Abstract

BackgroundBecause of the need to limit side-effects, nanoparticles are increasingly being studied as drug-carrying and targeting tools. We have previously reported on a scheme to produce protein-based self-assembling nanoparticles that can act as antigen display platforms. Here we attempted to use the same system for cancer-targeting, making use of a C-terminal bombesin peptide that has high affinity for a receptor known to be overexpressed in certain tumors, as well as an N-terminal polyhistidine tag that can be used for radiolabeling with technetium tricarbonyl.ResultsIn order to increase circulation time, we experimented with PEGylated and unPEGylated varities typo particle. We also tested the effect of incorporating different numbers of bombesins per nanoparticle. Biophysical characterization determined that all configurations assemble into regular particles with relatively monodisperse size distributions, having peaks of about 33 – 36 nm. The carbonyl method used for labeling produced approximately 80% labeled nanoparticles. In vitro, the nanoparticles showed high binding, both specific and non-specific, to PC-3 prostate cancer cells. In vivo, high uptake was observed for all nanoparticle types in the spleens of CD-1 nu/nu mice, decreasing significantly over the course of 24 hours. High uptake was also observed in the liver, while only low uptake was seen in both the pancreas and a tumor xenograft.ConclusionsThe data suggest that the nanoparticles are non-specifically taken up by the reticuloendothelial system. Low uptake in the pancreas and tumor indicate that there is little or no specific targeting. PEGylation or increasing the amount of bombesins per nanoparticle did not significantly improve targeting. In particular, the uptake in the spleen, which is a primary organ of the immune system, highlights the potential of the nanoparticles as vaccine carriers. Also, the decrease in liver and spleen radioactivity with time implies that the nanoparticles are broken down and cleared. This is an important finding, as it shows that the nanoparticles can be safely used as a vaccine platform without the risk of prolonged side effects. Furthermore, it demonstrates that technetium carbonyl radiolabeling of our protein-based nanoparticles can be used to evaluate their pharmacokinetic properties in vivo.

Highlights

  • Because of the need to limit side-effects, nanoparticles are increasingly being studied as drug-carrying and targeting tools

  • self-assembling protein nanoparticles (SAPN) design We wanted to test whether there was a difference in the biodistribution of the nanoparticles based on different density of the targeting peptide bombesin and different coupling modalities of poly(ethylene glycol) (PEG) to the nanoparticles

  • In the P6c-PbK peptide the lysine coupling site was engineered to the C-terminus at the in the P6cPbK peptide the lysine coupling site was engineered to the C-terminus at the very end of a peptide surface modification (GGSGDPPPPNPNDPPPPNPND) that previously produced very nice nanoparticles [24], it was moved from a less exposed site on the N-terminus to a wellexposed site on the C-terminus

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Summary

Introduction

Because of the need to limit side-effects, nanoparticles are increasingly being studied as drug-carrying and targeting tools. We have used coiled-coil domains as building blocks to engineer self-assembling protein nanoparticles (SAPN) [6,7,8] Such proteins can be produced with genetically engineered cells and can be extended at the N or C-terminus with freely selectable amino acid residues. If the peptide sequence of a known antigen is used, formation of the nanoparticle will result in the repetitive display of that antigen. We have used this concept to produce promising vaccine candidates for illnesses such as influenza, malaria, SARS, and HIV [9,10,11,12]

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