Kozo Miyazaki

Enhanced photodynamic cancer treatment by supramolecular nanocarriers charged with dendrimer phthalocyanine.

Photodynamic therapy (PDT) is a promising method for the localized treatment of solid tumors. In order to enhance the efficacy of PDT, we have recently developed a novel class of photosensitizer formulation, i.e., the dendrimer phthalocyanine (DPc)-encapsulated polymeric micelle (DPc/m). The DPc/m induced efficient and unprecedentedly rapid cell death accompanied by characteristic morphological changes such as blebbing of cell membranes, when the cells were photoirradiated using a low power halogen lamp or a high power diode laser. The fluorescent microscopic observation using organelle-specific dyes demonstrated that DPc/m might accumulate in the endo-/lysosomes; however, upon photoirradiation, DPc/m might be promptly released into the cytoplasm and photodamage the mitochondria, which may account for the enhanced photocytotoxicity of DPc/m. This study also demonstrated that DPc/m showed significantly higher in vivo PDT efficacy than clinically used Photofrin (polyhematoporphyrin esters, PHE) in mice bearing human lung adenocarcinoma A549 cells. Furthermore, the DPc/m-treated mice did not show skin phototoxiciy, which was apparently observed for the PHE-treated mice, under the tested conditions. These results strongly suggest the usefulness of DPc/m in clinical PDT.

Disulfide crosslinked polyion complex micelles encapsulating dendrimer phthalocyanine directed to improved efficiency of photodynamic therapy

Dendrimer phthalocyanine (DPc)-loaded polyion complex micelle (DPc/m) has been developed as photosensitizer (PS) formulation in photodynamic therapy (PDT). Incorporation of DPc into the micelle showed significant enhancement in the in vitro photocytotoxicity. Also, introduction of disulfide crosslinking in the micellar core further improved the in vitro PDT effect of DPc/m. Here, we aim to analyze the mechanism of the enhanced photocytotoxicity of DPc/m, particularly focusing on the photochemical reactions during photoirradiation. As a result, DPc/m has been shown to protect DPc from photobleaching induced by the reactions with serum proteins, although DPc were considerably quenched in the micellar core. Furthermore, the introduction of disulfide crosslinking into the micellar core has demonstrated to improve the efficiency of reactive oxygen species (ROS) production by DPc in the micellar core as well as more effectively prevent the photobleaching of DPc. These effects might lead to effective photochemical reactions by DPc/m, which may account for the enhanced photocytotoxicity. Our findings provide useful knowledge in designing PS formulations for effective PDT.

Light-induced cytosolic activation of reduction-sensitive camptothecin-loaded polymeric micelles for spatiotemporally controlled in vivo chemotherapy.

Nanomedicines capable of smart operation at the targeted site have the potential to achieve the utmost therapeutic benefits. Providing nanomedicines that respond to endogenous stimuli with an additional external trigger may improve the spatiotemporal control of their functions, while avoiding drawbacks from their inherent tissue distribution. Herein, by exploiting the permeabilization of endosomes induced by photosensitizer agents upon light irradiation, we complemented the intracellular action of polymeric micelles incorporating camptothecin (CPT), which can sharply release the loaded drug in response to the reductive conditions of the cytosol, as an effective strategy for precisely controlling the function of these nanomedicines in vivo, while advancing toward a light-activated chemotherapy. These camptothecin-loaded micelles (CPT/m) were stable in the bloodstream, with minimal drug release in extracellular conditions, leading to prolonged blood circulation and high accumulation in xenografts of rat urothelial carcinoma. With the induction of endosomal permeabilization with the clinically approved photosensitizer, Photofrin, the CPT/m escaped from the endocytic vesicles of cancer cells into the cytosol, as confirmed both in vitro and in vivo by real-time confocal laser microscopies, accelerating the drug release from the micelles only in the irradiated tissues. This spatiotemporal switch significantly enhanced the in vivo antitumor efficacy of CPT/m without eliciting any toxicity, even at a dose 10-fold higher than the maximum tolerated dose of free CPT. Our results indicate the potential of reduction-sensitive drug-loaded polymeric micelles for developing safe chemotherapies after activation by remote triggers, such as light, which are capable of permeabilizing endosomal compartments.

