Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer

Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. <i>In vitro</i> cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, <i>in vivo</i> studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient

Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer

Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. <i>In vitro</i> cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, <i>in vivo</i> studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient

Biocompatible Nanoparticles of KGd(H₂O)₂[Fe(CN)₆]·H₂O with Extremely High <i>T</i>₁‑Weighted Relaxivity Owing to Two Water Molecules Directly Bound to the Gd(III) Center

A simple one-step method for preparing biocompatible nanoparticles of gadolinium ferrocyanide coordination polymer KGd­(H₂O)₂[Fe­(CN)₆]·H₂O is reported. The crystal structure of this coordination polymer is determined by X-ray powder diffraction using the bulk materials. The stability, cytotoxicity, cellular uptake, and MR phantom and cellular imaging studies suggest that this coordination-polymer structural platform offers a unique opportunity for developing the next generation of <i>T</i>₁-weighted contrast agents with high relaxivity as cellular MR probes for biological receptors or markers. Such high-relaxivity MR probes may hold potential in the study of molecular events and may be used for in vivo MR imaging in biomedical research and clinical applications

Topical application of GB119 to heterotopic brain tumors results in rapid and specific <i>in vivo</i> activation of the probe.

<p>(a) Mice bearing Gli36Δ5EGFR flank tumors (n = 12) were treated with GB119 and imaged over time. The mean +/− standard deviation is plotted vs. time. Wilcoxon signed ranked test showed that the signal increased significantly above the time 0 signal at each of the times 1, 5, 15, 30, and 35 (p = 0.0005 at each time point). Inset is a representative mouse treated topically with GB119 and imaged across time. (b) Percent inhibition of GB119 activity by GB111-NH₂ was determined for each mouse (n = 4) and is presented as mean +/− standard deviation at 35 minutes. There was significant reduction in the signals when incubated with both GB119 and inhibitor at all time points except time 0 (two-sample t-test p<0.05). Inset represents GB119 (P) or a mixture of GB119 with 100-fold excess of GB111-NH₂ (P+I) at 35 minutes after treatment. Images are unmixed false colored maps of pixel intensity representing activated GB119.</p

Comparison of tumor cell labeling between topical application of GB119 and systemic administration of GB123.

<p>(<b>a–d</b>) Representative unmixed false colored maps of pixel intensity of coronal 2 mm sections (<b>a</b>,<b>b</b>) and dorsal surface tumors (<b>c</b>,<b>d</b>) comparing the pattern of activation of topical GB119 (left side) and systemic GB123 (right side). (<b>e,f</b>) H&E coronal sections demonstrating tumor mass. (<b>g–j</b>) Merged fluorescent microscopy of adjacent sections showing labeling in the center (<b>g,h</b>) and edge (<b>I,j</b>) of the tumor. Sections were immunostained with anti-human vimentin antibody to reveal the brain tumor xenograft (false colored green), covalently bound Cy5 (false colored red). Yellow represents co-localized staining. (<b>k & i</b>) H&E sections demonstrating tumor cells that migrated away from the main tumor mass (outlined) via the ventricular system (<b>k</b>) or along the cortical meninges (<b>i</b>). Boxed regions indicate invading cells (<b>m</b> & <b>n</b>). Merged images of boxed regions from panels k & i showing labeling of invading tumor cells. Vimentin positive tumor cells (false colored green) and Cy5 cells (false colored red) reveals that invading cells were identified by topical GB119 (yellow cells) (<b>m</b>) but not by systemic GB123 (<b>n</b>). DAPI staining for cellular orientation was also included in panels <b>m,n</b>. Scale bar, 1 mm (<b>e,f</b>), 500 µm (<b>k,l</b>), and 50 µm (<b>g–j, m, n</b>).</p

Topical application of GB119 to a brain tumor on the dorsal surface labels the tumor's edge more robustly than the tumor's center.

