Photochemical Internalization Research Articles

Enhanced efficacy of bleomycin in bladder cancer cells by photochemical internalization.

Bleomycin is a cytotoxic chemotherapeutic agent widely used in cancer treatment. However, its efficacy in different cancers is low, possibly due to limited cellular internalization. In this study, a novel approach known as photochemical internalization (PCI) was explored to enhance bleomycin delivery in bladder cancer cells (human T24 and rat AY-27), as bladder cancer is a potential indication for use of PCI with bleomycin. The PCI technique was mediated by the amphiphilic photosensitizer disulfonated tetraphenyl chlorin (TPCS2a) and blue light (435 nm). Two additional strategies were explored to further enhance the cytotoxicity of bleomycin; a novel peptide drug ATX-101 which is known to impair DNA damage responses, and the protease inhibitor E-64 which may reduce bleomycin degradation by inhibition of bleomycin hydrolase. Our results demonstrate that the PCI technique enhances the bleomycin effect under appropriate conditions, and importantly we show that PCI-bleomycin treatment leads to increased levels of DNA damage supporting that the observed effect is due to increased bleomycin uptake. Impairing the DNA damage responses by ATX-101 further enhances the efficacy of the PCI-bleomycin treatment, while inhibiting the bleomycin hydrolase does not.

Intradermal photosensitisation facilitates stimulation of MHC class-I restricted CD8 T-cell responses of co-administered antigen

The protection or treatment of several immunological disorders is dependent on the antigen-specific and cytotoxic CD8 T cells. However, vaccines aimed at stimulating CD8 T-cell responses are typically ineffective because vaccine antigens are primarily processed by the MHC class-II and not the MHC class-I pathway of antigen presentation: the latter requires cytosolic delivery of antigen. In order to facilitate targeting of antigen to cytosol, the antigen was combined with the photosensitiser TPCS2a (disulfonated tetraphenyl chlorin) and administered intradermally to mice. The photosensitiser was activated by illumination of the injection site. This photochemical internalization (PCI) strongly increased the stimulation of CD8 T-cell responses as measured by antigen-specific proliferation and secretion of pro-inflammatory cytokines. Fluorescence microscopy showed that delivery to cytosol was TPCS2a dependent and occurred by light-induced disruption of TPCS2a- and antigen-containing endosomes. PCI-based vaccination prevented growth of malignant B16 cells as compared with vaccination without PCI. In conclusion, PCI represents a potent tool for delivery of antigens to cytosol for stimulation of cytotoxic CD8 T-cell responses. This study demonstrated a first proof-of-principle for PCI-mediated immunisation with potential application in cancer immunotherapy.

The effect of photochemical internalization of bleomycin in the treatment of urothelial carcinoma of the bladder: An in vitro study

ObjectivesIn this in vitro study, we determined whether meso-tetraphenyl chlorin disulphonate (TPCS2a)-based photochemical delivery of bleomycin was able to potentiate the cytotoxicity of bleomycin on bladder cancer cells. Materials and methodsThe human RT4, RT112, 253J, T24, and rat AY-27 urothelial carcinoma cell lines were used. Cells were seeded in 96-well plates. TPCS2a was added to the growth medium and the plates were incubated overnight. Cells were then resuspended in TPCS2a-free culture medium and incubated for 3 hours. Subsequently, cells were treated for 60 minutes with increasing doses of epirubicin, gemcitabine, mitomycin C, or bleomycin followed by illumination for different periods. Cell viability was measured with a colorimetric assay after 72 hours. ResultsFor the single treatments, in all 5 cell lines a dose-dependent inhibition of cell proliferation was observed. This was seen both after treatment with TPCS2a-based photodynamic therapy (PDT), as well as after treatment with either bleomycin or one of the control chemotherapeutic agents.After treatment with PDT (240-s illumination), bleomycin 9.0µM, and the combination of these treatments, relative survival percentages were 89.2±13.0, 70.2±8.9, and 30.5±6.1, respectively, in the T24 cell line. After treatment with PDT (120-s illumination), bleomycin 27µM and the combination of these treatments, relative survival percentages were 93.6±15.7, 74.7±9.6, and 30.0±11.1, respectively, in the AY-27 cell line. In both cell lines, PDT combined with bleomycin showed significantly (P<0.001) higher cell kill than the sum of the single treatments, suggesting a photochemical internalization effect. ConclusionsTPCS2a-based photochemical internalization of bleomycin showed a significant, at least, additive antiproliferative activity against human and rat urothelial carcinoma cells in vitro. Thus, photochemical internalization may have therapeutic potential as an intravesical strategy against bladder cancer. As the effect is heterogeneous, biomarker studies are warranted to be able to predict the effects of a photochemical internalization–based treatment.