Multicompartment micelles with adjustable poly(ethylene glycol) shell for efficient in vivo photodynamic therapy

We describe the preparation of well-defined multicompartment micelles from polybutadiene-block-poly(1-methyl-2-vinyl pyridinium methyl sulfate)-block-poly(methacrylic acid) (BVqMAA) triblock terpolymers and their use as advanced drug delivery systems for photodynamic therapy (PDT). A porphyrazine derivative was incorporated into the hydrophobic core during self-assembly and served as a model drug and fluorescent probe at the same time. The initial micellar corona is formed by negatively charged PMAA and could be gradually changed to poly(ethylene glycol) (PEG) in a controlled fashion through interpolyelectrolyte complex formation of PMAA with positively charged poly(ethylene glycol)-block-poly(L-lysine) (PLL-b-PEG) diblock copolymers. At high degrees of PEGylation, a compartmentalized micellar corona was observed, with a stable bottlebrush-on-sphere morphology as demonstrated by cryo-TEM measurements. By in vitro cellular experiments, we confirmed that the porphyrazine-loaded micelles were PDT-active against A549 cells. The corona composition strongly influenced their in vitro PDT activity, which decreased with increasing PEGylation, correlating with the cellular uptake of the micelles. Also, a PEGylation-dependent influence on the in vivo blood circulation and tumor accumulation was found. Fully PEGylated micelles were detected for up to 24 h in the bloodstream and accumulated in solid subcutaneous A549 tumors, while non- or only partially PEGylated micelles were rapidly cleared and did not accumulate in tumor tissue. Efficient tumor growth suppression was shown for fully PEGylated micelles up to 20 days, demonstrating PDT efficacy in vivo.

A novel homogeneous irradiation fiber probe for whole bladder wall photodynamic therapy

We designed and fabricated a novel fiber probe that homogeneously illuminates the urinary bladder cavity for photodynamic therapy (PDT) of bladder cancer, which is known to occur in multifocal areas and to proliferate diffusely in the bladder mucosa.

Abstract 3469: Photodynamic diagnosis (PDD) and therapy (PDT) with dendrimer porphyrin micelle

[Background and purpose] We previously reported that the efficacy of photodynamic therapy (PDT) with dendrimer phthalocyanine-loaded micelles (DPc/m). However, monitoring of this photosensitizer by the clue of fluorescence is quite difficult because of the low intrinsic fluorescence originating from phthalocyanine, thus other fluorescence dye such as Alexa is required for the monitoring. For resolving this problem, we developed a novel micelle-based photosensitizer loading dendrimer porphyrin (DP). The DP-loaded micelles (DP/m) intensely emit fluorescence from porphyrin without an additional fluorescence labeling. The aim of this study was to verify practical effectiveness of DP/m by comparing that of DP on PDD and PDT. [Materials and Methods] For in vitro cytotoxicity tests, rat bladder carcinoma cells (AY-27) were incubated with DP/m for 4 h and were exposed to laser light (635 nm, 100 J/cm2). One day after PDT, cell viability was measured by WST-8 assay. Distribution of DP/m in cellular organelles was investigated using fluorescence markers of cellular organelles by a confocal laser scanning microscope. In addition, we examined in vivo fluorescence imaging using an orthotopic bladder tumor model established by inoculating AY27 cells in female Fischer F344 rats via transurethral catheterization. Four hours after intravenous administration of DP/m, the bladders were extracted and fluorescence image was obtained of bladder. [Results] DP and DP/m without light irradiation were basically non-toxic (low dark toxicity), showing cell viability being > 85% at the concentration up to 17 μM. However, photoirradiated DP and DP/m induced the high level of cytotoxicity: survival rate was less than 5%. PDT using DP/m showed an approximately 15 times of cytotoxicity when compared with PDT using DP. The results suggested that DP/m highly accumulate in cancer cells and can provide a significant photodynamic effect leading to cell damage. Confocal laser scanning microscopic studies showed that DP/m exhibited preferential accumulation in lysosomes through endocytosis and retention in the cells. When DP/m were injected (i.v.) into the bladder tumor animal models, fluorescence from the DP/m were clearly visualized in accordance with tumor distribution. We detected fluorescence even when a size of tumor was less than 1 mm, thus enabling optical imaging with excellent contrast between tumor and surrounding normal tissue. A study of PDT in vivo is now carried out. [Conclusion] We developed dendrimer porphyrin-loaded micelles (DP/m), which were effective for both PDD and PDT. An excellent detection of tumor was achieved owing to the intense fluorescence of DP/m and highly accumulation in tumors. The preferable DP/m uptake in tumors resulted in effective phototoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3469. doi:1538-7445.AM2012-3469

