Design and Evaluation of Granzyme-B Loaded Albumin Nanoparticles for Cancer Immunotherapy

Immunotherapy has been presented as a less invasive alternative for cancer treatment. However, the efficacy of immunotherapy may be compromised by challenges associated with  drug administration, the compatibility of treatment with the patient, and the immunosuppressive nature of the tumor microenvironment. Granzyme B is a serum protease whose main function is to induce the death of target cells that are recognized by cytotoxic T lymphocytes. Various studies demonstrate the role of granzyme B in autoimmune diseases and in cancer therapy.  One of the main obstacles to the efficient therapeutic use of this enzyme is the need for protected transport and targeted delivery to the intracellular environment. To overcome these limitations, in this study, human serum albumin (HSA) nanocages were designed as a vehicle for the delivery of granzyme B for application in cancer immunotherapy. Protein nanoparticles (HSA:GrB) were successfully synthesized by reduction intramolecular disulfide bonds of HSA and were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and enzyme activity assays. With an average particle size of 40 nm, the encapuslated granzyme B presented improved stability in enzyme activity, with an encapsulation of active enzyme efficiency of 52%. Successful cellular internalization of HSA:GrB in MDA-MB-231 cell line was confirmed by confocal microscopy.  H₂O₂ was used as an oxidizing agent to break the HSA:GrB disulfide bonds allowing the contact between GrB and the intracellular enviroment.  Cell viability assay revealed a cytotoxic effect in MDA-MB-231 (24.49% of viability) after treatment with HSA:GrB followed by H₂O₂ treatment (15 min, 3mM) to accelerate the disassembly of the nanoparticles intracellularly, while the cells treated only with 3 mM H₂O₂  and free GrB did not show statistically significant differences with the negative control (untreated cells). HSA:GrB in its highest concentration without H₂O₂ showed 60% of cellular viability.  These results confirm the encapsulation efficiency and preservation of the enzymatic activity of granzyme B, the successful intracellular delivery of Granzyme B, as well as its cytotoxic effect on breast cancer cells, demonstrating its immunotherapeutic potential in cancer treatment.