Prostate-Specific Membrane Antigen Targeted Deep Tumor Penetration of Polymer Nanocarriers

Abstract

Tumoral uptake of large-size nanoparticles is mediated by the enhanced permeability and retention (EPR) effect, with variable accumulation and heterogenous tumor tissue penetration depending on the tumor phenotype. The performance of nanocarriers via specific targeting has the potential to improve imaging contrast and therapeutic efficacy in vivo with increased deep tissue penetration. To address this hypothesis, we designed and synthesized prostate cancer-targeting starPEG nanocarriers (40 kDa, 15 nm), [⁸⁹Zr]PEG-(DFB)₃(ACUPA)₁ and [⁸⁹Zr]PEG-(DFB)₁(ACUPA)₃, with one or three prostate-specific membrane antigen (PSMA)-targeting ACUPA ligands. The in vitro PSMA binding affinity and in vivo pharmacokinetics of the targeted nanocarriers were compared with a nontargeted starPEG, [⁸⁹Zr]PEG-(DFB)₄, in PSMA+ PC3-Pip and PSMA- PC3-Flu cells, and xenografts. Increasing the number of ACUPA ligands improved the in vitro binding affinity of PEG-derived polymers to PC3-Pip cells. While both PSMA-targeted nanocarriers significantly improved tissue penetration in PC3-Pip tumors, the multivalent [⁸⁹Zr]PEG-(DFB)₁(ACUPA)₃ showed a remarkably higher PC3-Pip/blood ratio and background clearance. In contrast, the nontargeted [⁸⁹Zr]PEG-(DFB)₄ showed low EPR-mediated accumulation with poor tumor tissue penetration. Overall, ACUPA conjugated targeted starPEGs significantly improve tumor retention with deep tumor tissue penetration in low EPR PC3-Pip xenografts. These data suggest that PSMA targeting with multivalent ACUPA ligands may be a generally applicable strategy to increase nanocarrier delivery to prostate cancer. These targeted multivalent nanocarriers with high tumor binding and low healthy tissue retention could be employed in imaging and therapeutic applications.

Keywords: deep tumor penetration; enhanced permeability and retention (EPR) effect; polymer nanocarriers; positron emission tomography (PET) imaging; prostate-specific membrane antigen (PSMA).

Figure 1

Graphical representation of PSMA-targeted polymer nanocarriers tagged with ⁸⁹Zr radioisotope, demonstrating significantly improved deep tumor penetration in PSMA+ prostate cancer xenograft.

Figure 2

Representative chemical structures of ⁸⁹Zn-labeled starPEG nanocarriers to evaluate the PSMA-targeted PET imaging of prostate cancer. [⁸⁹Zr]PEG-(DFB)₄, previously reported nontargeted nanocarrier, was used in this study as a baseline control.

Scheme 1. Synthesis of the Azido Derivatives (a) Azido-ACUPA, (b) Azido-DFB, and Their Conjugation to starPEG Nanocarriers to Produce (c) PEG-(DFB)₃(ACUPA)₁ and (d) PEG-(DFB)₁(ACUPA)₃

Detailed synthetic routes with chemical structures have been provided in the Supporting Information. Individual synthetic steps with intermediate structures for the targeted nanocarriers have been presented in Supporting Information Schemes S1 and S2.

Figure 3

Cell-binding assays with starPEG nanocarriers in PSMA+ PC3-Pip and PSMA– PC3-Flu cell lines demonstrate efficient cell binding and uptake of PSMA-targeted nanocarriers. (a) IC₅₀ of nonradiolabeled starPEGs, Azido-ACUPA, and 2-PMPA determined by ⁶⁸Ga-PSMA-11-based in vitro competitive radioligand binding assay in PSMA+ PC3-Pip cells (^NSP > 0.05, Student’s t-test). (b) K_d measurement of ⁸⁹Zr-labeled starPEGs in the PSMA+ PC3-Pip cell line by a saturation binding assay. (c) Blocking assay of ⁸⁹Zr-labeled starPEGs (100 nM) in PSMA+ PC3-Pip cells using PSMA-2 as the blocking agent at 1 h (%AD = percentage added dose). Detailed blocking assays at different concentrations and incubation times are presented in the Supporting Information (Figures S17–S19). (d) Membrane-bound and internalization assay of the ⁸⁹Zr-labeled starPEGs at 1 h in PSMA+ PC3-Pip cells (%AD = percentage added dose). The membrane-bound activity was collected by 5 min of acid wash with a cold mixture of 50 mM glycine and 150 mM NaCl. Membrane-bound and internalization assays at later time points are presented in the Supporting Information (Figure S20).

Figure 4

In vivo μPET/CT imaging. (a) Representation of experimental design for in vivo evaluation of the ⁸⁹Zr-labeled starPEGs in mice bearing dual xenografts of PSMA+ PC3-Pip (left flank) and PSMA– PC3-Flu (right flank). (b) Maximum intensity projection (MIP) μPET/CT, axial μPET/CT, and axial CT images obtained at 216 h following administration of ⁸⁹Zr-labeled starPEGs reveal high tumor accumulation with low background tissue retention of [⁸⁹Zr]PEG-(DFB)₁(ACUPA)₃ over time. Respective coronal CT and coronal μPET/CT images are presented in the Supporting Information (Figure S21). ROIs on the heart and tumors are presented in the Supporting Information (Figure S22).

Figure 5

Ex vivo organ biodistribution of ⁸⁹Zr-labeled starPEGs. (a) Tumor biodistribution of [⁸⁹Zr]starPEGs at 216 h postinjection of the nanocarriers (n = 4, mean ± SD, **P < 0.01, Student’s t-test). (b) Ratio of PC3-Pip to PC3-Flu tumor biodistribution of [⁸⁹Zr]starPEGs at 216 h postinjection of the nanocarriers (n = 4, mean ± SD, *P < 0.05, Student’s t-test). (c) Tumor to the muscle and (d) tumor to blood of [⁸⁹Zr]starPEGs at 216 h postinjection of the nanocarriers. Ex vivo biodistribution of ⁸⁹Zr-labeled starPEGs on selected major organs is presented in the supporting information (Figure S23).

Figure 6

Autoradiography images of 20 μm tumor slices of PSMA+ PC3-Pip and PSMA– PC3-Flu tumors collected after 216 h postinjection of ⁸⁹Zr-labeled nanocarriers.