<p>(<b>a</b>) Monochromatic image of a whole brain with a tumor growing near the dorsal surface (arrow) showing the treated areas (outlines). (<b>b</b>) Unmixed false colored map of pixel intensity representing activated GB119 at 35 minutes post application. (<b>c</b>) Horizontal H&E stained section showing higher magnification of square area in panel b demonstrating tumor mass and normal surrounding brain. (<b>d</b>) Adjacent sections revealing human vimentin positive cells labeling tumor edge and center (false colored green), (<b>e</b>) activated GB119 (Cy5, false colored red) revealing infiltrating cells, the tumor margin and to a lesser degree the interior of the tumor and (<b>f</b>) merged imaged demonstrating co-registration (yellow) of GB119-labeled cells and vimentin positive cells within the tumor mass (arrows) and outside of the main tumor mass, arrow head. (<b>g</b>) GB119 labeled cells at the tumor's edge more robustly than at the tumor's center, although not significantly, p = 0.124. (<b>h</b> and <b>i</b>) reveal that GB119 is associated with CD11b-positive cells, but only when they are tumor associated, (<b>h</b>) is a typical image observed from within the tumor interior and (<b>i</b>) shows the images typical of the tumor – brain interface. Note that only CD11b positive cells associated with normal brain do not activate the probe. Section thickness 25 µm (<b>d–f</b>) and 10 µm (<b>h</b>,<b>i</b>). Scale bar, 5 µm (<b>a</b>,<b>b</b>), and 100 µm (<b>c–f, h,i</b>).</p

<i>Ex vivo</i> activation of GB119 in orthotopic brain tumors.

<p>(<b>a</b>) Representative 2-mm sections from normal and tumor-bearing brains (see arrow for main tumor mass). Monochromatic images are shown on left and unmixed false colored maps of pixel intensity representing activated GB119 at 35 minutes are shown on the right. (<b>b</b>) The averages of total signal/area/volume from the slices for each animal is plotted over time for normal (n = 3) and tumor (n = 6) groups. The groups did not differ at time 0 (p = 0.65). Using Bonferroni correction with overall significance level 0.05, the groups differed significantly at times 5, 15, 30 and 35 minutes. (<b>c</b>) Inhibition of GB119 activation in an orthotopic brain tumor model using opposing brain slices (opened like a book) containing tumor (arrow, n = 11). (<b>d</b>) Percent inhibition of GB119 activity was determined for each mouse and is presented as mean +/− standard deviation at 35 minutes. Using Bonferroni correction for multiple testing at times the groups did not differ at time 0 (p = 0.86) but differed significantly at times 1, 15, 30 and 35 minutes (p<0.0003). Scale bar, 5 mm.</p

GB119 specifically labels cells <i>in vitro</i> and tumor tissue <i>ex vivo</i>.

<p>(<b>a</b>) Gli36Δ5EGFR cells were incubated with JPM-OEt, a general papain-family inhibitor (+, lanes 2,4 and 6) or 0.1% DMSO vehicle control (−, lanes 1,3 and 5) before the addition of the indicated probe GB119, GB123, or GB125 (the control probe, GB125, contains the Cy5 fluorescence label with a non-reactive amide in place of the acyloxymethylketone (AOMK) “warhead”(22)). (<b>b</b>) <i>In vitro</i> cell labeling with NIRF-APBs. C2C12/ras cells were incubated with JPM-OEt, a general papain-family inhibitor (+) or 0.1% DMSO as a vehicle control (−) before the addition of the indicated probe. Samples were analyzed by SDS-PAGE and Cy5 fluorescence was measured by scanning the gels with a Typhoon scanner. Specific bands are indicated as follows: cathepsin L single-chain form (Cat L sc); cathepsin L heavy-chain (Cat L hc), and cathepsin B (Cat B). (<b>c</b>) Gli36D5EGFR flank tumor tissue was immersed in GB119 for the indicated time or DMSO vehicle control (D) for 30 minutes. Specific cathepsin bands are indicated as follows: cathepsin L single-chain form (Cat L sc,); cathepsin L heavy-chain (Cat L hc); and cathepsin B (Cat B).</p

Topical probe administration reveals remaining tumor tissue in incomplete tumor resection.

<p>(<b>a</b>) Gross resection of tumor growing on dorsal surface of brain was performed and GB119 was added directly to resulting cavity and imaged over time. H&E stained section demonstrating the resulting tumor cavity with remaining tumor cells (<b>right</b>). (<b>b</b>) Adjacent sections demonstrating GB119-labeled tissue (false-colored red, <b>left</b>) is associated with vimentin-positive tumor xenograft (false-colored green, <b>right</b>) but not with normal brain tissue. (<b>c</b>) Higher magnification of cavity edge from (b). Cell nuclei are labeled with DAPI (false-colored blue) in (<b>b and c</b>). Section thickness 10 µm. Scale bar, 5 mm (<b>a, left</b>) 1 mm (<b>a, right</b>), 500 µm (<b>b</b>), 100 µm (<b>c</b>).</p