Peptide-Derivatized Shell-Cross-Linked Nanoparticles. 2.\nBiocompatibility Evaluation

Abstract

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell cross-linked (SCK) nanoparticles is a method to facilitate cell surface binding and transduction. In the\nprevious report, the preparation, derivatization, and characterization of peptide-functionalized SCK\nnanoparticles were reported in detail. Following assembly, the constructs were evaluated in vitro\nand in vivo to obtain a preliminary biocompatibility assessment. The effects of SCK exposure on cell\nviability were evaluated using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2<i>H</i>-tetrazolium\nbromide (MTT) and a fluorescent apoptosis assay. Furthermore, stages of apoptosis were quantified\nby flow cytometry. Although higher levels of peptide functionalization resulted in decreased metabolic\nfunction as measured by MTT assay, significant apoptosis was not observed below 500 mg/L for all\nthe samples. To evaluate the potential immunogenic response of the peptide-derivatized constructs,\na real-time polymerase chain reaction (RT-PCR) system that allows for the in vitro analysis and\nquantification of the cellular inflammatory responses tumor necrosis factor alpha (TNF-α) and\ninterleukin-1 beta (IL1-β) was utilized. The inflammatory response to the peptide-functionalized SCK\nnanoparticles as measured by RT-PCR show statistically significant increases in the levels of both\nTNF-α and IL1-β relative to tissue culture polystyrene (TCPS). However, the measured cytokine levels\ndid not preclude the further testing of SCKs in an in vivo mouse immunization protocol. In this limited\nassay, measured increases in immunoglobulin G (IgG) concentration in the sera were minimal with\nno specific interactions being isolated, and more importantly, none of the mice (>50) subjected to the\nthree 100 μg immunization protocol have died. Additionally, no gross morphological changes were\nobserved in postmortem organ histology examinations.