fig5

Figure 5. Characterization of siADAM9 encapsulation, stability, and target gene silencing mediated by EV-PSMA^scFv. (A) Quantification of siRNA copy number per EV particle following encapsulation of siADAM9 variants (#1 and #2). ND; (B) Encapsulation efficiency of siADAM9 in EV-PSMA^scFv determined by qPCR analysis, relative to input naked siRNA; (C) RNase protection assay. Naked or EV-encapsulated siADAM9 was exposed to exogenous RNase prior to qPCR analysis. EV encapsulation protected siADAM9 from RNase-mediated degradation, as indicated by lower Cq values compared with naked siRNA controls; (D) RT-qPCR analysis of ADAM9 mRNA expression in LNCaP and CWR22Rv1 cells following treatment with EV-PSMA^scFv loaded with siADAM9 or siNC. Unloaded EVs and mock-treated cells served as controls; (E) Immunoblot analysis of ADAM9 protein expression in LNCaP and CWR22Rv1 cells following treatment with siADAM9-loaded EV-PSMA^scFv. β-Actin served as the loading control. Relative protein expression levels normalized to β-actin are indicated below the blots. Experiments were conducted in three independent biological repeats (n = 3), each using an independently prepared EV batch, with analyses performed in triplicate where applicable. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Šídák’s multiple-comparisons test for the indicated comparisons. ns, not significant; ^***P < 0.001. EVs: Extracellular vesicles; PSMA: prostate-specific membrane antigen; siRNA: small interfering RNA; siADAM9: ADAM9-targeting small interfering RNA; siNC: non-targeting small interfering RNA control; ND: not detected; RNase: ribonuclease; qPCR: quantitative polymerase chain reaction; Cq: quantification cycle; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SD: standard deviation; ANOVA: analysis of variance; β-actin: beta-actin; EV-PSMA^scFv: extracellular vesicles displaying PSMA-targeting single-chain variable fragments.