Photochemical internalisation as a delivery tool to improve chemotherapy for oral squamous cell carcinoma

Photochemical internalisation as a delivery tool to improve chemotherapy for oral squamous cell carcinoma

Circumvention of resistance to photodynamic therapy in doxorubicin-resistant sarcoma by photochemical internalization of gelonin

A wide range of anti-cancer therapies have been shown to induce resistance upon repetitive treatment and such adapted resistance may also cause cross-resistance to other treatment modalities. We here show that MES-SA/Dx5 cells with adapted resistance to doxorubicin (DOX) are cross-resistant to photodynamic therapy (PDT). A DOX-induced increased expression of the reactive oxygen species (ROS)-scavenging proteins glutathione peroxidase (GPx) 1 and GPx4 in MES-SA/Dx5 cells was indicated as the mechanism of resistance to PDT in line with the reduction in PDT-generated ROS observed in this cell line. ROS-induced p38 activation was, in addition, shown to be reduced to one-third of the signal of the parental MES-SA cells 2h after PDT, and addition of the p38 inhibitor SB203580 confirmed p38 activation as a death signal after PDT in the MES-SA cells. The MES-SA/Dx5 cells were also cross-resistant to ionizing radiation in agreement with the increased GPx1 and GPx4 expression. Surprisingly, PDT-induced endo/lysosomal release of the ribosome-inactivating protein gelonin (photochemical internalization (PCI)) was more effective in the PDT-resistant MES-SA/Dx5 cells, as measured by synergy calculations in both cell lines. Analysis of death-inducing signaling indicated a low activation of caspase-3 and a strong PARP I cleavage after PDT and PCI in both cell lines. The PARP I activation was, however, stronger after PCI than after PDT in the MES-SA cells, but not in the MES-SA/Dx5 cells, and therefore cannot explain the strong PCI effect in the MES-SA/Dx5 cells. In conclusion PCI of recombinant gelonin circumvents ROS resistance in an apoptosis-independent manner.

Enhancing Nucleic Acid Delivery By Photochemical Internalization

Photochemical internalization (PCI) is a method for releasing macromolecules from endosomal and lysosomal compartments. The PCI approach uses a photosensitizer that localizes to endosomal and lysosomal compartments, and a light source with appropriate light spectra for excitation of the photosensitizer. Upon photosensitizer excitation, endosomal and lysosomal membranes are destroyed, due to the formation of reactive oxygen species, followed by release of the endocytosed material. PCI has been demonstrated to enhance and control (site- and time-specific) delivery of various macromolecules such as viruses, proteins, chemotherapeutics, nucleic acid, and so on. In this Review we present past and current studies of PCI-controlled delivery of natural and artificial nucleic acids, such as peptide nucleic acids, siRNA molecules, mRNA molecules and plasmids. We also discuss critical aspects to further the possibilities for successful gene targeting in space and time.

Vascular endothelial cells as targets for photochemical internalization (PCI)

Cancer treatment can be exerted by targeting both cancer cells and the vasculature supplying solid tumors. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, but recent data on contrast-enhanced MRI have indicated that the method also reduces blood perfusion in HT1080 fibrosarcoma xenografts. The present report aims to investigate if PCI may exert direct cytotoxic effects on endothelial cells. PCI of saporin was performed on endothelial human umbilical vein endothelial cell (HUVEC) and fibrosarcoma cells (HT1080) using two PCI-relevant photosensitizers, TPPS2a and AlPcS2a. A 22- and 13-fold higher photosensitizer uptake was detected in the endothelial cells compared with the HT1080 cells for AlPcS2a and TPPS2a, respectively. PCI of saporin was, however, found more effective in HT1080 cells. For HT1080 cells, PCI with saporin increased cell killing 1.9-fold over photodynamic therapy alone, but under the same conditions, only increased HUVEC cell killing by 1.6- and 1.3-fold with AlPcS2a and TPPS2a , respectively. Saporin uptake was higher in HUVECs than in the HT1080 cells, hence did not reflect the cell line differences in PCI efficacy. This is the first report on PCI-mediated kill of endothelial cells and lays the foundation for further preclinical evaluation of the PCI technology as an antivascular strategy to ablate tumors.

