for tracking. - Properties: Size ~200 nm; zeta potential ~40 mV; spherical by SEM. RGD conjugation did not substantially alter nanoparticle formation. Selected formulation: RGD-CH-NP 5 (1.45 µg RGD/mg chitosan).

Approach: In vitro: SKOV3ip1 (αvβ3-positive), A2780ip2 (αvβ3-negative), HeyA8, A2780, and murine ovarian endothelial cells. - In vivo: Female athymic nude mice; orthotopic ovarian cancer via intraperitoneal injection of SKOV3ip1, HeyA8, or A2780 cells. Treatment began 1 week after tumor injection; n = 10 mice/group. - Dosing: siRNA/RGD-CH-NP or siRNA/CH-NP given twice weekly by i.v. injection at 150 µg/kg body weight. Docetaxel 100 µg i.p. once weekly. Mice sacrificed when moribund. - Delivery study: Single i.v. injection of Alexa555 siRNA/RGD-CH-NP or CH-NP into SKOV3ip1-bearing mice; tissues harvested after 48 h.

Key methods: ¹H-NMR and FITC-labeled RGD for conjugation/quantification. - Dynamic light scattering and zeta potential; SEM for morphology. - Flow cytometry, fluorescence microscopy, confocal microscopy, and TEM for binding/uptake. - In vivo fluorescence imaging for tumor/organ distribution. - Western blot and qRT-PCR for target gene silencing. - Immunohistochemistry for Ki67 (proliferation), CD31 (microvessel density), TUNEL (apoptosis), and POSTN; dual immunofluorescence for CD31/TUNEL and CD31/PLXDC1. - Tumor weight and nodule counts.

Key results: Nanoparticle binding: In αvβ3-positive SKOV3ip1 cells, binding increased with RGD concentration; siRNA/RGD-CH-NP 5 showed highest binding, with 94.25% binding efficiency versus CH-NP. Little binding occurred in αvβ3-negative A2780ip2 cells. - In vivo delivery: Alexa555 siRNA/RGD-CH-NP localized in >80% of tumor fields and showed up to 3-fold higher tumor localization than CH-NP. It colocalized with αvβ3 integrin and showed minimal accumulation in liver, kidney, spleen, lung, heart, and brain compared with CH-NP. - POSTN silencing: Single i.v. POSTN siRNA/RGD-CH-NP reduced POSTN expression by >51% versus control siRNA/CH-NP and >20% versus CH-NP at 24 h. Immunohistochemistry confirmed greater POSTN inhibition. - SKOV3ip1 efficacy: POSTN siRNA/RGD-CH-NP + PBS reduced tumor growth by 24% versus POSTN siRNA/CH-NP + PBS (P < 0.04) and 71% versus control siRNA/CH-NP + PBS (P < 0.001). Combination with docetaxel showed greatest inhibition: 32% reduction versus control siRNA/CH-NP + docetaxel (P < 0.006) and 22% versus CH-NP + docetaxel (P < 0.01). - A2780 efficacy: POSTN siRNA/RGD-CH-NP + PBS reduced tumor growth by 73% versus control siRNA/CH-NP + PBS (P < 0.01), but no additional benefit over POSTN siRNA/CH-NP (P < 0.22). Combination with docetaxel was effective. - FAK silencing in HeyA8: FAK siRNA/RGD-CH-NP + PBS significantly inhibited tumor growth versus FAK siRNA/CH-NP + PBS (P < 0.04) and control (P < 0.001); combination with docetaxel was greatest (P < 0.02). - Vascular targeting in A2780: PLXDC1 siRNA/RGD-CH-NP reduced tumor growth by 87% versus control siRNA/CH-NP (P < 0.001) and was superior to PLXDC1 siRNA/CH-NP (P < 0.01). It increased apoptosis in tumor vasculature and completely silenced PLXDC1 in tumor endothelial cells. - Toxicity: No differences in body weight, feeding, or behavior; no overt therapy-related toxicities.

Interpretation: The authors conclude that RGD-CH-NP is a novel, highly selective siRNA delivery system that targets both tumor cells and tumor vasculature. It enables efficient silencing of multiple growth-promoting genes and produces significant antitumor efficacy in preclinical ovarian cancer models, supporting broad potential for targeted RNAi therapy in human disease.

Limitations: Preclinical mouse models only; no human or large-animal validation. - Efficacy was tested in ovarian cancer models; broader applicability remains to be demonstrated. - αvβ3 integrin expression is heterogeneous; A2780 tumor cells were negative, though vasculature was positive. - No long-term survival, immune memory, or detailed pharmacokinetic/biodistribution analysis reported. - Potential off-target effects, immunogenicity, and chronic toxicity were not deeply evaluated. - RGD peptide may bind other integrins in addition to αvβ3.