Skip to main content
Log in

In Vitro Evaluation and Biodistribution Studies of HPMA Copolymers Targeting the Gastrin Releasing Peptide Receptor in Prostate Cancer

  • RESEARCH PAPER
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

The development of diagnostic and therapeutic agents utilizing small peptides (e.g., bombesin (BBN)) to target the overexpression of the gastrin-releasing peptide receptor (GRPR) in cancers has been widely investigated. Herein, we examine the capabilities of BBN-modified HPMA copolymers to target the GRPR.

Methods

Four positive, four negative, and two zwitterionic BBN HPMA copolymer conjugates of varying peptide content and charge were synthesized. In vitro and in vivo studies were conducted in a GRPR-overexpressing prostate cancer cell line (PC-3) and a normal CF-1 mouse model, respectively.

Results

Cellular uptake of the conjugates were found to be charge and BBN density dependent. The positively-charged conjugates illustrated a direct relationship between the extent of cellular internalization, ranging from 0.7 to 20%, and BBN-incorporation density. The negative and zwitterionic conjugates showed low PC-3 uptake values. Blocking studies confirmed the GRPR-targeting effect of the positively-charged constructs. In vivo studies of the positively-charged copolymers resulted in rapid blood clearance by the mononuclear phagocyte system (MPS)-associated tissues (e.g., liver and spleen).

Conclusion

Positively-charged BBN-HPMA copolymer conjugates demonstrated good GRPR-targeting and internalization in vitro. However, the impact of peptide density and charge on in vivo MPS recognition are parameters that must be optimized in future agent development.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BBN:

Bombesin

BBN-EE:

Bombesin peptide modified with two negatively charged amino acids

BBN-RR:

Bombesin peptide modified with two positively charged amino acids

GRPR:

Gastrin releasing peptide receptor

P-D-RR:

HPMA copolymer modified with D-BBN-RR

P-EE:

HPMA copolymer modified with BBN-EE

P-RR:

HPMA copolymer modified with BBN-RR

P-RREE:

HPMA copolymer modified with BBN-RR and BBN-EE

References

  1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70(1):7–30.

    PubMed  Google Scholar 

  2. Markwalder R, Reubi JC. Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res. 1999;59(5):1152–9.

    CAS  PubMed  Google Scholar 

  3. Yu Z, Ananias HJ, Carlucci G, Hoving HD, Helfrich W, Dierckx RA, et al. An update of radiolabeled bombesin analogs for gastrin-releasing peptide receptor targeting. Curr Pharm Des. 2013;19(18):3329–41.

    CAS  PubMed  Google Scholar 

  4. Moreno P, Ramos-Alvarez I, Moody TW, Jensen RT. Bombesin related peptides/receptors and their promising therapeutic roles in cancer imaging, targeting and treatment. Expert Opin Ther Targets. 2016;20(9):1055–73.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Aloj L, Attili B, Lau D, Caraco C, Lechermann LM, Mendichovszky IA, Harper I, Cheow H, Casey RT, Sala E. The emerging role of cell surface receptor and protein binding radiopharmaceuticals in cancer diagnostics and therapy. Nuclear Medicine and Biology. 2020.

  6. Accardo A, Galli F, Mansi R, Del Pozzo L, Aurilio M, Morisco A, et al. Pre-clinical evaluation of eight DOTA coupled gastrin-releasing peptide receptor (GRP-R) ligands for in vivo targeting of receptor-expressing tumors. EJNMMI Res. 2016;6(1):17.

    PubMed  PubMed Central  Google Scholar 

  7. Hoppenz P, Els-Heindl S, Beck-Sickinger AG. Identification and stabilization of a highly selective gastrin-releasing peptide receptor agonist. J Pept Sci. 2019;25(12):e3224.

    CAS  PubMed  Google Scholar 

  8. Kurth J, Krause BJ, Schwarzenbock SM, Bergner C, Hakenberg OW, Heuschkel M. First-in-human dosimetry of gastrin-releasing peptide receptor antagonist [(177)Lu]Lu-RM2: a radiopharmaceutical for the treatment of metastatic castration-resistant prostate cancer. Eur J Nucl Med Mol Imaging. 2020;47(1):123–35.

