Single-Cell Nanoencapsulation for Immune Enhancement: An Experimental Study on the Immunomodulatory Effects of CuO Nanoparticles Bio-Conjugated with BCC in Cisplatin-Treated Rats

Abstract

This study aimed to biosynthesize a Nano-therapeutic vaccine to alleviate cisplatin-induced immunosuppression and toxicity in rats. Copper oxide nanoparticles (CuONPs) were synthesized using human serum albumin (HSA) as a stabilizing agent and bio-conjugated with breast cancer cell lysates (BCC) to generate CuONPs-BCC, a nanomaterial designed as a therapeutic vaccine candidate. The prepared copper nanoparticles were characterized using UV–Visible spectroscopy, Fourier Transform Infrared spectroscopy (FTIR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), and Energy Dispersive X-ray (EDX) analysis. Further, in-vitro biological activities of prepared nanoparticles were assessed, antioxidant activity was assessed using the ferric reducing antioxidant power (FRAP) assay, while anti-inflammatory potential was investigated through anti-hemolytic and protein denaturation inhibition assays. For in-vivo study, female Wistar albino rats were randomly divided into four groups (n = 5): Control rat group, cisplatin-treated rat group, immune-depressed rat group treated with CuONPs-BCC, and immune-depressed rat group treated with BCC. The rats were received treatments via intraperitoneal route for three weeks. Cisplatin was used to induce immunodepression and organ toxicity. Different investigations were performed, including hematological parameters; biochemical parameters, such as blood glucose and C-reactive protein (CRP); oxidative stress biomarkers in different organs, including lipid peroxidation and antioxidant defense markers; and histopathological examination of liver, kidney, heart, and spleen tissues. Immunological evaluation was also carried out to assess the immunomodulatory effects of the therapeutic formulation. The obtained results confirmed the successful formation of stable copper nanoparticles with very small crystallite sizes, approximately 8 nm for CuONPs and 8.3 nm for CuONPs-BCC .For in-vitro study results, in the FRAP assay, CuONPs-BCC exhibited the strongest antioxidant activity with an IC₅₀ value of 66 µg/mL, followed by CuONPs (80 µg/mL. Anti-hemolytic activity results showed that CuONPs displayed the highest protective effect against hemolysis with an IC₅₀ value of approximately 7 µg/mL, outperforming CuONPs-BCC (14 µg/mL). In contrast, the protein denaturation assay revealed that CuONPs-BCC possessed the strongest inhibitory activity, with an IC₅₀ value (70 µg/mL). On the other hand, toxicity test results showed that the nanoparticles can be used safely, as they did not exhibit any toxicity to Rats at high concentrations. In vivo findings demonstrated that cisplatin administration caused significant increases (p < 0.05) in blood glucose and C-reactive protein (CRP) levels, associated with alterations in hematological parameters. Furthermore, an immunosuppressive effect was observed, as evidenced by changes in leukocyte counts and IgG levels. Oxidative stress markers were also significantly affected (p < 0.05) by elevated malondialdehyde (MDA) levels, along with decreased reduced glutathione (GSH) content and superoxide dismutase (SOD) activity in study organs. Histopathological analysis also showed severe tissue damage characterized by necrosis, inflammatory infiltration, hemorrhage, and disruption of normal tissue architecture in the liver, kidney, heart, and spleen. In contrast, treatment with CuNPs-BCC markedly enhanced immune function, improved hematological and biochemica