Photochemical internalisation as a delivery tool to improve chemotherapy for oral squamous cell carcinoma

Photochemical internalisation as a delivery tool to improve chemotherapy for oral squamous cell carcinoma

The photosensitizer disulfonated aluminum phthalocyanine reduces uptake and alters trafficking of fluid phase endocytosed drugs in vascular endothelial cells—Impact on efficacy of photochemical internalization

Targeting cancer vasculature is an emerging field in cancer treatment. Photochemical internalization (PCI) is a drug delivery technology based on photochemical lysis of drug-bearing endocytic vesicles originally designed to target cancer cells. Recent investigations have revealed a lower PCI efficacy in vascular endothelial cells (HUVECs) in vitro than in HT1080 fibrosarcoma cells. This manuscript aims to explore the limiting factor for the PCI effect in HUVECs. Cellular uptake of the photosensitizers AlPcS2a and TPPS2a, and a model compound for macromolecular drugs taken up by fluid phase endocytosis, Alexa488-dextran, was explored by flow cytometry. The uptake of AlPcS2a and TPPS2a was 3.8-fold and 37-fold higher in HUVECs than in HT1080 cells, respectively, while the Alexa488-dextran uptake was 50% lower. AlPcS2a (but not TPPS2a) was shown to reduce Alexa488-dextran uptake in a concentration-dependent manner, resulting in 66% and 33% attenuation of Alexa488-dextran uptake at 20μg/ml AlPcS2a in HUVECs and HT1080 cells respectively. Studies of intracellular localization of Alexa488-dextran and AlPcS2a by confocal microscopy in HUVECs uncovered a concentration-dependent AlPcS2a-induced inhibition of Alexa488-dextran trafficking into AlPcS2a-stained and acidic vesicles. The localization of Alexa488-dextran to AlPcS2a-localizing compartments was reduced by 40% when the AlPcS2a concentration was increased from 5 to 20μg/ml. The treatment dose of AlPcS2a was found to influence on the efficacy of PCI of saporin, but to a lesser extent than expected considering the data from cellular uptake and intracellular trafficking of Alexa488-dextran. The implications of these results for further development of vascular targeting-PCI are discussed.

Tat-tetanus toxin fragment C: a novel protein delivery vector and its use with photochemical internalization

Protein delivery vectors can be grouped into two classes, those with specific membrane receptors undergoing conventional endocytosis and cell penetrating peptides (CPP) that have the capacity to cross cell or endosomal membranes. For both forms of vectors, translocation across a membrane is usually an inefficient process. In the current study, a novel vector combining the widely used CPP, Tat and the non-toxic neuronal binding domain of tetanus toxin (fragment C or TTC) was assessed for its capacity to deliver GFP as a test cargo protein to human neural progenitor cells (NPCs). These two functional membrane interacting domains dramatically enhanced internalization of the conjugated cargo protein. Tat-TTC-GFP was found to be bound or internalized at least 83-fold more than Tat-GFP and 33-fold more than TTC-GFP in NPCs by direct fluorimetry, and showed enhanced internalization by quantitative microscopy of 18 - and 14-fold, respectively. This preferential internalization was observed to be specific to neuronal cell types. Photochemical internalization (PCI) was utilized to facilitate escape of the endosome-sequestered proteins. The combined use of the Tat-TTC delivery vector with PCI led to both enhancement of neural cell type specific delivery to an endosomal target, followed by the option of efficient release to the cytosol.

