ABSTRACT
Photochemical internalization (PCI) is a modified form of photodynamic therapy (PDT) that enhances the efficacy of therapeutic agents in a site and temporal specific manner in both in vitro and in vivo publications. The purpose of the study reported here was to evaluate the benefits of a modified PCI protocol in a 3D rat glioma spheroid model. In the modified protocol, F98 glioma cells were incubated with photosensitizer (AlPcS2a) prior to spheroid generation, as opposed to post-spheroid formation photosensitizer exposure commonly used in conventional protocols. The efficacy of both bleomycin and doxorubicin PCI was evaluated using either the conventional or modified protocols. The formed spheroids were then exposed to light treatment from a diode laser, λ= 670 nm. Spheroid growth was monitored for a period of 14 days. The results of spheroid growth assays showed that there was no statistically significant difference in PCI efficacy between the conventional and modified protocols for both of the drugs tested. The direct PDT effect was significantly reduced using the modified protocol. Therefore, due to its several advantages, the modified protocol is recommended for evaluating the efficacy of PCI in tumor spheroid models.
Subject(s)
Glioma , Photochemotherapy , Animals , Bleomycin/therapeutic use , Cell Line, Tumor , Glioma/drug therapy , Photosensitizing Agents/pharmacology , Photosensitizing Agents/therapeutic use , RatsABSTRACT
Fibrin glue (FG) has potential as a delivery vehicle for photosensitizer directly to the resection cavity, so it may bypass the blood-brain barrier (BBB) and increase the concentration of successfully delivered photosensitizer. A specialized form of photodynamic therapy (PDT), photochemical internalization (PCI), which involves both photosensitizer and chemotherapeutic agent internalization, can locally inhibit the growth of cells. This will allow the reduction of recurrence of malignant gliomas around surgical resection. This study will look at the efficacy of FG loaded with drugs in mediating both PDT and PCI in inhibiting 3-dimensional tumor spheroid growth in vitro. Experiments were conducted on spheroids comprised of F98 glioma cells using photosensitizer AlPcS2a and chemotherapeutic drug bleomycin (BLM). At 2-, 24-, 48-, and 72-h increments, supernatant covering an FG layer within a well was collected and replaced by fresh medium, then added to spheroid-containing wells, which contained the respective chemicals for PDT and PCI. The wells were then exposed to light treatment from a diode laser, and after, spheroid growth was monitored for a period of 14 days. Significant spheroid growth inhibition was observed in both PDT and PCI modalities, but was far greater in PCI. Additionally, complete growth suppression was achieved via PCI at the highest radiant exposure. Achieving a slow photosensitizer release, significant F98 spheroid inhibition was observed in FG-mediated PDT and PCI. The present study showed BLM-PCI was the most efficacious of the two modalities.
Subject(s)
Blood-Brain Barrier/metabolism , Drug Carriers/chemistry , Fibrin Tissue Adhesive/chemistry , Photosensitizing Agents/chemistry , Photosensitizing Agents/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Biological Transport , Bleomycin/chemistry , Bleomycin/metabolism , Bleomycin/pharmacology , Blood-Brain Barrier/drug effects , Cell Line, Tumor , Humans , Indoles/chemistry , Indoles/metabolism , Indoles/pharmacology , Lasers, Semiconductor , Organometallic Compounds/chemistry , Organometallic Compounds/metabolism , Organometallic Compounds/pharmacology , Photosensitizing Agents/pharmacologyABSTRACT
Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. The procedure involves administration of a photosensitizing agent followed by irradiation at a wavelength corresponding to an absorbance band of the sensitizer. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature, and induction of a local inflammatory reaction. Clinical studies revealed that PDT can be curative, particularly in early stage tumors. It can prolong survival in patients with inoperable cancers and significantly improve quality of life. Minimal normal tissue toxicity, negligible systemic effects, greatly reduced long-term morbidity, lack of intrinsic or acquired resistance mechanisms, and excellent cosmetic as well as organ function-sparing effects of this treatment make it a valuable therapeutic option for combination treatments. With a number of recent technological improvements, PDT has the potential to become integrated into the mainstream of cancer treatment.
