Nanoparticles Yield Increased Drug Uptake and Therapeutic Efficacy upon Sequential Near-Infrared Irradiation

Abstract

Nanoparticles offer great opportunities for precision medicine. However, the use of nanoparticles as smart photosensitizers that target tumor biomarkers and are responsive to the tumor microenvironment has yet to be explored. Herein, prostate cancer (PCa)-selective theranostic gold nanoparticles (AuNPs) for precise cancer imaging and therapy are developed. Silicon phthalocyanine, Pc158, was synthesized and deactivated by conjugating it to AuNPs via a biocleavable linker. In vitro and in vivo, the targeted AuNPs show excellent selectivity for PSMA-positive tumor cells. Triggered release of the therapeutic, Pc158, followed by sequential photodynamic therapy (PDT) results in significant inhibition of tumor growth. Further, we demonstrate that multiple sequential PDT greatly enhances nanoparticle uptake and therapeutic efficacy. PSMA is highly expressed in the neovasculature of most other solid tumors in humans, as well as PCa, making this approach of great practical interest for precision PDT in a wide range of cancers.

Keywords: PSMA; nanoparticles; photodynamic therapy; prostate cancer; sequential irradiation.

Figure 1.

Activatable AuNPs-Pc158 conjugates for selective photodynamic therapy. (a) Schematic representation of PSMA-targeted AuNPs-Pc158 conjugates with AuNP core as quencher. Activation occurs with cathepsin, which cleaves the GLFGC linker, releasing Pc158 for PDT. (b) Table shows the size of nanoparticles and Pc158 loading.

Figure 2.

In vitro cell targeting, intracellular Pc158 release and phototoxicity. (a) Selective uptake and intracellular Pc158 release in PC3pip cells after 1, 6, and 24 h incubation times. Lysosomes (magenta), mitochondria (green), and nuclei (blue) were stained with LysoOrange, MitoGreen, and DAPI, respectively, and Pc158 fluorescence (red) was imaged directly. Overlay of Pc158 and lysosomes (pink) at 6 h indicates cleavage of Pc158 into lysosomes and at 24 h, free Pc158 was released from lysosomes to mitochondria (yellow) (for more details see Figure S17). Silver staining assay revealed that there was significant AuNPs-Pc158 uptake by PC3pip cells as early as 1 h. (b) Schematic representation of the uptake sequence of AuNP-Pc158 conjugates and intracellular Pc158 release from lysosomes to mitochondria. (c) Confocal images showing intracellular ROS generation after PDT at 6 and 24 h after incubation with AuNPs-Pc158 conjugates. Intracellular ROS (green) was stained with DCFH-DA (transformed into fluorescent DCF⁻ by ROS) and nuclei were stained with DAPI. (d,e) Phototoxicity shows selective killing of PC3pip cells (PSMA+) over PC3flu cells (PSMA−) at (d) 6 h and (e) 24 h. Data are presented as mean ± SD (n = 5), and differences between groups are compared with two-tailed t-tests, **p ≤ 0.01.

Figure 3.

In vivo tumor targeting of AuNPs-Pc158 conjugates, intratumoral Pc158 release and PDT of 100 mm³ sized tumors. (a) Black and white image showing mouse with both PC3pip (right) and PC3flu (left) tumors, left; Maestro fluorescence image (at 48 h), middle; and 3D CT image (at 8 h) showing good selectivity of AuNPs-Pc158 conjugates, right. (b) Kinetics of Pc158 fluorescence intensity (top panel) and quantitative CT signals (HU) (bottom panel) of PC3pip and PC3flu tumors shows that Pc158 fluorescence peaked at 48 h and Au accumulation peaked at 8 h (n = 3). (c) Maestro fluorescence images show intratumoral ROS generation. (d) Quantitative Pc158 and ROS fluorescence intensity before and after PDT (n = 3). (e) Representative tumor H&E and immunochemistry images showing the damage by PDT. AuNPs in tumor tissue were stained with silver (red arrows). (f) Photodynamic therapy for small sized tumor (~100 mm³) (n = 5). Inset images show the tumor size before PDT (left) and 30 days after PDT (right). Data are presented as mean ± SD, and tumor growth inhibition are compared with a two-tailed t-test, **p ≤ 0.01.

Figure 4.

Sequential irradiation induces intracellular Pc158 release and enhances efficacy. (a) Light-induced release of protease liberated Pc158 escaping from lysosomes to mitochondria. Confocal image (left) shows mitochondria containing Pc158, yellow, and free Pc158, red. NIR was irradiated (670 nm, 1 J) at 6 h, and lysosome was disrupted showing no staining compared to Figure 2a at 6 h with no PDT. Right image diagrams lysosomal release and accumulation into mitochondria after PDT. (b) Phototoxicity of PC3pip cells incubated with AuNPs-Pc158 showing an enhanced PDT efficacy by carrying out a second irradiation 30 min after the first light exposure. (c) Scheme shows the timeline of the repeated PDT treatments in mice with 500 mm³ tumors. (d) Maestro fluorescence images of mice injected with AuNP-Pc158 and PSMA-Pc413 conjugates before and after each PDT treatment (150 J/cm²). (e) Normalized Pc158 fluorescence intensity for mice injected with AuNPs-Pc158 and PSMA-Pc413 before and after each NIR irradiation. (f) In vivo pharmacokinetics of AuNPs in blood over 7 days. (g) AuNP uptake in tumors before and after each PDT showing a PDT enhanced AuNP accumulation in irradiated tumors compared to tumors without PDT. For all studies, data are presented as mean ± SD (n = 3). Differences of Au content in tumors was compared with a two-tailed t-test, *p ≤ 0.05, **p ≤ 0.01.

Figure 5.

Multiple photodynamic therapy enhances the eradication of large tumors. (a) Effects of PDT on growth kinetics of larger tumors (around 500 mm³). (b) Tumor weight for each of the groups at the end of growth monitoring. Data are presented as mean ± SD (n = 5), and tumor growth inhibition is compared with two-tailed t-test, **p ≤ 0.01. (c) Tumor H&E and immunochemistry images showing the damage by PDT. Upper panel shows the H&E staining at low magnification; middle panel shows the silver stained tissue from the highlighted areas (black boxes). The AuNPs were stained black (red arrows); bottom panel shows the immunohistochemistry staining (TUNEL) of tumor tissues taken from the blue box regions demonstrating increase apoptosis with increased iterations of PDT.