Photochemical internalization of CD133-targeting immunotoxins efficiently depletes sarcoma cells with stem-like properties and reduces tumorigenicity

BackgroundThe normal stem cell marker CD133 is also a putative marker of cancer stem cells (CSCs) in different types of cancers. Hence, a major challenge when targeting CD133-expressing CSCs is to prevent depletion of the normal stem cell pool. We hypothesized that the site-specific and light-controlled drug delivery method photochemical internalization (PCI) may have the potential to enhance selectivity and endosomal escape of CD133-targeting immunotoxins in stem-like sarcoma cells. MethodsWe have used a sarcoma model, SW872 cells isolated from xenografts harboring CSCs within a ~2% CD133high subpopulation to investigate the potential of PCI of CD133-targeting toxin as a novel strategy to kill CSCs. Model immunotoxins were generated by binding the ribosome-inactivating protein toxin saporin to each of the monoclonal antibodies CD133/1 (AC133) or CD133/2 (293C), specific for individual CD133-epitopes. Cellular targeting, intracellular co-localization with the PCI photosensitizer, disulfonated meso-tetraphenylchlorin (TPCS2a), and cytotoxic efficacy of PCI of the CD133-targeting toxins were evaluated. ResultsPCI of CD133–saporin efficiently targets CD133-expressing SW872 and HT1080 sarcoma cells and results in loss of cell viability. Following sub-toxic treatment, surviving SW872 cells, depleted of the CD133-expressing population, display reduced proliferative capacity and attenuated CSC properties, such as reduced colony-forming ability and tumorigenicity. ConclusionHere we present a proof-of-concept study, where PCI enables light-triggered delivery of CD133-targeting antibody-drug conjugates, resulting in decreased sarcoma tumor-initiating capacity. General significancePCI of CD133-targeting toxins may be used as a minimal invasive strategy in the treatment of sarcomas, and potentially as a therapeutic for other solid tumors expressing CD133.

Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins

Membranes are vital barriers by which cells control the flux of molecules and energy between their exterior and interior and also between their various intracellular compartments. While numerous transport systems exist for ions and small molecules, the cytosolic uptake of larger biological molecules and in particular antibody-targeted drugs, is a big challenge. Inducing leakage of the plasma membrane is unfavorable since the target cell specificity mediated by the antibody would likely be lost in this case. After binding and internalization, the antibody drug conjugates reach the endosomes. Thus, enforcing the endosomal escape of anti-tumor toxins without affecting the integrity of other cellular membranes is of paramount importance. Different strategies have been developed in the last decades to overcome endosomal accumulation and subsequent lysosomal degradation of targeted protein-based drugs. In this review we summarize the various efforts made to establish efficient techniques to disrupt the endosomal membrane barrier including the use of molecular ferries such as cell penetrating peptides or viral membrane fusion proteins, endosomal leakage inducing molecules such as saponins or monensin and physicochemical methods as represented by photochemical internalization.

Abstract 1606: Photochemical internalization (PCI): a novel technology for site-specific drug delivery.

Abstract The utilization of macromolecules in therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. To achieve the expected biological effect of these macromolecules in many cases internalisation to the cell cytosol is crucial. At an intracellular level, the most fundamental obstruction for cytosolic delivery of therapeutic macromolecule is the membrane-barrier of the endocytic vesicles. Photochemical internalisation (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces rupture of the endocytic vesicles and thereby release of the macromolecules into the cytosol. PCI has been shown to enhance the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus, oligonucleotides and the chemotherapeutic agent bleomycin. For clinical utilization a novel photosensitizer has been developed and evaluated for PCI of bleomycin. Preclinical results with this combination will be presented. Furthermore, a phase I/II dose-escalating clinical trial has been finalized and results from this trial will also be presented. Citation Format: Kristian Berg, Colin Hopper, Anette Weyergang, Pål K. Selbo, Anders Høgset. Photochemical internalization (PCI): a novel technology for site-specific drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1606. doi:10.1158/1538-7445.AM2013-1606

Abstract 5665: New alternatives to photodynamic therapy based on light-enabled drug delivery.

