Photodynamic Therapy Combined with Gemcitabine for Prostate Cancer Using Nanoprobe for in-vivo CEST Imaging.

Background: Current clinical treatment is confronted with challenges such as low drug delivery efficiency, lagging efficacy evaluation, and a high tumor recurrence rate. However, chemical exchange saturation transfer (CEST) imaging, with its molecular specificity and non-invasive monitoring advantages, provides a new path for real-time tracking of the metabolokinetics of nanoprobes. Objectives: Drug-loaded nanoprobes that self-assembled and had CEST activation imaging capabilities were created, and there in vivo and in vitro CEST activation imaging efficacy and the value of photodynamic sensitization for pyroptosis treatment of prostate cancer cells were evaluated. Methods: A self-assembly strategy was adopted to construct a nanoprobe (GC) coloaded with gemcitabine (Gem) and the photosensitizer chlorin e6 (Ce6), and a scanning electron microscope was used. Scanning electron microscope (SEM), dynamic light scattering (DLS), etc., were used to characterize the in vitro CEST activation imaging efficacy of the nanoprobe, and the pH, concentration-, and time-dependent drug release were observed. GC was combined with 2',7'-dichlorodihydrogen fluorescein diacetate ester (2',7'-dichlorodihydrofluorescein diacetate) after laser treatment of prostate cancer cells in mice. The generation of reactive oxygen species (ROS) was detected with a DCFH-DA probe. By using ELISA, the amounts of inflammatory factors such as interleukin-1β (IL-1β) and IL-18 were determined, and calreticulin was detected via immunofluorescence. CRT and high mobility group box 1 protein (HMGB1) were used to evaluate pyroptosis-mediated immunogenic cell death (ICD) effects. A mouse prostate cancer tumor model was constructed to observe the CEST-specific activation effect of the GC nanoprobes in vivo, and its tumor volume was measured, together with the detection of inflammatory substances and ICD markers, in order to assess its pyroptosis-based anticancer activity. Results: Scanning electron microscope and DLS analyses revealed that the GC nanoprobes had a uniform spherical-like structure. The release of the drug Gem by this drug-loaded nanoprobe under acidic conditions at pH 5.0 was as high as 80%, which was significantly greater than that at pH 7.4 (P = 0.003). DCFH-DA fluorescence staining indicated that photosensitizing pyroptosis mediated by nanoprobes could generate a large amount of ROS. The detection of pyroptosis-related factors revealed that the levels of IL-1β and IL-18 significantly increased (all P < 0.05), whereas the fluorescence of the ICD marker CRT increased and that of HMGB1 decreased. The results of in vivo experiments indicated that the CEST signal at the tumor site was significantly enhanced and reached its peak 4 hours after tail vein injection of the GC nanoprobes. In addition, the results of antitumor treatment in vivo revealed that, compared with those in the control group treated with PBS, the levels of the inflammatory factors IL-1β and IL-18 in the experimental group treated with GC combined with laser irradiation increased, the ICD markers HMGB1 and CRT significantly changed, and the tumors were significantly inhibited (all P < 0.05). The CEST imaging of GC nanoprobes demonstrates excellent diagnostic accuracy in the detection of prostate cancer. The sensitivity reaches 92.3%, the specificity is 88.7%, and the area under the curve (AUC) is 0.95, which is significantly superior to traditional imaging methods. The specific enhancement of CEST signals at the tumor site reaches its peak 4 hours after injection, which is highly consistent with the histopathological results, confirming its clinical value in the early diagnosis and precise treatment guidance of prostate cancer. Conclusions: The GC nanoprobes successfully prepared in this study can specifically activate CEST imaging, guide the photodynamic sensitization of prostate cancer tumor cells to pyroptosis, and promote the precise ablation of prostate cancer.