Novel theranostic nanoporphyrins for photodynamic diagnosis and trimodal therapy for bladder cancer

Abstract

The overall prognosis of bladder cancer has not been improved over the last 30 years and therefore, there is a great medical need to develop novel diagnosis and therapy approaches for bladder cancer. We developed a multifunctional nanoporphyrin platform that was coated with a bladder cancer-specific ligand named PLZ4. PLZ4-nanoporphyrin (PNP) integrates photodynamic diagnosis, image-guided photodynamic therapy, photothermal therapy and targeted chemotherapy in a single procedure. PNPs are spherical, relatively small (around 23 nm), and have the ability to preferably emit fluorescence/heat/reactive oxygen species upon illumination with near infrared light. Doxorubicin (DOX) loaded PNPs possess slower drug release and dramatically longer systemic circulation time compared to free DOX. The fluorescence signal of PNPs efficiently and selectively increased in bladder cancer cells but not normal urothelial cells in vitro and in an orthotopic patient derived bladder cancer xenograft (PDX) models, indicating their great potential for photodynamic diagnosis. Photodynamic therapy with PNPs was significantly more potent than 5-aminolevulinic acid, and eliminated orthotopic PDX bladder cancers after intravesical treatment. Image-guided photodynamic and photothermal therapies synergized with targeted chemotherapy of DOX and significantly prolonged overall survival of mice carrying PDXs. In conclusion, this uniquely engineered targeting PNP selectively targeted tumor cells for photodynamic diagnosis, and served as effective triple-modality (photodynamic/photothermal/chemo) therapeutic agents against bladder cancers. This platform can be easily adapted to individualized medicine in a clinical setting and has tremendous potential to improve the management of bladder cancer in the clinic.

Keywords: Bladder cancer; Nanotechnology; Photodynamic therapy; Photothermal therapy.

Figure 1. Illustration and characterization of PLZ4-Nanoporphyrins (PNPs)

(A)Diagram of PNPs spontaneously assembled by the Pyropheophorbide a -containing-telodendrimer(PEG^5k-Por₄-CA₄) and PLZ4 conjugated telodendrimer (PLZ4-PEG^5k-CA₈). Moreover, PLZ4-micelle (PM) was a mixture of PLZ4-PEG^5k-CA₈ and PEG^5k-CA₈, while nanoporphyrin(NP) was a mixture of PEG^5k-CA₈ and PEG^5k-Por₄-CA₄ (B)Transmission electron microscopy images and (C) particle size distribution of PNPs and PNP-DOX. (D) The drug release profiles of free DOX, PM-DOX, and PNPs loaded with DOX (PNP-DOX). (E) Near infrared fluorescence (NIRF, left Y-axis) and singlet oxygen production (indicator: SOSG, right Y-axis) of PNPs in PBS (intact) or SDS (dissociated) after light exposure (20 J/cm²). Solid triangle: NIRF of PNPs in SDS; solid circle: NIRF of PNPs in PBS. Open triangle: SOSG production of PNPs in SDS; open circle: SOSG production of PNPs in PBS. (F) The relationship between temperature change and NIRF of PNPs in PBS or SDS as well as corresponding pyropheophorbide a (Ppa) in DMSO after light exposure (20J/cm²). (G) Pharmacokinetics of DOX after administration of free DOX and PNP-DOX (5mg Dox/kg). (H) Fluorescence microscopic observation for selective uptake of PNPs in the normal canine bladder urothelial cells (URO: no fluorescence, large polygonal cells with abundant cytoplasm) co-cultured with 5637 bladder cancer cells (BC: DiO pre-labeled, green). (160×, Bar=150 μm).

Figure 2. Photodynamic diagnosis of orthotopic mouse bladder cancer in vivo

(A) PNPs mediated intravesical photodynamic diagnosis using Kodak imaging station in orthotopic mouse bladder cancer model. Mouse with established MB49-GFP-Luc bladder cancers were intravesically injected with 10 mg/ml PNPs (pyropheophorbide a: 2 mg/ml) using 26G blunt needle via urethra. After 2 hours, bladder and other major organs were harvested and imaged in the GFP (tumor cells) and NIRF channel (PNP uptake) using Kodak imaging station. Yellow line: cut line for histopathology; red circles: tumor locations. (B) Large scale 3-D confocal imaging (Leica) for PNP mediated photodynamic diagnosis in orthotopic mouse MB49 bladder cancer model. (C) Uptake of PNPs in an orthotopic PDX BL269 and other organs after intravesical administration of PNPs. (D) The penetration depth analysis between lumen exposed tumor sites and normal urothelium Cryosection was performed on bladder BL269 PDX samples. The fluorescence intensity was measured using Metamorph image analysis software at different spots and depth from lumen surface of tumor (such as red arrow) versus normal urothelial areas (such as green arrow). (p<0.01, t-test). (E) A representative cryosection showing selective uptake of PNPs by PDX bladder cancers (red arrow) but not normal urothelial cells (yellow lines).

Figure 3. In vitro antitumor efficacy and cytotoxic mechanisms of PNPs against bladder cancer cells

(A) left: the viability of 5637 bladder cancer cells at 24 hours post PNP treatment and illumination with different doses of light (Pyropheophorbide a: 2 μg/ml for 2 hours) and different PNP and 5-ALA concentrations (right) (Light dose: 4.2 J/cm²). (B) Cell morphology changes at 3 hours post PNP-mediated photodynamic therapy. (Hema3™, 1000× oil). (C) Intracellular ROS production and (D) Glutathione (GSH) levels in 5637 cells upon photodynamic therapy. (E) Mitochondria membrane potential (DiOC₆(3): green) and cell integrity/viability (PI : red) 24 hours post treatment. Cells were incubated with DiOC₆(3) and PI for 20 minute. DiOC₆(3) ^low referred to loss of membrane potential, while PI + (red) stained dead cell nucleus. Bar=150 μm. (F) Apoptosis/necrosis assay, and (G) caspase 3/7 activation of 5637 cells at 24 hours post photodynamic therapy. PTX (1μg/ml) treated groups were served as positive control. (PI+/Annexvin V+ : late apoptosis; PI-/Annexvin V+: early apoptosis; PI+/Annexvin V− : Necrosis). (n=3, t-test, * p<0.05).