Abstract 3468: Endoscopic diagnosis and treatment of esophageal tumor using a novel DDS-type photoactivatable agent

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Photodynamic diagnosis (PDD) and therapy (PDT) have been clinically used for the early phase cancer of hollow organs. We are advancing the photomedicine to the next stage, in which treatment is carried out immediately after the detection of lesions. For achieving the simultaneous diagnosis and treatment, we established a novel DDS-type photoactivatable agent, micelle-based photosensitizer loading dendrimer porphyrin (DP/M). A micelle-based shell structure is expected to be selectively accumulated in tumors. An intensely emitted fluorescence from porphyrin can be used for lesion detection and a highly quantum yield of porphyrin promises an effective PDT. Apart from the agent, we recently developed a fluorometric system equipped with an ultrathin endoscope that has a spatial resolution of 15,000 pixels even with a diameter of 0.8 mm. The aim of this study was to verify practical effectiveness of the novel photosensitizer combined with the endoscopic fluorometric system for simultaneous diagnosis and treatment. In this study, we targeted esophageal cancer because satisfactory results are not always obtained with conventional therapies and an endoscopic approach is applicable. Materials and Methods: A rat orthotopic esophageal tumor model was established in female Fischer F344 rats by orally administered with NMBuA (N-Nitroso Methylbutylamine) solved in drinking water. After intravenous administration of DP/M, PDD was carried out using the endoscopic fluorometric system (ex. 405 nm, em. > 630 nm). After the detection of lesions, tumors were irradiated using a diffuser tip fiber connected to a 635nm LD laser. Results: About 6 weeks after the administration of NMBuA, polypoid tumors were observed in the esophagus. Pathological analysis revealed that proliferated polymorphonuclear cells were seen: a part of which reached the muscular layer of mucosa. A selective accumulation of DP/M in the tumors was confirmed with the endoscopic fluorometric system. This modality enabled repeated observations of tumors without sacrifice. The following PDT provided a promising result. Conclusions: DP/M combined with the endoscopic fluorometric system brought the simultaneous diagnosis and treatment for the orthotopic rat esophagus tumor models. Acknowledgment: This research is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3468. doi:1538-7445.AM2012-3468

Abstract 5235: Fluorometric imaging system for visualizing submillimeter level tumors using an ultrathin endoscope

