Abstract
Background: In nuclear medicine, developing diagnostic and therapeutic agents targeted to the gastrin releasing-peptide receptor (GRPR), which is overexpressed in many cancers including prostate, ovarian, breast and gastrointestinal stormal cancers, is an active area of research. These GRPR-targeted agents are typically small radiolabeled bombesin (BBN) peptide conjugates which have nM binding affinity to the receptor. However, the targeting efficacy of macromolecular conjugates modified with BBN analogues is still largely unexplored. Owing to the advantages of large polymeric systems over small peptide conjugates such as in longevity in blood circulation and carrier capacity, our goal was to investigate the targeting efficacy of BBN-conjugated polymeric system in vitro and in vivo. Methods: HPMA copolymer construct was synthesized using RAFT polymerization. Active (L-form) and inactive (D-form) analogues of BBN (7-14) peptides modified with two positively charged amino acids to improve solubility were synthesized by solid phase peptide synthesis. Using maleimide-thiol click chemistry, four concentration, 2, 5, 10 and 15 mol% of BBN peptide, was conjugated to the polymeric platform. As a control, 10 mol% D-BBN-HPMA was synthesized similarly. The inhibitory concentration (IC50) for L-BBN and D-BBN were determined. In addition, the 1 hr cellular internalization studies for all peptide-polymer conjugates and 4 hr cellular internalization studies for the 10% L-BBN-HPMA and 10% D-BBN-HPMA were performed using the PC-3 human prostate cancer cell line. Confocal imaging studies at 4 hr were performed for 10% L-BBN-HPMA and 10% D-BBN-HPMA. Finally, biodistribution studies for all peptide-HPMA conjugates were performed. Results: The IC50 for L-BBN was found to be 1.36 nM, and no inhibition was found for D-BBN after incubating with the highest substrate concentration (3.33 µM). After 1 hr, cellular internalization studies showed high uptake of 10% L-BBN-HPMA by around 13.76% internalized activity compared to 0.61%, 3.58%, 6.00% and 9.35% for 2% L-BBN-HPMA, 5% L-BBN-HPMA, 10% D-BBN-HPMA and 15% L-BBN-HPMA , respectively. Similarly, after 4 hr, 10% L-BBN-HPMA showed higher internalized radioactivity (16.96%) compared to 10% D-BBN-HPMA (9.59%).The confocal imaging study showed higher fluorescent signal for 10% L-BBN-HPMA compared to 10% D-BBN-HPMA by two folds. Surprisingly, all peptide-HPMA conjugates showed higher retention in MPS organs (liver and spleen) except 2% L-BBN-HPMA. Interestingly, the retention in spleen was found to be directly proportional to the concentration of peptide/polymer. Since the peptides are positively charged, a relationship between the relative charge of the peptide-polymer construct and the spleen retention was observed. Conclusion: Incorporating of BBN peptides in the HPMA copolymer construct showed enhanced internalization into PC-3 cells, with 10% molar concentration being the optimum concentration. However, due to high retention in MPS organs (liver and spleen), further modifications to the BBN-HPMA conjugate are needed.