(T1330-06-32) Bombesin-Tagged Nanobubbles as Ultrasound Contrast Agents for Prostate Cancer Diagnosis

Purpose: Prostate cancer (Pca) is the second leading cause of death in men.¹ The increase in mortality rate is directly associated with the lack of accurate detection and grading of Pca. The standard diagnosis of Pca includes transrectal ultrasound-guided prostate biopsies.² However, due to the random nature of biopsies, this diagnostic technique has 20-30% false negative rates.² Therefore, sensitive tools capable of not only early PCa detection but also specific enough for distinguishing aggressive and indolent PCa are required. Nanobubbles (NBs) are nano-scale ultrasound contrast agents that can extravasate through leaky vasculature and accumulate at the tumor site.³ NBs have been functionalized to target prostate-specific membrane antigen (PSMA); a biomarker overexpressed in Pca.⁴ G-protein coupled gastrin-releasing peptide receptor (GRPR) is another biomarker overexpressed in the early stages of Pca.⁵ Therefore, this study proposes to develop bombesin-tagged NBs, a ligand targeting GRPR,⁶ as ultrasound contrast agents for early detection of Pca.

Methods: Bombesin (BBN) peptide was conjugated to the lipid DSPE-PEG_2k-maleimide using cysteine-maleimide chemistry (Figure 1A). The conjugation was evaluated using HPLC and MALDI-TOF mass spectrometry. Plain (untargeted) NBs containing perfluoropropane as a gas core were prepared using a previously established method.⁷ BBN-NBs were prepared by adding the conjugate, BBN-mDSPE-PEG_2k (25 µg/ml), to the precursor lipid emulsion prior to activating the NBs. The size and surface charge of the formulations were determined using dynamic light scattering. The stability and echogenicity of prepared formulations were tested in an agarose phantom using an ultrasound system (FUJIFILM VisualSonics Vevo 2100) using nonlinear contrast mode imaging at 18 MHz, 4% power, and 1 fps. All measurements were collected in triplicates and reported as mean ± SD.

Results: The successful conjugation of BBN to DSPE-PEG_2k-maleimide was validated with the increase in molecular weight observed using a MALDI-TOF mass spectrometry. BBN peptides containing NBs were successfully formulated (Figure 1B). BBN NBs showed a narrow size distribution and average intensity-weighted size increased to 325.90 ±15.98 nm, as compared to plain NBs (299.02 ± 3.89 nm) (Figure 1C). The addition of BBN peptide to the NBs decreased the zeta potential from -40.36 ± 0.68 mV (plain NBs) to -36.12 ±1.36 mV (BBN NBs) (Figure 1D). The nonlinear contrast ultrasound signal intensities of plain NBs and BBN NBs were comparable and were stable for 300 s under ultrasound (Figure 1E and 1F).

Conclusion: BBN peptide was successfully conjugated to DSPE-mPEG_2k phospholipid polymer. BBN-tagged NBs were successfully formulated. The incorporation of the peptide to the surface of the NBs increased the particle size and reduced the surface charge. The addition of BBN did not alter the stability and echogenicity of the NBs. Further studies for in vitro and in vivo targetability, efficacy, and safety of the formulation for GRPR-expressing prostate cells and mice models are currently under investigation.

References: (1) CDC. Prostate cancer statistics. https://www.cdc.gov/prostate-cancer/statistics/index.html (accessed 2024 July 13).
(2) Harvey, C. J.; Pilcher, J.; Richenberg, J.; Patel, U.; Frauscher, F. Applications of transrectal ultrasound in prostate cancer. Br J Radiol 2012, 85 Spec No 1 (Spec Iss 1), S3-17. DOI: 10.1259/bjr/56357549
(3) Jin, J.; Yang, L.; Chen, F.; Gu, N. Drug delivery system based on nanobubbles. Interdisciplinary Materials 2022, 1 (4), 471-494. DOI: https://doi.org/10.1002/idm2.12050.
(4) Perera, R. H.; de Leon, A.; Wang, X.; Wang, Y.; Ramamurthy, G.; Peiris, P.; Abenojar, E.; Basilion, J. P.; Exner, A. A. Real time ultrasound molecular imaging of prostate cancer with PSMA-targeted nanobubbles. Nanomedicine 2020, 28, 102213. DOI: 10.1016/j.nano.2020.102213
(5) Mansi, R.; Fleischmann, A.; Macke, H. R.; Reubi, J. C. Targeting GRPR in urological cancers--from basic research to clinical application. Nat Rev Urol 2013, 10 (4), 235-244. DOI: 10.1038/nrurol.2013.42
(6) de Visser, M.; Bernard, H. F.; Erion, J. L.; Schmidt, M. A.; Srinivasan, A.; Waser, B.; Reubi, J. C.; Krenning, E. P.; de Jong, M. Novel 111In-labelled bombesin analogues for molecular imaging of prostate tumours. Eur J Nucl Med Mol Imaging 2007, 34 (8), 1228-1238. DOI: 10.1007/s00259-006-0356-3
(7) Perera, R. H.; Wu, H.; Peiris, P.; Hernandez, C.; Burke, A.; Zhang, H.; Exner, A. A. Improving performance of nanoscale ultrasound contrast agents using N,N-diethylacrylamide stabilization. Nanomedicine 2017, 13 (1), 59-67. DOI: 10.1016/j.nano.2016.08.020

Acknowledgements:

Funding:
1) DoD grant # W81XWH2210931
2) National Institutes of Health grant # R01EB025741

Figure 1. BBN-tagged NBs preparation and characterizations. (A) Conjugation of BBN to DSPE-mPEG2k-maleimide, (B) Schematic representation of BBN NBs. Comparison of plain NB to BBN NBs based on (C) Intensity-weighted particle size, (D) Surface charge, (E) Nonlinear contrast ultrasound signals (yellow circles depicting the region of interest), scale bar = 0.7 mm. (F) Stability of formulations under ultrasound for 300 s. Figures created with BioRender.com.