Prostate-Specific Membrane Antigen Targeted StarPEG Nanocarrier for Imaging and Therapy of Prostate Cancer

Abstract

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7 nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192 h. Single doses of 18.5 MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138 days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation.

Keywords: enhanced permeability and retention; polymer nanocarriers; prostate cancer; prostate‐specific membrane antigen (PSMA); radioligand therapy; single photon excited computed tomography (SPECT) imaging.

Figure 1.

Graphical representation of PSMA-targeted μSPECT/CT imaging and therapy of prostate cancer using ¹⁷⁷Lu-labeled StarPEG nanocarrier.

Figure 2.

In vitro 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 theranostic agents. (a) IC₅₀ of nonradiolabeled theranostic StarPEGs, previously evaluated diagnostic nanocarrier PEG-(DFB)₁(ACUPA)3 and Azido-ACUPA determined by [⁶⁸Ga]PSMA-11-based in vitro competitive radioligand binding assay in PSMA+ PC3-Pip cells. (b) K_d measurement of ¹⁷⁷Lu-labeled StarPEGs in the PSMA+ PC3-Pip cell line by a saturation binding assay. (c,d) Blocking assay of [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ labeled starPEGs (5 nM) in PSMA+ PC3-Pip and PSMA− PC3-Flu cells using PSMA-2 (10 μM) as the blocking agent at 1 h, 4 h and 24h (%AD = percentage added dose). Blocking assays with the nontargeting [¹⁷⁷Lu]PEG-(DOTA)₁ have been presented in the Supporting Information (Figure S6). (e,f) Membrane-bound and internalization assay of the [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ at 1 h, 4 h and 24 h in PSMA+ PC3-Pip and PSMA− PC3-Flu cells (%AD = percentage added dose). Membrane-bound and internalization assay with the nontargeting [¹⁷⁷Lu]PEG-(DOTA)₁ have been presented in the Supporting Information (Figure S7). Overall, the ACUPA conjugated StarPEG nanocarrier demonstrated excellent PSMA targeted binding affinity in PSMA+ PC3-Pip cells. (n=3, mean ± SD)

Figure 3.

In vivo μSPECT/CT imaging demonstrates PSMA-targeted accumulation of StarPEG nanocarriers in PSMA+ PC3-Pip subcutaneous tumors. (a) Representation of experimental design for in vivo evaluation of the ¹⁷⁷Lu-labeled starPEGs in mice bearing dual xenografts of PSMA+ PC3-Pip (left flank) and PSMA− PC3-Flu (right flank). (b) Maximum intensity projection (MIP) μSPECT/CT and coronal μSPECT/CT images obtained on day 24 h, 72 h, 144 h and 192 h post-injection of ¹⁷⁷Lu-labeled starPEGs reveal high tumor accumulation of targeted nanocarriers in PSMA+ PC3-Pip. (c-d) Quantification of in vivo tumors’ accumulation by drawing ROIs on the respective tumors at 24 h, 72 h, 144 h and 192 h post-injection of ¹⁷⁷Lu-labeled starPEGs. ROIs on the heart at respective imaging time points are presented in the Supporting Information (Figure S9). Overall, the in vivo μSPECT/CT imaging demonstrated excellent PSMA targeted tumor accumulation of the ACUPA conjugated StarPEG nanocarrier in PSMA+ PC3-Pip xenografts. (n=2, mean ± SD)

Figure 4.

Ex vivo organ biodistribution of ¹⁷⁷Lu-labeled starPEG nanocarriers. Organ biodistribution of (a) [¹⁷⁷Lu]PEG-(DOTA)₁ and (b) [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ at 72 h and 192 h post-injection of the nanocarriers (n = 3, mean ± SD). The ratio of (c) PC3-Pip to PC3-Flu, (d) PC3-Pip to the muscle and (e) PC3-Pip to the blood of [¹⁷⁷Lu]starPEGs at 72 h and 192 h post-injection of the nanocarriers (n=3, mean ± SD). The supporting information presents ex vivo organ biodistribution of [¹⁷⁷Lu]starPEGs in % ID/Organ (Figure S10). Overall, the ex vivo organ biodistribution demonstrated excellent PSMA targeted tumor accumulation of the ACUPA conjugated StarPEG nanocarrier in PSMA+ PC3-Pip xenografts.

Figure 5.

Autoradiography images of 20 μm tumor slices of PSMA+ PC3-Pip and PSMA− PC3-Flu tumors collected after 72 h and 192 h post-injection of [¹⁷⁷Lu]StarPEG nanocarriers. Overall, the targeted nanocarrier demonstrated superior tissue penetration in PSMA+ PC3-Pip xenografts.

Figure 6.

In vivo treatment study of [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ in nude mice models bearing PSMA+ PC3-Pip tumors. (a) Representation of experimental design for in vivo treatment efficacy and toxicity of ¹⁷⁷Lu-labeled starPEGs in mice models bearing PSMA+ PC3-Pip subcutaneous tumors. Tumor volume (b), body weight (c) and survival (d) of mice models bearing PC3-Pip tumors and treated with different doses (0, 4.6, 9.2 and 18.5, MBq) of either [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ or 9.2 MBq of [¹⁷⁷Lu]PSMA-617. Overall, a single 18.5 MBq dose of [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ demonstrated highly efficient suppression of the tumor volume without any regrowth of tumors with 80% survival at 138 days post drug injection. (n=10, mean ± SD)

Figure 7.

[¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ was effective in suppressing the growth of metastatic prostate cancer in the PC3-PiP intracardiac metastatic model. (a) Representation of experimental design for in vivo treatment study of the ¹⁷⁷Lu-labeled starPEGs in mice bearing PSMA+ PC3-Pip metastatic tumors. (b) Multiple time points [⁶⁸Ga]PSMA-11 μPET/CT imaging of the nude mice bearing PC3-Pip metastatic tumors before and after treatment of 9.2 MBq [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ (n=4, mean ± SD). (c) Body weight measurement of the treated and control mice up to 51 days post-injection of 9.2 MBq [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃. (d) Overall survival of mice models bearing PC3-Pip metastatic tumors and treated with/without 9.2 MBq) of [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃. (e,f) [⁶⁸Ga]PSMA-11 organ biodistribution of the nude mice bearing PC3-Pip metastatic tumors on 35 and 50 days post-treatment of 9.2 MBq [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ (n=10, mean ± SD). Overall, a single 9.2 MBq dose of [¹⁷⁷Lu]PEG-(DOTA)₁(ACUPA)₃ demonstrated highly efficient suppression of PC3-Pip metastatic tumors with 100% survival at 51 days post drug injection.

Scheme 1.

Synthetic routes to the StarPEG theranostic conjugates (a) PEG-(DOTA)₁ and (b) PEG-(DOTA)₁(ACUPA)₃ evaluated in PSMA+ subcutaneous as well metastatic tumor models. Detailed synthetic protocols are provided in the method section.