In-vitro and in-vivo efficacy of hollow gold nanoparticles encapsulating horseradish peroxidase: Oxidative stress-mediated tumor cell killing

This article reports the protocol for the synthesis of hollow gold nanoparticles using harmless template-material calcium phosphate nanoparticles for encapsulating enzyme to overcome the problem associated with antibody-directed enzyme pro-drug therapy (ADEPT) or chemotherapy against cancer. Horseradish peroxidase (HRP) could be effectively loaded inside the cavity of hollow gold nanoparticles. The high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS) analysis confirmed monodispersed formulation demonstrating spherical morphology with an average diameter of 50 nm. Aqueous dispersion of these nanoparticles was highly stable under cold conditions. HRP loaded in the cavity of hollow gold nanoparticles showed more stability in response to change in temperature as compared to unloaded/free enzyme and followed Michaelis-Menten kinetics. From the in-vitro MTT assay, it is concluded that the prodrug - Indole acetic acid (IAA) is non-toxic, however, after oxidative decarboxylation by HRP, it became active/toxic. The cell death mechanism by IAA/entrapped-HRP combination confirmed apoptosis as demonstrated by DNA fragmentation and AO/PI staining. For assessing in-vivo efficacy, the hollow gold nanoparticles with HRP was topically administered into the xenographted tumor in mice. The benign substrate IAA was given either orally or by intra-peritoneal injection. It reached the tumor tissue through systemic circulation to get ox- 0nbvfcxidized by HRP. This created severe and lethal oxidative stress to the cancer cells resulting in regression of tumor. The uniqueness and novelty of our work lies in the fact that we have used benign template to generate/create hollow formulation loaded with HRP, which could enhance the treatment efficacy by multi-fold as compared to the free HRP or solid formulation as more active sites are available due to internal mobility of the enzyme for interaction with the prodrug. From the survival percent graph, we concluded that all mice died after ∼32 days in case of free HRP/IAA combination but in case of HGNP-HRP/IAA combination, 100% mice were alive after ∼30 days and ∼50% of tumor bearing mice remained alive even after 60 days. In conclusion, both in vitro and in vivo experiments revealed that the combination of IAA/HRP entrapped in hollow gold nanoparticles could be efficiently and effectively used for cancer therapy.