    CAS  PubMed  Google Scholar 

  9. Zhang J, Niu G, Fan X, Lang L, Hou G, Chen L, et al. PET using a GRPR antagonist (68)Ga-RM26 in healthy volunteers and prostate Cancer patients. J Nucl Med. 2018;59(6):922–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Wieser G, Mansi R, Grosu AL, Schultze-Seemann W, Dumont-Walter RA, Meyer PT, et al. Positron emission tomography (PET) imaging of prostate cancer with a gastrin releasing peptide receptor antagonist--from mice to men. Theranostics. 2014;4(4):412–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Rizzo LY, Theek B, Storm G, Kiessling F, Lammers T. Recent progress in nanomedicine: therapeutic, diagnostic and theranostic applications. Curr Opin Biotechnol. 2013;24(6):1159–66.

    CAS  PubMed  Google Scholar 

  12. Lammers T, Aime S, Hennink WE, Storm G, Kiessling F. Theranostic nanomedicine. Acc Chem Res. 2011;44(10):1029–38.

    CAS  PubMed  Google Scholar 

  13. Soares S, Sousa J, Pais A, Vitorino C. Nanomedicine: principles, properties, and regulatory issues. Front Chem. 2018;6:360.

    PubMed  PubMed Central  Google Scholar 

  14. Maeda H. Toward a full understanding of the EPR effect in primary and metastatic tumors as well as issues related to its heterogeneity. Adv Drug Deliv Rev. 2015;91:3–6.

    CAS  PubMed  Google Scholar 

  15. Bertrand N, Wu J, Xu X, Kamaly N, Farokhzad OC. Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology. Adv Drug Deliv Rev. 2014;66:2–25.

    CAS  PubMed  Google Scholar 

  16. Chanda N, Kattumuri V, Shukla R, Zambre A, Katti K, Upendran A, et al. Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity. Proc Natl Acad Sci U S A. 2010;107(19):8760–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Lee C-M, Jeong H-J, Cheong S-J, Kim E-M, Kim DW, Lim ST, et al. Prostate cancer-targeted imaging using magnetofluorescent polymeric nanoparticles functionalized with bombesin. Pharm Res. 2010;27(4):712–21.

    CAS  PubMed  Google Scholar 

  18. Li R, Gao R, Wang Y, Liu Z, Xu H, Duan A, et al. Gastrin releasing peptide receptor targeted nano-graphene oxide for near-infrared fluorescence imaging of oral squamous cell carcinoma. Sci Rep. 2020;10(1):1–12.

    Google Scholar 

  19. Cai H, Xie F, Mulgaonkar A, Chen L, Sun X, Hsieh J-T, Peng F, Tian R, Li L, Wu C. Bombesin functionalized 64Cu-copper sulfide nanoparticles for targeted imaging of orthotopic prostate cancer. Nanomedicine. 2018(0), 13.

  20. Kirpotin DB, Drummond DC, Shao Y, Shalaby MR, Hong K, Nielsen UB, et al. Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models. Cancer Res. 2006;66(13):6732–40.

    CAS  PubMed  Google Scholar 

  21. Schmidt MM, Wittrup KD. A modeling analysis of the effects of molecular size and binding affinity on tumor targeting. Mol Cancer Ther. 2009;8(10):2861–71.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Chytil P, Koziolová E, Etrych T, Ulbrich K. HPMA copolymer–drug conjugates with controlled tumor-specific drug release. Macromol Biosci. 2018;18(1):1700209.

    Google Scholar 

  23. Kopeček J, Kopečková P. HPMA copolymers: origins, early developments, present, and future. Adv Drug Deliv Rev. 2010;62(2):122–49.

    PubMed  Google Scholar 

  24. Fan W, Zhang W, Jia Y, Brusnahan SK, Garrison JC. Investigation into the biological impact of block size on Cathepsin S-degradable HPMA copolymers. Mol Pharm. 2017;14(5):1405–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Fan W, Shi W, Zhang W, Jia Y, Zhou Z, Brusnahan SK, et al. Cathepsin S-cleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer. Biomaterials. 2016;103:101–15.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Pike DB, Ghandehari H. HPMA copolymer-cyclic RGD conjugates for tumor targeting. Adv Drug Deliv Rev. 2010;62(2):167–83.