Abstract Conventional photodynamic therapy (PDT) aims to achieve tumor drug targeting by selectively killing cells upon illumination. PDT serves as a non-invasive treatment option for many types of non-metastatic, light-accessible cancers such as those involving the head and neck, lung, bladder, prostate, or esophagus. The various types of PDT all use the same basic strategy for killing cells; photosensitizing molecules are taken up by cancer cells, and, upon illumination, singlet oxygen is generated in the vicinity of the photosensitizer, killing the cell. A new alternative to PDT is photochemical internalization, in which singlet oxygen generated by a photosensitizer disrupts endosomes containing a toxin, releasing it into the cytoplasm and killing the cell. A drawback to both of these approaches is that moderate levels of oxygen are required, yet tumors are often hypoxic environments. Moreover, during PDT treatment, oxygen levels drop even further as the singlet oxygen is consumed; this can compromise treatment effectiveness. We have sought to address these challenges by developing new strategies for light-based treatment of cancer that are oxygen independent. In the first approach, which we call photocaged permeability, we have attached the anticancer drug doxorubicin to a cell impermeable small molecule via a light-cleavable linker. We show using flow cytometry and confocal microscopy that this conjugate is cell impermeable in the dark, yet upon illumination, doxorubicin is released and enters the cells. The IC50 for our conjugate in JH-EsoAd1 cells using non-cytotoxic doses of light is 1.6 μM, identical to that of doxorubicin alone. No cytotoxicity is observed at doses 30-fold higher in the dark. In a second approach, we have attached doxorubicin to folate receptor and PSMA ligands via a light cleavable linker in order to enable dual cancer cell targeting with both light and ligand specificity. The ability of the resulting conjugates to selectively kill cells expressing the requisite receptors with light will be described. Citation Format: M. Michael Dcona, Deboleena Mitra, Rachel W. Goehe, David A. Gewirtz, Deborah A. Lebman, Matthew C.T. Hartman. New alternatives to photodynamic therapy based on light-enabled drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5665. doi:10.1158/1538-7445.AM2013-5665

Abstract 5611: Photochemical internalization of VEGF121/rGel: a strategy for optimizing tumor-vasculature targeting.

Abstract Photochemical internalization (PCI) is a modality for intracellular delivery of drugs which lack an effective transport mechanism into the cell. Such drugs are taken up by means of endocytosis and are transported to the lysosomes where they are degraded before they have exerted their biological effect. PCI utilizes photosensitizers which accumulate in the membranes of these endo/lysosomal compartments. Light exposure causes rupture of these membranes and subsequent cytosolic release of the drug which freely can interact with its target. PCI of the conventional cytostatic drug Bleomycin has been shown to be highly effective in vivo and is currently evaluated in clinical trials with promising results. Bleomycin is, however, probably not optimal for PCI mediated delivery, and therefore cannot fully take use of the technology. Recombinant targeted toxins may be designed to possess all characteristics for an optimal drug to be delivered by PCI: (1) Exclusive uptake in tumor cells, (2) Intracellular target, (3) Not able to penetrate the plasma membrane, (4) Suboptimal escape from endocytic compartments without PCI, (5) Able to diffuse into the tumor tissue. We here present PCI of a recombinant fusion toxin composted of VEGF121, and gelonin, a type I ribosome inactivating protein toxin. VEGF121/rGel administration has previously been shown to be effective in suppressing tumor xenografts and metastasis. Severe adverse effects may, however, limit the possibilities to obtain complete responses with VEGF121/rGel monotherapy. PCI was shown to increase the selectivity of VEGF121/rGel in VEGFR1 and VEGFR2 transfected porcine endothelial cells (PAE) in vitro. The PCI treatment also resulted in a dramatic reduction in the dosage of VEGF121/rGel to 1/100 as measured by a LD90 of 10 nM for the fusion toxin alone compared to 100 pM with the PCI treatment in VEGFR2 expressing PAE cells. For in vivo experiments the murine colon cell line CT26.CL25 was injected subcutaneously in the left hip of BALB c mice and PCI of VEGF121/rGel was administered one week after implantation. By day 24 after PCI all animals in the non-treatment control group were sacrificed due to tumor size (&amp;gt;1000m3),while a 50% complete response (CR) was found with PCI of VEGF121/rGel. The animals were followed up to day 60 when the experiment was terminated. No tumor regression was observed in CR animals in this time-frame. About 50%CR was also detected in PCI of bleomycin-treated animals. Animals receiving PCI of bleomycin showed an average weight loss of 15% compared to no significant weight loss in the PCI of the VEGF121/rGel treated group. This is the first report on PCI of a tumor vascular targeting drug. In conclusion, the present results indicate PCI of VEGF121/rGel as a highly selective method for destroying tumor vasculature. The project is supported by the Norwegian Cancer Society. Research was conducted, in part, by the Clayton Foundation for Research Citation Format: Anette Weyergang, Lawrence H. Cheung, Michael Rosenblum, Khalid Mohamedali, Johannes Waltenberger, Kristian Berg. Photochemical internalization of VEGF121/rGel: a strategy for optimizing tumor-vasculature targeting. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5611. doi:10.1158/1538-7445.AM2013-5611