Figure 4. Anti-bladder cancer efficacy study of PNPs and PNP-DOX in an orthotopic PDX mouse model

(A) B-mode and microbubble enhanced ultrasound images, gross, and histopathology of mice carrying orthotopic PDX bladder cancer after treatments. Mice were implanted with BL645 inside bladder after pre-conditioned with acid. Mice were treated with PBS control, 1 mg/ml free DOX, or 5 mg/ml PNPs (1 mg/ml pyropheophorbide a) for 1 hour. After wash, PNP groups were further received whole bladder illumination (0.2 W for 3 minutes) via optical fiber. One month later, mice were imaged with ultrasound, and B-mode and contrast enhanced images were collected before and after microbubble injection to facilitate bladder cancer evaluation. After imaging, mice were sacrificed. Bladders were pre-filled with formalin (gross) before removal and submitted for histopathology evaluation (H&E stain, 1× and 4×). (B) The comparison of the wall thickness and (C) bladder functional area ratio of PDX bearing mice after treatment (n=3, one-way ANOVA tests, *p<0.05, **p<0.01, ***P<0.001).

Figure 5. Intracellular delivery of DOX and potential synergistic cytotoxic effect of PNP-DOX against bladder cancer cells

(A) Subcellular distribution of PNP-DOX at 15 minutes, 1 and 3 hours after treatment. (DOX: green; PNPs: red) (630×, oil). (B) Viability assay on 5637 cells treated with PNPs, PM-DOX, and PNP-DOX for 2 hours followed by different light exposure. (n=3, t-test, * p<0.05). (C) Apoptosis assay on 5637 cells treated with PNPs, PM-DOX, and PNP-DOX for 2 hours followed by light exposure. (D) Intra-nucleus DOX fluorescence was visualized by confocal microscope after 2 hour incubation, and (E) quantitative imaging analysis of intranuclear DOX was performed using Image J. (n=3, t-test, * p<0.05)

Figure 6. NIRF imaging of PDX mice models bearing subcutaneous and orthotopic bladder cancer

(A) In vivo NIFR imaging of NSG mice bearing subcutaneous PDX BL293 up to 24 hours after intravenously administration of 5-ALA (100 mg/kg), PNPs (Pyropheophorbide a 5 mg/kg), and PNP-DOX (Pyropheophorbide a 5mg/kg and DOX 2.5 mg/kg). Right: ex vivo NIFR imaging for BL293 tumors and other major organs. (5-ALA induced protoporphyrin IX ex/em = 633/650–710nm; PNP ex/em = 680/690nm. Kodak imaging system 650/700nm) (B) Biodistribution and tumor retention of PNP-DOX at different time points after injection. (C) The NIRF imaging of mice bearing orthotopic BL440 PDX model (red arrow) 24 hours after the administration of PNP. (Left panel: In vivo whole mouse imaging; Right panel: ex vivo imaging)

Figure 7. In vivo efficacy study of PNPs and PNP-DOX in PDX mouse model

(A) The tumor volume changes and (B) survival curve of mice bearing subcutaneous PDX BL293 tumor after treatment. Black arrows: intravenous injection; red arrows: light treatment (90 J/cm², 690 nm) (n=6). Tumors larger than 1000 mm³ were considered as end-point. Graph ended when one mouse in each group reached its end point. (*p<0.05, One-way ANOVA). (C) Histopathology evaluation of BL293 tumors at 24 hours post illumination. H&E stain, bar = 300 μm; insert: bar = 60 μm). (D) Intratumoral ROS production in mice bearing BL293 tumors after PNPs and PBS (control) followed by light exposure 90 J/cm²). (n=5, t-test, * p<0.01) (E) Time course of tumor temperatures before and after laser irradiation. Light dose low(L) (90 J/cm²) and high dose (H) (180 J/cm²). (yellow area: lights on; n=3, t-test, *p<0.05, **p<0.01) (F) Representative tumor surface temperature captured in the central spot by FLIR thermal camera.

Figure 7. In vivo efficacy study of PNPs and PNP-DOX in PDX mouse model

(A) The tumor volume changes and (B) survival curve of mice bearing subcutaneous PDX BL293 tumor after treatment. Black arrows: intravenous injection; red arrows: light treatment (90 J/cm², 690 nm) (n=6). Tumors larger than 1000 mm³ were considered as end-point. Graph ended when one mouse in each group reached its end point. (*p<0.05, One-way ANOVA). (C) Histopathology evaluation of BL293 tumors at 24 hours post illumination. H&E stain, bar = 300 μm; insert: bar = 60 μm). (D) Intratumoral ROS production in mice bearing BL293 tumors after PNPs and PBS (control) followed by light exposure 90 J/cm²). (n=5, t-test, * p<0.01) (E) Time course of tumor temperatures before and after laser irradiation. Light dose low(L) (90 J/cm²) and high dose (H) (180 J/cm²). (yellow area: lights on; n=3, t-test, *p<0.05, **p<0.01) (F) Representative tumor surface temperature captured in the central spot by FLIR thermal camera.