    CAS  PubMed  Google Scholar 

  27. Allmeroth M, Moderegger D, Gündel D, Buchholz HG, Mohr N, Koynov K, et al. PEGylation of HPMA-based block copolymers enhances tumor accumulation in vivo: a quantitative study using radiolabeling and positron emission tomography. J Control Release. 2013;172(1):77–85.

    CAS  PubMed  Google Scholar 

  28. Allmeroth M, Moderegger D, Biesalski B, Koynov K, Rösch F, Thews O, et al. Modifying the body distribution of HPMA-based copolymers by molecular weight and aggregate formation. Biomacromolecules. 2011;12(7):2841–9.

    CAS  PubMed  Google Scholar 

  29. Omelyanenko V, Kopeckova P, Gentry C, Kopecek J. Targetable HPMA copolymer-adriamycin conjugates. Recognition, internalization, and subcellular fate. J Control Release. 1998;53(1–3):25–37.

    CAS  PubMed  Google Scholar 

  30. Li C, Winnard PT, Takagi T, Artemov D, Bhujwalla ZM. Multimodal image-guided enzyme/prodrug cancer therapy. J Am Chem Soc. 2006;128(47):15072–3.

    CAS  PubMed  Google Scholar 

  31. Buckway B, Frazier N, Gormley AJ, Ray A, Ghandehari H. Gold nanorod-mediated hyperthermia enhances the efficacy of HPMA copolymer-90Y conjugates in treatment of prostate tumors. Nucl Med Biol. 2014;41(3):282–9.

    CAS  PubMed  Google Scholar 

  32. Yi Y, Kim HJ, Mi P, Zheng M, Takemoto H, Toh K, et al. Targeted systemic delivery of siRNA to cervical cancer model using cyclic RGD-installed unimer polyion complex-assembled gold nanoparticles. J Control Release. 2016;244:247–56.

    CAS  PubMed  Google Scholar 

  33. Yang M, Gao H, Zhou Y, Ma Y, Quan Q, Lang L, et al. 18F-labeled GRPR agonists and antagonists: a comparative study in prostate cancer imaging. Theranostics. 2011;1:220–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang W, Fan W, Ottemann BM, Alshehri S, Garrison JC. Development of improved tumor-Residualizing, GRPR-targeted agents: preclinical comparison of an Endolysosomal trapping approach in agonistic and antagonistic constructs. J Nucl Med. 2020;61(3):443–50.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Mccormick LA, Seymour LC, Duncan R, Kopecek J. Interaction of a cationic N-(2-hydroxypropyl) methacrylamide copolymer with rat visceral yolk sacs cultured in vitro and rat liver in vivo. J Bioact Compat Polym. 1986;1(1):4–19.

    CAS  Google Scholar 

  36. Gustafson HH, Holt-Casper D, Grainger DW, Ghandehari H. Nanoparticle uptake: the phagocyte problem. Nano Today. 2015;10(4):487–510.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Montet X, Weissleder R, Josephson L. Imaging pancreatic cancer with a peptide− nanoparticle conjugate targeted to normal pancreas. Bioconjug Chem. 2006;17(4):905–11.

    CAS  PubMed  Google Scholar 

  38. Etrych T, Šubr V, Strohalm J, Šírová M, Říhová B, Ulbrich K. HPMA copolymer-doxorubicin conjugates: the effects of molecular weight and architecture on biodistribution and in vivo activity. J Control Release. 2012;164(3):346–54.

    CAS  PubMed  Google Scholar 

  39. Baratto L, Jadvar H, Iagaru A. Prostate cancer theranostics targeting gastrin-releasing peptide receptors. Mol Imaging Biol. 2018;20(4):501–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. de Aguiar FC, Fuscaldi LL, Townsend DM, Rubello D, de Barros ALB. Radiolabeled bombesin derivatives for preclinical oncological imaging. Biomed Pharmacother. 2017;87:58–72.

