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

2026-08-07

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Saja Adil Yasir

Cheng Heng Wong

Matthew Tze Jian Wong

Kumitaa Theva Das *

DOI：10.1007/s12668-026-02773-5

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Human immunodeficiency virus (HIV) persists in long-lived latent reservoirs and are not eliminated by antiretroviral therapy (ART). Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a powerful gene-editing tool with significant potential for HIV therapy, highlighting the need for curative strategies using safe non-viral delivery systems. 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. At 3 mg/kg, minimal physiological changes were detected, with slight body weight increases (AgNPs: +1.00%; AgNPs-CRISPR: +3.81%) comparable to control (+ 10.18%). At 4 mg/kg, AgNPs induced a modest weight reduction (− 1.66%, p < 0.05), whereas AgNPs-CRISPR maintained a slight increase (+ 1.84%). More pronounced weight loss occurred at 5 mg (AgNPs: −3.51%; AgNPs-CRISPR: −3.42%, p < 0.01) and 6 mg (AgNPs: −4.84%; AgNPs-CRISPR: −4.62%, p < 0.01). Histopathological analysis demonstrated normal hepatic morphology at 3 mg/kg, early inflammatory changes at 4 mg/kg, and progressive hepatocellular degeneration and fibrosis at 5–6 mg/kg. Flow cytometric analysis revealed a dose-dependent increase in apoptosis, with significant reductions in viable cells and marked increases in late apoptotic and necrotic populations at ≥ 5 mg (p < 0.001). IL-6–positive cells increased from 9.63% in controls to 33.31% at 5 mg (p < 0.01) and 37.49% at 6 mg (p < 0.01) in AgNP-treated mice, with consistently lower levels observed in AgNPs-CRISPR groups. Similarly, TNF-α expression increased significantly at higher doses, reaching 1.725-fold (+ 238%, p < 0.01), while Bax expression showed a marked upregulation up to 1.81-fold (+ 522%, p < 0.001) at 6 mg. Across comparable doses, AgNPs-CRISPR consistently exhibited reduced cytotoxicity, inflammatory responses, and apoptotic progression compared with AgNPs alone, indicating partial mitigation of nanoparticle-induced toxicity. 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 application.

Keywords:

Cas9

CRISPR

Gene Therapy

HIV

Silver Nanoparticle

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

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