In Vivo Safety Evaluation of Silver Nanoparticle Encapsulated CRISPR/Cas9: Implications for HIV Gene Therapy

Human immunodeficiency virus (HIV) persists in long-lived latent reservoirs that are not eliminated by antiretroviral therapy (ART), highlighting the need for curative strategies such as CRISPR/Cas9-mediated gene editing delivered through safe non-viral vectors. This study evaluated the short-term in vivo safety, physiological tolerance, hepatic effects, inflammatory responses, and apoptotic gene activation associated with silver nanoparticles (AgNPs) and AgNPs-CRISPR in BALB/c mice. Thirty mice were assigned to control, AgNPs (3–6 mg/kg), or CRISPR/Cas9–AgNPs groups and received a single intraperitoneal injection, followed by a 14-day monitoring period. Behavioural parameters, body weight, hepatic histopathology, apoptosis (Annexin V–FITC/PI), intracellular IL-6 expression, and hepatic TNF-α and Bax gene expression were assessed. No mortality or behavioural abnormalities were observed. Minimal physiological changes occurred at 3 mg/kg, while dose-dependent reductions in body weight appeared at higher concentrations. Histopathological analysis revealed normal hepatic morphology at 3 mg/kg, early inflammatory alterations at 4 mg/kg, and progressive hepatocellular degeneration and fibrosis at 5–6 mg/kg. Flow cytometry demonstrated a dose-dependent increase in apoptosis, accompanied by elevated IL-6, TNF-α, and Bax expression at higher concentrations. Across comparable doses, AgNPs-CRISPR consistently produced milder pathological effects than AgNPs alone. These findings identify 3–4 mg/kg as a short-term safe dose range for AgNPs-mediated CRISPR delivery and provide foundational safety evidence supporting further investigation of this platform for HIV gene-therapy applications.