    Google Scholar 

  41. Li L, Yang Q, Zhou Z, Zhong J, Huang Y. Doxorubicin-loaded, charge reversible, folate modified HPMA copolymer conjugates for active cancer cell targeting. Biomaterials. 2014;35(19):5171–87.

    CAS  PubMed  Google Scholar 

  42. Liu J, Bauer H, Callahan J, Kopeckova P, Pan H, Kopecek J. Endocytic uptake of a large array of HPMA copolymers: elucidation into the dependence on the physicochemical characteristics. J Control Release. 2010;143(1):71–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. He C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials. 2010;31(13):3657–66.

    CAS  PubMed  Google Scholar 

  44. Gu F, Zhang L, Teply BA, Mann N, Wang A, Radovic-Moreno AF, et al. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc Natl Acad Sci. 2008;105(7):2586–91.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Varasteh Z, Mitran B, Rosenström U, Velikyan I, Rosestedt M, Lindeberg G, et al. The effect of macrocyclic chelators on the targeting properties of the 68Ga-labeled gastrin releasing peptide receptor antagonist PEG2-RM26. Nucl Med Biol. 2015;42(5):446–54.

    CAS  PubMed  Google Scholar 

  46. Volková M, Mandikova J, Lázníčková A, Lázníček M, Bárta P, Trejtnar F. The involvement of selected membrane transport mechanisms in the cellular uptake of 177Lu-labeled bombesin, somatostatin and gastrin analogues. Nucl Med Biol. 2015;42(1):1–7.

    PubMed  Google Scholar 

  47. Gibbens-Bandala B, Morales-Avila E, Ferro-Flores G, Santos-Cuevas C, Meléndez-Alafort L, Trujillo-Nolasco M, et al. 177Lu-Bombesin-PLGA (paclitaxel): a targeted controlled-release nanomedicine for bimodal therapy of breast cancer. Mater Sci Eng C. 2019;105:110043.

    CAS  Google Scholar 

  48. Patel S, Kim J, Herrera M, Mukherjee A, Kabanov AV, Sahay G. Brief update on endocytosis of nanomedicines. Adv Drug Deliv Rev. 2019;144:90–111.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Shan D, Li J, Cai P, Prasad P, Liu F, Rauth AM, et al. RGD-conjugated solid lipid nanoparticles inhibit adhesion and invasion of α v β 3 integrin-overexpressing breast cancer cells. Drug delivery and translational research. 2015;5(1):15–26.

    CAS  PubMed  Google Scholar 

  50. Alkilany AM, Zhu L, Weller H, Mews A, Parak WJ, Barz M, et al. Ligand density on nanoparticles: a parameter with critical impact on nanomedicine. Adv Drug Deliv Rev. 2019;143:22–36.

    CAS  PubMed  Google Scholar 

  51. Nanda PK, Pandey U, Bottenus BN, Rold TL, Sieckman GL, Szczodroski AF, et al. Bombesin analogues for gastrin-releasing peptide receptor imaging. Nucl Med Biol. 2012;39(4):461–71.

    CAS  PubMed  Google Scholar 

  52. Salouti M, Saghatchi F. BBN conjugated GNPs: a new targeting contrast agent for imaging of breast cancer in radiology. IET Nanobiotechnol. 2017;11(5):604–11.

    PubMed  PubMed Central  Google Scholar 

  53. Shi W, Ogbomo SM, Wagh NK, Zhou Z, Jia Y, Brusnahan SK, et al. The influence of linker length on the properties of cathepsin S cleavable 177Lu-labeled HPMA copolymers for pancreatic cancer imaging. Biomaterials. 2014;35(22):5760–70.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jered C. Garrison.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 3290 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alshehri, S., Fan, W., Zhang, W. et al. In Vitro Evaluation and Biodistribution Studies of HPMA Copolymers Targeting the Gastrin Releasing Peptide Receptor in Prostate Cancer. Pharm Res 37, 229 (2020). https://doi.org/10.1007/s11095-020-02952-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11095-020-02952-3

Key words

Navigation