Subject(s)
Neoplasms/drug therapy , Photochemotherapy , Humans , Photochemotherapy/instrumentation , Photochemotherapy/methods , Photosensitizing Agents/therapeutic useABSTRACT
Light-based treatment modalities such as photothermal therapy (PTT) or photochemical internalization (PCI) have been well documented both experimentally and clinically to enhance the efficacy of chemotherapy. The main purpose of this study was to examine the cytotoxic effects of silica-gold nanoshell (AuNS)-loaded macrophage-mediated (MaNS) PTT and bleomycin BLM-PCI on monolayers of squamous cell carcinoma cells. The two modalities were applied separately and in simultaneous combination. Two different wavelengths of light were employed simultaneously, one to activate a highly efficient PCI photosensitizer, AlPcS2a (670 nm) and the other for the MaNS-mediated PTT (810 nm), to evaluate the combined effects of these modalities. The results clearly demonstrated that macrophages could ingest sufficient numbers of silica-gold nanoshells for efficient near infrared (NIR) activated PTT. A significant synergistic effect of simultaneously applied combined PTT and PCI, compared to each modality applied separately, was achieved. Light-driven therapies have the advantage of site specificity, non-invasive and non-toxic application, require inexpensive equipment and can be given as repetitive treatment protocols.
Subject(s)
Hyperthermia, Induced , Macrophages/metabolism , Phototherapy , Animals , Bleomycin , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/therapy , Cell Line, Tumor , Endocytosis , Gold/chemistry , Humans , Nanoshells/chemistry , Photosensitizing Agents/pharmacology , Polyethylene Glycols/chemistry , RatsABSTRACT
Four amphiphilic covalently linked meso-tetraphenylchlorin-chitosan nanoconjugates were synthesized and evaluated for use in photochemical internalization (PCI) in vitro and in vivo. The synthetic protocol for the preparation of two different hydrophobic chlorin photosensitizers, 5-(4-aminophenyl)-10,15,20-triphenylchlorin and 5-(4-carboxyphenyl)-10,15,20-triphenylchlorin, was optimized. These monofunctional photosensitizers were covalently attached to carrier chitosan via silyl-protected 3,6-di-O-tert-butyldimethylsilyl-chitosan (Di-TBDMS-chitosan) with 0.10 degree of substitution per glucosamine (DS). Hydrophilic moieties such as trimethylamine and/or 1-methylpiperazine were incorporated with 0.9 DS to give fully water-soluble conjugates after removal of the TBDMS groups. A dynamic light scattering (DLS) study confirmed the formation of nanoparticles with a 140-200 nm diameter. These nanoconjugates could be activated at 650 nm (red region) light, with a fluorescence quantum yield (ΦF) of 0.43-0.45, and are thus suitable candidates for use in PCI. These nanoconjugates were taken up and localized in the endocytic vesicles of HCT116/LUC human colon carcinoma cells, and upon illumination they substantially enhanced plasmid DNA transfection. The nanoconjugates were also evaluated in preliminary in vivo experiments in tumor-bearing mice, showing that the nanoconjugates could induce a strong photodynamic therapy (PDT) and also PCI effects in treatment with bleomycin.
Subject(s)
Chitosan/chemistry , Endosomes/drug effects , Nanoconjugates/chemistry , Photosensitizing Agents/chemistry , Animals , Bleomycin , Female , HCT116 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Spectroscopy , Mice , Mice, Nude , Photochemistry , Piperazines/chemistry , Polymers/chemistry , Porphyrins/chemistry , Spectroscopy, Fourier Transform Infrared , Transfection , Transport Vesicles/drug effects , Transport Vesicles/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The possibility of using photochemical internalization (PCI) to enhance the effects of the cytotoxic drug bleomycin is investigated, together with photophysical determination and outlines of a possible treatment for intravesical therapy of bladder cancer. In vitro experiments indicated that the employment of PCI technology using the novel photosensitizer TPCS2a® can enhance the cytotoxic effect of bleomycin in bladder cancer cells. Furthermore, experiments in an orthotopic in vivo bladder cancer model show an effective reduction in both the necrotic area and the bladder weight after TPCS2a based photodynamic therapy (PDT). The tumor selectivity and PDT effects may be sufficient to destroy tumors without damaging the detrusor muscle layer. Our results present a possible new treatment strategy for non-muscle invasive bladder cancer, with the intravesical instillation of the photosensitizer and bleomycin followed by illumination through an optic fiber by using a catheter.