[Background and purpose] Direct observation of pathological lesions by a relatively noninvasive approach is important for understanding pathophysiological mechanism and for assessing the objective response to therapeutic interventions. On the other hand, it is very difficult to perform direct and relatively noninvasive observation of minimal lesions inside a diseased model using a small live animal. However, recent technological developments in imaging, especially significant progress in fluorescent methods and in endoscopic examinations, have enabled detection and tracing of micro-lesions inside small animal models. We have developed a fluorescence imaging technique using high-resolution ultrathin endoscope, which is accessible to hollow organs of small animals. In addition, we have established an orthotopic rat bladder cancer model in which the cancer cells express a fluorescent protein. The aim of this study was to establish a high-resolution imaging method for visualizing the vascular distribution and mucosal morphology of a bladder tumor. [Materials and Methods] GFP-expressing bladder tumor in a rat orthotopic model: Firstly, we established a cell line with stable integration of GFP constructs that were produced by transfection of pTurboGFP into rat bladder cells (AY27). The stable AY27+tGFP populations were purified by repeated selections using Geneticin (G418). AY27-tGFP cells were implanted in the bladders of female Fischer F344 rats, resulting in development of submillimeter-sized bladder tumors. Development of fluorescence ultrathin endoscopy: We developed a fluorescence ultrathin endoscopic system installed with a 180 W Xenon lamp with excitation filters and 3CCD camera with emission filters. The outer diameter of the ultrathin endoscopic fiber is only 0.8 mm, but its number of pixels reaches 15,000, resulting in a spatial resolution of 50μm. [Results] Fluorescence from AY27-tGFP cells implanted in the bladder was clearly visualized by the ultrathin endoscope placed in the bladder by the transurethral approach. Furthermore, when fluorescence-probed tumor-directive micelles were injected (i.v.) into the animals as a model of drug delivery, fluorescence from the probe agents was visualized in accordance with tumor distribution. In both cases, we detected fluorescence from tumors of less than 1 mm in size. [Conclusion] We established an orthotopic GFP-tumor rat model using a GFP-expressing tumor cell line (AY27-tGFP). Submillimeter tumors in this model were visualized by the newly developed ultrathin endoscopic system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5235.

Abstract 501: A novel PDT system consisting of a DDS-type photosensitizer and a newly developed fiber probe for homogenous irradiation

Introduction: Photodynamic therapy (PDT) has already been established as anti-cancer therapy. However, since some types of cancer proliferate multifocally and/or diffusely, topical irradiation of light by a conventional optical fiber results in an insufficient PDT effect. Moreover, using a conventional fiber, we cannot irradiate minimal tumors that escape from detection by conventional diagnostic approaches. When PDT is applied to the lesions of hollow organs, if we homogenously irradiate a wide area of the organ wall, undetectable minimal tumors and multifocally proliferating tumors can be treated simultaneously. In order to realize this concept, we have developed a novel fiber probe that provides homogenous light irradiation on the mucosa of hollow organs. In this study, we fabricated a homogenous irradiation fiber probe (HIFP) for illuminating the bladder by the use of state-of-the-art processing technology (torsional deformation of a fiber core, conical shaping of fiber tip, etc). We evaluated the PDT effect on a rat orthotopic bladder tumor model using the HIFP and a dendrimer phthalocyanine micelle (DPc/m) that we developed as a DDS-type photosensitizer. Materials and Methods: The property of spatial distribution of light emitted from the HIFP was measured. Using the HIFP, PDT was carried out on a rat orthotopic bladder tumor model 24 h after administration of a photosensitizer (Photofrin or DPc/M) and PDT effects were pathologically evaluated. Results: Preliminarily examination of ultrasonic imaging revealed that the shape of the rat bladder approximates an ellipsoidal body with a long radius of 7.5 mm and a short radius of 5 mm. Analysis of spatial distribution showed that the light emitted from the HIFP covered 90% of the whole luminal area of the approximate ellipsoidal body, while the light emitted from a control fiber (CF) (NA=0.41; flat cleaved end) covered only 15%. Pathological study of the PDT-treated rat using the CF revealed that an anti-tumor effect occurred only in the local area proximate to the focal point of the CF with no anti-tumor effect in tumors in other areas of the bladder wall. In contrast, pathological study of the PDT-treated rat using the HIFP revealed that light was distributed over 60 −80% of the mucosa of the bladder and tumor proliferation was widely suppressed. Conclusion: Widespread and homogenous irradiation in the mucosa of the bladder was achieved using the newly developed fiber probe. PDT using a combination of the fiber probe and DPc/M enhanced the anti-tumor effect without bladder shrinking. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 501.