111In- and IRDye800CW-Labeled PLA-PEG Nanoparticle for Imaging Prostate-Specific Membrane Antigen-Expressing Tissues

Abstract

Targeted delivery of drug-encapsulated nanoparticles is a promising new approach to safe and effective therapeutics for cancer. Here we investigate the pharmacokinetics and biodistribution of a prostate-specific membrane antigen (PSMA)-targeted nanoparticle based on a poly(lactic acid)-polyethylene glycol copolymer by utilizing single photon emission computed tomography (SPECT) and fluorescence imaging of a low-molecular-weight, PSMA-targeting moiety attached to the surface and oriented toward the outside environment. Tissue biodistribution of the radioactive, PSMA-targeted nanoparticles in mice containing PSMA(+) PC3 PIP and PSMA(-) PC3 flu (control) tumors demonstrated similar accumulation compared to the untargeted particles within all tissues except for the tumor and liver by 96 h postinjection. For PSMA(+) PC3 PIP tumor, the targeted nanoparticle demonstrated retention of 6.58% injected dose (ID)/g at 48 h and remained nearly at that level out to 96 h, whereas the untargeted nanoparticle showed a 48 h retention of 8.17% ID/g followed by a significant clearance to 2.37% ID/g at 96 h (P < 0.02). On the other hand, for control tumor, both targeted and untargeted particles displayed similar 48 h retentions and rates of clearance over 96 h. Ex vivo microscopic analysis with near-infrared versions of the nanoparticles indicated retention within PSMA(+) tumor epithelial cells as well as tumor-associated macrophages for targeted particles and primarily macrophage-associated uptake for the untargeted particles. Retention in control tumor was primarily associated with tumor vasculature and macrophages. The data demonstrate the utility of radioimaging to assess nanoparticle biodistribution and suggest that active targeting has a modest positive effect on tumor localization of PSMA-targeted PLA-PEG nanoparticles that have been derivatized for imaging.

Figure 1

Longitudinal in vivo SPECT-CT of ¹¹¹In-TNP and ¹¹¹In-UNP in xenograft-bearing mice. Male athymic nude mice bearing both a PC-3 PIP (PSMA(+), red circles) and PC-3 flu (PSMA(−), yellow circles) xenograft were injected with ∼31 MBq of labeled TNP or UNP and scanned side-by-side at the indicated times. Images are scaled to the same maximum. TNP uptake at 48 h shows tumor uptake in both the PSMA(+) PC-3 PIP and PSMA(−) PC-3 flu xenografts, while both tumors in the UNP mouse remain comparatively low in uptake. After 72 h, TNP is still present along the edges of both PSMA(+) PC-3 PIP and PSMA(−) PC-3 flu xenografts and mediastinal uptake is apparent, possibly within lymphatic tissue (arrowhead). UNP uptake at 72 h remains comparatively low. By 96 h, TNP uptake is still present at the edges of both tumor types, and some UNP uptake is apparent within the PSMA(+) PC-3 PIP tumor (upper panel). Distal spleen uptake is visible within the slices and is depicted by white arrows.

Figure 2

Ex vivo NIRF imaging of TNP and UNP at 72 h postinjection. Panel A depicts NIRF imaging of male athymic nude mice after sacrifice, each bearing a PSMA(+) PC-3 PIP xenograft (red dotted circles, arrow) and PSMA(−) PC-3 flu xenograft (yellow circles, arrow) 72 h after IV injection with fluorescent TNP or UNP. Both images are normalized to exposure time and show similarly luminescent tumors. Panel B shows high resolution NIRF images of 20 μm sections of the tumors in panel A. Tumor types in dotted boxes are labeled as in panel A. TNP distribution in PSMA(+) PC-3 PIP tumors is slightly more intense and is distributed throughout the tumor as compared with particle uptake in all of the other tumor sections. PSMA(−) PC-3 flu sections is also distributed throughout the tumor. All tumor section measurements in panel B were acquired simultaneously and are scaled to the same maximum.

Figure 3

Epifluorescence micrographs of PSMA(+) PC-3 PIP (top panel) and PSMA(−) PC-3 flu (bottom panel) frozen sections containing ex vivo TNPs after 72 h of uptake. (A) Tumor rim (dotted line) of indicated xenografts showing TNP (red) accumulation (arrows) within abundant CD68+ macrophages (green). (B) Tumor parenchyma of indicated sections showing TNP (red) colocalization with anti-PSMA antibody staining (green) in PSMA(+) PC-3 PIP (top row) and within a linear pattern in PSMA(−) PC-3 flu (bottom row). Tumor sections were probed with anti-PSMA antibody (green) to define PSMA(+) tissues and Hoechst 33342 (blue) to reveal nuclei. These micrographs demonstrate delivery of TNPs to PSMA(+) tumor parenchyma (B, top row), and to PSMA(+) cells in a linear orientation, possibly within a vessel (B, bottom row). Colocalized TNP with PSMA is yellow (B, PIP and flu). Tumor sections are 20 μm thick, scale bar = 50 μm.

Figure 4

Ex vivo epifluorescence micrographs of PSMA(+) PC-3 PIP and PSMA(−) PC-3 flu frozen sections containing TNPs. Tumor sections were subsequently probed with anti-CD68 antibody to delineate macrophages and Hoechst 33342 to reveal nuclei. TNPs (red) predominantly colocalize with CD68 (green) suggesting substantial phagocytosis of TNPs by macrophages. Tumor sections are 20 μm thick, scale bar = 50 μm.

Figure 5

Ex vivo epifluorescence micrograph of cultured PSMA(+) PC-3 PIP cells containing TNPs. This magnification shows the intracellular distribution of TNPs (red) with PSMA (green), tubulin (white), and nuclei (blue). White arrows indicate colocalization of TNPs with endocytosed PSMA at the centrosome. Scale bar = 10 μm.

Scheme 1

Two-Step Radiolabeling Method for the PSMA-Targeted Nanoparticles