Photochemical internalization (PCI) of immunotoxins targeting CD133 is specific and highly potent at femtomolar levels in cells with cancer stem cell properties

CD133 is a putative cancer stem cell (CSC) marker for a number of different cancers and is suggested to be a therapeutic target. Since also normal stem cells express CD133 it is of paramount importance that targeting strategies provide a specific and efficient delivery of cytotoxic drugs in only CD133-positive CSCs. In this study, we have employed photochemical internalization (PCI), a minimally invasive method for light-controlled, specific delivery of membrane-impermeable macromolecules from endocytic vesicles to the cytosol, to specifically target CD133-positive cancer cells. We demonstrate that PCI increases the cytotoxic effect of an immunotoxin (IT) targeting CD133-expressing cancer cells of colon (WiDr and HCT116) and pancreas (BxPC-3) origin. The IT consisted of the mAb CD133/1 (AC133) bound to the ribosome inactivating plant toxin saporin (anti-CD133/1-sap). We show that TPCS2a-PCI of anti-CD133/1-sap is specific, and highly cytotoxic at femto-molar concentrations. Specific binding and uptake of CD133/1, was shown by fluorescence microscopy and co-localization with TPCS2a in endosomes/lysosomes was determined by confocal microscopy. CD133high WiDr cells, isolated by fluorescence activated cell sorting, had a 7-fold higher capacity to initiate spheroids than CD133low cells (P<0.001) and were resistant to photodynamic therapy (PDT). However, PDT-resistance was bypassed by the PCI strategy. Tumor initiation and aggressive growth in athymic nude mice was obtained with only 10 CD133high cells in contrast to CD133low cells where substantially higher cell numbers were needed.The excellent high efficacy and selectivity of eliminating CD133-expressing cells by PCI warrant further pre-clinical evaluations of this novel therapeutic approach.

591 THE EFFECT OF PHOTOCHEMICAL INTERNALIZATION OF BLEOMYCIN IN THE TREATMENT OF UROTHELIAL CARCINOMA: AN IN VITRO STUDY

You have accessJournal of UrologyBladder Cancer: Basic Research (I)1 Apr 2013591 THE EFFECT OF PHOTOCHEMICAL INTERNALIZATION OF BLEOMYCIN IN THE TREATMENT OF UROTHELIAL CARCINOMA: AN IN VITRO STUDY Harm C. Arentsen, Jos Falke, Anders Høgset, Egbert Oosterwijk, and J. Alfred Witjes Harm C. ArentsenHarm C. Arentsen Nijmegen, Netherlands More articles by this author , Jos FalkeJos Falke Nijmegen, Netherlands More articles by this author , Anders HøgsetAnders Høgset Oslo, Norway More articles by this author , Egbert OosterwijkEgbert Oosterwijk Nijmegen, Netherlands More articles by this author , and J. Alfred WitjesJ. Alfred Witjes Nijmegen, Netherlands More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1987AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Photochemical internalization (PCI) is a novel light-directed drug delivery system. It triggers the release of endocytosed drugs into the cytosol. Thus the drugs can reach their intracellular target of action, realizing their therapeutic potential, instead of being degraded by lysosomal hydrolases. The PCI effect is achieved with photosensitizing compounds specifically localized in the membranes of endocytic vesicles, destroying these membranes by an oxidative process after illumination. Bleomycin is used in multiple standard cancer chemotherapy regimens. The relatively large and hydrophilic chemical structure limits its ability to penetrate membranes, which causes accumulation in endocytic vesicles. PCI of bleomycin can induce a synergistic inhibition of tumor growth in different animal tumor models. In this in vitro study we determined whether meso-tetraphenyl chlorin disulphonate (TPCS2a)-based photochemical delivery of bleomycin was able to potentiate the cytotoxicity of bleomycin on bladder cancer cell lines. Epirubicin, gemcitabine and mitomycin C were used as controls. METHODS The human RT4, RT112, 253J, T24 and rat AY-27 urothelial carcinoma cell lines were used in this study. Cells were seeded in 96-well plates. TPCS2a was added to the growth medium and the plates were incubated overnight. Cells were then resuspended in TPCS2a-free culture medium and incubated for 3 h. Subsequently, cells were treated for 60 min with increasing doses of epirubicin, gemcitabine, mitomycin C or bleomycin followed by illumination for different periods (0-5 min) to obtain dose-response curves. Cell viability was measured with a colorimetric assay after 72 hours. RESULTS For the single treatments, in all five cell lines a dose dependent inhibition of cell proliferation was observed. This was seen both after treatment with TPCS2a-based PDT alone as well as after treatment with either bleomycin or one of the control chemotherapeutic agents alone. For the combination treatments, a significant (p<0,001) synergistic effect, i.e. photochemical internalization effect, was only observed for PDT combined with bleomycin in the human T24 cell line and the rat AY-27 cell line. CONCLUSIONS TPCS2a-based photochemical internalization of bleomycin showed a significant synergistic antiproliferative activity against human and rat urothelial carcinoma cells in vitro. Thus, PCI may have therapeutic potential as an intravesical strategy against NMIBC. Since the effect is heterogeneous, further animal studies to investigate the in vivo effects seem justified. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e242 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Harm C. Arentsen Nijmegen, Netherlands More articles by this author Jos Falke Nijmegen, Netherlands More articles by this author Anders Høgset Oslo, Norway More articles by this author Egbert Oosterwijk Nijmegen, Netherlands More articles by this author J. Alfred Witjes Nijmegen, Netherlands More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Light-Controlled Modulation of Gene Expression Using Polyamidoamine Formulations

