Peptide-derivatized shell-cross-linked nanoparticles. 2. Biocompatibility 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 previous report, the preparation, derivatization, and characterization of peptide-functionalized SCK nanoparticles were reported in detail. Following assembly, the constructs were evaluated in vitro and in vivo to obtain a preliminary biocompatibility assessment. The effects of SCK exposure on cell viability were evaluated using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and a fluorescent apoptosis assay. Furthermore, stages of apoptosis were quantified by flow cytometry. Although higher levels of peptide functionalization resulted in decreased metabolic function as measured by MTT assay, significant apoptosis was not observed below 500 mg/L for all the samples. To evaluate the potential immunogenic response of the peptide-derivatized constructs, a real-time polymerase chain reaction (RT-PCR) system that allows for the in vitro analysis and quantification of the cellular inflammatory responses tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL1-beta) was utilized. The inflammatory response to the peptide-functionalized SCK nanoparticles as measured by RT-PCR show statistically significant increases in the levels of both TNF-alpha and IL1-beta relative to tissue culture polystyrene (TCPS). However, the measured cytokine levels did not preclude the further testing of SCKs in an in vivo mouse immunization protocol. In this limited assay, measured increases in immunoglobulin G (IgG) concentration in the sera were minimal with no specific interactions being isolated, and more importantly, none of the mice (>50) subjected to the three 100 microg immunization protocol have died. Additionally, no gross morphological changes were observed in postmortem organ histology examinations.