In Vivo Toxicity Assessment of Biosynthesized Copper and Zinc Oxide Nanoparticles Administered via Intraperitoneal and Intravenous Routes in Rats

Abstract

Copper (Cu) and zinc oxide (ZnO) nanoparticles (NPs) are widely applied as antimicrobial agents in cosmetics, food additives and medicine. However, their potential toxicity to humans and ecosystems remains a critical concern. This study investigated the in‑vivo fate and safety profile of biosynthesized CuNPs (Bio‑CuNPs) and ZnONPs (Bio‑ZnONPs) in male Wistar rats, administered either intraperitoneally (i/p) or intravenously (i/v). Doses ranged from 6.1–19.82 µg kg⁻¹ for Bio‑CuNPs and 11.14–30.3 µg kg⁻¹ for Bio‑ZnONPs, reflecting concentrations used in antibacterial studies. Over 28 days, we monitored mortality, body weight, behavior, hematology, serum biochemistry (creatinine, ALT, AST, ALP), organ weights, and histopathology of liver, kidney, spleen, and brain. No deaths occurred in either group. Bio‑ZnONPs, irrespective of route, did not alter liver or kidney biomarkers, and body weight changes were minimal. In contrast, Bio‑CuNPs at 9.5–11.7 µg kg⁻¹ (i/v) produced mild to moderate elevations in serum ALT, AST, and ALP that persisted to day 28, accompanied by reduced spleen and liver indices and histological evidence of hepatic vacuolation and renal tubular damage. The i/p route mitigated these effects. Overall, Bio‑ZnONPs exhibited a more favorable safety profile, suggesting their potential suitability for intravenous biomedical applications.

Background

Copper and zinc are essential trace elements, yet excessive exposure to their nanoparticulate forms can trigger hemolysis, gastrointestinal irritation, and organ toxicity. With the rapid expansion of metal‑based nanoparticles in consumer and medical products, understanding their biological interactions and potential hazards has become paramount. Previous in‑vivo studies have examined intranasal, intratracheal, and oral exposure to CuNPs and ZnONPs, but data on systemic routes—intravenous (i/v) and intraperitoneal (i/p)—remain sparse. This work addresses that gap by evaluating the toxicokinetics and organ‑level effects of 16–96 nm biosynthesized CuNPs and ZnONPs in male Wistar rats, using dose ranges aligned with antibacterial efficacy studies.

Methods

Biosynthesis

CuNPs and ZnONPs were produced extracellularly using non‑pathogenic Enterococcus faecalis supernatant, yielding spherical particles of 12–90 nm for CuNPs and 16–96 nm for ZnONPs (FeSEM and TEM confirmation).

Animal Study Design

Sixteen‑ to 17‑week‑old male Wistar rats (160–200 g) were acclimatized for one week before dosing. Rats were randomly assigned to six groups per route (i/p and i/v), each containing six animals. Doses were prepared in sterile water (50 mg mL⁻¹ stock) and diluted to final concentrations of 6.1–19.82 µg kg⁻¹ (CuNPs) and 11.14–30.3 µg kg⁻¹ (ZnONPs). Vehicle controls received distilled water. All procedures followed Good Laboratory Practice and were approved by the institutional Animal Ethics Committee (Approval no. 346/CPCSEA).

Observations

Clinical signs were recorded daily; food and water intake, body weight, and behavior were monitored every two days. Hematology (RBC, WBC, hemoglobin, platelets) and serum biochemistry (creatinine, ALT, AST, ALP) were measured on days 14 and 28. Organ weights were obtained post‑mortem, and organ indices were calculated as: O_X = (organ weight/animal weight) / (control organ weight/control animal weight). Histological sections of liver, kidney, spleen, and brain were stained with H&E and examined microscopically.

Statistical Analysis

Data are expressed as mean ± SD (N = 6 per group). Statistical significance was assessed by ANOVA with post‑hoc tests; p < 0.05 was considered significant.

Results and Discussion

Body Weight and Clinical Observations

All rats survived the 28‑day period. Body weight reductions were modest (<5 %) and transient for both nanoparticle types. Bio‑ZnONPs produced the smallest weight changes, whereas Bio‑CuNPs at the highest i/v dose (11.7 µg kg⁻¹) exhibited a statistically significant decline by day 28 (p < 0.05).

Hematology

Day 14 hematology revealed slight, non‑significant decreases in RBC, WBC, and platelet counts for Bio‑CuNPs (i/v) at IC₅₀ and TLC. By day 28, hematologic parameters returned to baseline, indicating no persistent hematopoietic toxicity. Bio‑ZnONPs did not alter any hematologic indices at any dose or route.

Serum Biochemistry

Creatinine levels remained within normal limits for all groups. Bio‑CuNPs (i/v) caused modest elevations in ALT (up to 67.7 mg dL⁻¹), AST (70 IU L⁻¹), and ALP (128 IU L⁻¹) that persisted to day 28, reflecting hepatocellular and cholestatic injury. In contrast, Bio‑ZnONPs (both i/v and i/p) did not significantly alter ALT, AST, or ALP at any dose. The i/p route mitigated hepatic enzyme elevations for Bio‑CuNPs, underscoring route‑dependent toxicity.

Organ Indices and Histopathology

Organ indices for liver and spleen were significantly reduced in Bio‑CuNPs (i/v) at IC₅₀ and TLC (p < 0.05), while Bio‑ZnONPs produced no detectable changes. Histology corroborated these findings: Bio‑CuNPs (i/v) induced hepatic cytoplasmic vacuolation, central vein congestion, and renal tubular necrosis, whereas Bio‑ZnONPs preserved normal architecture. Spleen sections from Bio‑CuNPs (i/v) displayed reduced red pulp cellularity, whereas splenic structure remained intact after Bio‑ZnONPs exposure.

Route‑Dependent Toxicity

Intravenous administration consistently elicited greater biochemical and histological alterations for Bio‑CuNPs compared with intraperitoneal delivery. Bio‑ZnONPs were well tolerated via both routes, with only minor, reversible weight changes and no organ damage.

Conclusion

This comprehensive in‑vivo assessment demonstrates that biosynthesized ZnONPs exhibit a superior safety profile than CuNPs when administered systemically to rats. While CuNPs induced dose‑dependent hepatotoxicity and renal injury—especially via the i/v route—ZnONPs caused negligible adverse effects at comparable concentrations. These results support further exploration of intravenous ZnONPs for biomedical applications, with CuNPs requiring careful dose and route optimization to mitigate organ toxicity.

Abbreviations

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

ALP

Alkaline phosphatase

Bio‑CuNPs

Biogenic copper nanoparticles

Bio‑ZnONPs

Biogenic zinc oxide nanoparticles

IC₅₀

Inhibitory concentration 50%

NOAEC

No observable adverse effect concentration

TLC

Total lethal concentration

i/p

Intraperitoneal route

i/v

Intravenous route