PSMA-Targeted Theranostic Nanocarrier for Prostate Cancer

Abstract

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin β1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin β1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size.

Keywords: PSMA; peptide; polymeric nanoparticles; prostate cancer..

Figure 1

(A) Chemical structure of a cleavable Folate-Doxorubicin probe. (B) Mechanism of fluorescent activation upon PSMA-mediated cell internalization. The fluorescence of Doxorubicin (Doxo) is quenched by the close proximity of the folic acid (folate) ligand that act as both quencher and targeting ligand. Upon PSMA-mediated internalization of the probe, the disulfide bond that links Doxo and Folate is cleaved by reduced glutathione in the cytoplasm, activating the fluorescence of doxorubicin. Fluorescence microscopy images of (C) LNCaP, (D) PC3 and (E) PSMA(+) PC3 prostate cancer cell lines upon a 12 hour incubation with the activated probe Folate-s-s-Doxo (1.2 uM at 37C). Internalization and fluorescence activation of the probe is observed in the PSMA expressing PCa cells (LNCaP and PSMA(+) PC3), but not in wild type PC3, that does not express PSMA.

Figure 2

(A) Diagram depicting the conjugation of folate to HBPE(CT20p) NPs using ethylene diamine as a non-cleavable linker. For clarity, only one ligand is depicted attached to the nanoparticle, but multiple folate ligands are attached, yielding a multivalent folate conjugated nanoparticle (B). Scanning transmission electron micrograph (STEM) image of the Folate-HBPE(CT20p) NPs. (C) Release profile of the CT20p peptide from the HBPE nanoparticle at pH 5.0 and 7.4. The stability of CT20p encapsulation at physiological pH is shown by the lack of peptide release at pH 7.4. However, at pH 5, the typical lysosomal pH, a time-dependent release of the peptide is observed.

Figure 3

Assessment of targeting and PSMA-mediated cell internalization of Folate-HBPE(DiI)-NPs by flow cytometry analysis. (A) Control, non -treated LNCaP and PC-3 (inset) cell lines, (B) LNCaP, (C) LNCaP pre-treated with PMPA. (D) PC-3, (E) PSMA(+) PC-3, (F) PSMA(+) PC-3 pre-treated with PMPA. Inset in B-E: Fluorescence microscopy images of the corresponding cells. Internalization of Folate-HBPE(DiI)-NPs and fluorescence labeling is observed in the PSMA expressing PCa cells (LNCaP and PSMA(+) PC3), but not in wild type PC3, that does not express PSMA.

Figure 4

Cell associated fluorescence of various tumor cell lines upon incubation with an anti-PSMA antibody (Fluorescein-J591) (A) or Folate-HBPE(DiI) (B). Note the strong cell associated fluorescence in the cell lines treated with either the anti-PSMA antibody or the folate nanoparticle, indicating that the folate nanoparticle can discriminate between cells that express PSMA from those that do not.

Figure 5

(A) Levels of CCTβ protein in LNCaP, PC3 and PSMA(+) PC3 cell lines by Western Blot analysis. Green bands indicate the presence of CCT-β in the samples. Red bands indicate total protein stain, loading control. (B) Genomic analysis of CCTβ mRNA levels in LNCaP, PC3 cell lines and (C) patients with neuroendocrine prostate cancer. Genomic analysis was performed using a Prostate Cancer TCGA database.

Figure 6

Dose- (a - c) and time- (d - f) dependent cytotoxicity assay (MTT) of prostate cancer cells treated with Folate HBPE(CT20p)-NPs and Folate-HBPE. LNCaP (A,D), PSMA(+) PC3 (B,E) and PC3 (C,F). A reduction in cell viability is observed in the cells that express PSMA when treated with Folate HBPE(CT20p)-NPs.

Figure 7

Cell viability assay via the assessment of cell membrane permeability (SYTOX AADvanced staining) and cell membrane asymmetry (F2N12S dyes staining). (A) Diagram showing the quadrants representing the population of viable (live), apoptotic and necrotic cells based on the relative uptake of Sytox and F2N12S. As cells undergo apoptosis and necrosis, they will become more permeable and lose membrane symmetry, resulting in shifting of the cell population to the left and up. (B) Untreated LNCaP cells, control. (C) LNCaP cells treated with Folate-HBPE(CT20p). (D) LNCaP cells pre-incubated with PMPA and then treated with Folate-HBPE(CT20p). Incubation time is 48h in all experiments.

Figure 8

Cell viability (Sytox AADvanced/F2N12S) assay of: (A) untreated mouse macrophages (RAW), control, (B) RAW cells treated with Folate-HBPE(CT20p), (C) RAW cells treated with doxorubicin (D) RAW cells treated with Folate s-s-Doxo. Incubation time is 48h in all experiments.

Figure 9

Levels of integrin β1 (CD29) in: (A) PC3, (B) PSMA(+) PC3 and (C) LnCaP cells treated with Folate-HBPE(CT20p). Corresponding cells were incubated with an anti-integrin β1 antibody conjugated with a phycoerythrin fluorescent dye. Binding to the corresponding cells was determined by flow cytometry to assess the levels of cell surface integrin β1 in the corresponding cells. UNT CTRL (red lines) represent the level of fluorescence of the corresponding to the untreated cells and correspond to the basal levels of integrin β1 on the cells surface. Isotype CTRL (black lines) represent the level of fluorescence corresponding to the non-specific binding of an antibody that does not bind to integrin β1. Upon incubation of the corresponding cells with Folate-HBPE(CT20p), a decrease in the levels of cell surface integrin β1 was determined by a movement to the left of the fluorescence peak, toward the Isotype CTRL peak. Corresponding percentages of cell adhesion in (D) PC3, (E) PSMA(+) PC3 and (F) LnCaP cells treated with Folate-HBPE(CT20p), determined using a crystal violet assay.

Figure 10

Targeting and treatment of prostate cancer tumors expressing PSMA with Folate-HBPE(CT20p). Mice (n=3) were injected subcutaneously (SC) with PSMA (+) (right flank) or PSMA(-) (left flank) prostate cancer cells. Upon tumor detection (~2 weeks), mice were injected intravenously (IV) with Folate-HBPE-NPs (2mg/kg/dose) containing (A) DiR, a near IR dye or (B) CT20p. Mice were imaged after 24 hours using a fluorescence in vivo imaging system (IVIS) to assess the specific targeting of the folate conjugated nanoparticles to PSMA expressing tumors (A). Ultrasound imaging was performed to assess tumor regression of mice treated with the Folate-HBPE(CT20p). Growth curves of (C) PSMA(+) PC3 or (D) wild type PC3 tumors with or without treatment with Folate-HBPE(CT20p).

Figures 11

(A) Growth curves of LNCaP tumors treated with Folate-HBPE(CT20p) or PBS. (B) Average weight of the PBS-treated and Folate-HBPE(CT20p) treated mice (n = 4) at the end of the experiment. (C) Tumor size comparison after necropsy. (D) Average weight of the PBS-treated and Folate-HBPE(CT20p) tumors at the end of the experiment.