A promising method that offers both time- and site-specific delivery of macromolecules is photochemical internalization technology (PCI). Here, we have characterized various polyamidoamine (PAMAM) carriers [generation (G) 0-7], for light-directed delivery of nucleic acids in vitro by the use of PCI technology. A number of parameters for optimal delivery of nucleic acids into human cancer cells, that is, various light-doses, carrier-doses, and small interfering RNA (siRNA)/messenger RNA (mRNA) doses were investigated for either up- or down-regulation of enhanced green fluorescent protein (EGFP) gene expression. In summary, our results showed in an osteosarcoma cell line (OHS) [EGFP] model system the possibility for efficient light-directed siRNA silencing (>80% silencing) when using PAMAM G3 to G7 as carriers. Surprisingly, no EGFP mRNA up-regulation was detected either with or without PCI after EGFP mRNA/PAMAM (G0-G7) transfection in standard OHS cells. We have here identified properties for PAMAM formulations enabling light-directed siRNA delivery with the aim of developing a site-specific strategy for delivery of nucleic acids in vivo.

Photophysical and photobiological properties of a sulfonated chlorin photosensitiser TPCS2a for photochemical internalisation (PCI)

This study investigated the photophysical and photobiological properties of a new amphiphilic chlorin photosensitiser, disulfonated tetraphenylchlorin (TPCS(2a)), for photochemical internalisation (PCI). The absorption and fluorescence spectra of TPCS(2a) were examined in a range of solvents together with fluorescence lifetime measurements. The fluorescence lifetime of TPCS(2a) was found to be 8.5 ns in methanol, whereas non-exponential decays were observed in distilled water due to sensitiser dimerisation. The singlet oxygen quantum yield of TPCS(2a) was determined as 0.62 in deuterated methanol by direct observation of singlet oxygen phosphorescence. In a human oral squamous carcinoma (HN5) cell line, intracellular co-localisation of TPCS(2a) and Alexa488-labelled saporin, a macromolecular toxin, was observed corresponding predominantly to a lysosomal distribution. Intracellular fluorescence redistribution of TPCS(2a) and Alexa488-saporin was observed after 405 nm irradiation. Using two-photon confocal microscopy at 840 nm, and fluorescence lifetime imaging (FLIM), the lifetime was measured as 6 ns in HN5 cells. PCI using TPCS(2a) was shown to be very effective, and a synergistic increase in saporin toxicity was achieved in HN5 cells where viability was significantly reduced after light exposure compared to saporin (25 nM) treatment alone. The results demonstrate the favourable photophysical and photobiological properties of TPCS(2a) for PCI, which induces the relocalisation of a macromolecular anti-cancer toxin inside cells and significantly enhances cell death.

153 The effect of photochemical internalization of bleomycin in the treatment of urothelial carcinoma of the bladder: An in vitro study

153 The effect of photochemical internalization of bleomycin in the treatment of urothelial carcinoma of the bladder: An in vitro study