Subject(s)
Antineoplastic Agents/pharmacology , Bleomycin/pharmacology , Disease Models, Animal , Light , Photosensitizing Agents/pharmacology , Urinary Bladder Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemistry , Bleomycin/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Female , Photochemical Processes , Photosensitizing Agents/chemistry , Rats , Rats, Inbred F344 , Tumor Cells, Cultured , Urinary Bladder Neoplasms/pathologyABSTRACT
BACKGROUND: Photochemical internalisation, a novel minimally invasive treatment, has shown promising preclinical results in enhancing and site-directing the effect of anticancer drugs by illumination, which initiates localised chemotherapy release. We assessed the safety and tolerability of a newly developed photosensitiser, disulfonated tetraphenyl chlorin (TPCS2a), in mediating photochemical internalisation of bleomycin in patients with advanced and recurrent solid malignancies. METHODS: In this phase 1, dose-escalation, first-in-man trial, we recruited patients (aged ≥18 to <85 years) with local recurrent, advanced, or metastatic cutaneous or subcutaneous malignancies who were clinically assessed as eligible for bleomycin chemotherapy from a single centre in the UK. Patients were given TPCS2a on day 0 by slow intravenous injection, followed by a fixed dose of 15â000 IU/m(2) bleomycin by intravenous infusion on day 4. After 3 h, the surface of the target tumour was illuminated with 652 nm laser light (fixed at 60 J/cm(2)). The TPCS2a starting dose was 0·25 mg/kg and was then escalated in successive dose cohorts of three patients (0·5, 1·0, and 1·5 mg/kg). The primary endpoints were safety and tolerability of TPCS2a; other co-primary endpoints were dose-limiting toxicity and maximum tolerated dose. The primary analysis was per protocol. This study is registered with ClinicalTrials.gov, number NCT00993512, and has been completed. FINDINGS: Between Oct 3, 2009, and Jan 14, 2014, we recruited 22 patients into the trial. 12 patients completed the 3-month follow-up period. Adverse events related to photochemical internalisation were either local, resulting from the local inflammatory process, or systemic, mostly as a result of the skin-photosensitising effect of TPCS2a. The most common grade 3 or worse adverse events were unexpected higher transient pain response (grade 3) localised to the treatment site recorded in nine patients, and respiratory failure (grade 4) noted in two patients. One dose-limiting toxicity was reported in the 1·0 mg/kg cohort (skin photosensitivity [grade 2]). Dose-limiting toxicities were reported in two of three patients at a TPCS2a dose of 1·5 mg/kg (skin photosensitivity [grade 3] and wound infection [grade 3]); thus, the maximum tolerated dose of TPCS2a was 1·0 mg/kg. Administration of TPCS2a was found to be safe and tolerable by all patients. No deaths related to photochemical internalisation treatment occurred. INTERPRETATION: TPCS2a-mediated photochemical internalisation of bleomycin is safe and tolerable. We identified TPCS2a 0·25 mg/kg as the recommended treatment dose for future trials. FUNDING: PCI Biotech.
Subject(s)
Antineoplastic Agents/therapeutic use , Bleomycin/therapeutic use , Neoplasm Recurrence, Local/drug therapy , Neoplasms/drug therapy , Photosensitizing Agents/chemistry , Porphyrins/chemistry , Adult , Aged , Aged, 80 and over , Dose-Response Relationship, Drug , Female , Follow-Up Studies , Humans , Light , Male , Maximum Tolerated Dose , Middle Aged , Neoplasm Metastasis , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Neoplasms/pathology , Photosensitizing Agents/pharmacokinetics , Porphyrins/pharmacokinetics , Prognosis , Tissue DistributionABSTRACT
Here we evaluate the photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a) in survival studies of rat glioma cancer cells in combination with the novel photochemical internalization (PCI) technique. The tested anticancer drugs were bleomycin (BLM) and temozolomide (TMZ). Glioma cells were incubated with TPCS2a (0.2 µg ml(-1), 18 h, 37 °C) before BLM or TMZ stimulation (4 h) prior to red light illumination (652 nm, 50 mW cm(-2)). The cell survival after BLM (0.5 µm)-PCI (40 s light) quantified using the MTT assay was reduced to about 25% after 24 h relative to controls, and to 31% after TMZ-PCI. The supplementing quantification by clonogenic assays, using BLM (0.1 µm), indicated a long-term cytotoxic effect: the surviving fraction of clonogenic cells was reduced to 5% after light exposure (80 s) with PCI, compared to 70% in the case of PDT. In parallel, structural and morphological changes within the cells upon light treatment were examined using fluorescence microscopy techniques. The present study demonstrates that PCI of BLM is an effective method for killing F98 glioma cells, but smaller effects were observed using TMZ following the "light after" strategy. The results are the basis for further in vivo studies on our rat glioma cancer model using PDT and PCI.
Subject(s)
Antineoplastic Agents/metabolism , Bleomycin/metabolism , Dacarbazine/analogs & derivatives , Glioma/metabolism , Photochemical Processes , Animals , Antineoplastic Agents/chemistry , Bleomycin/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Dacarbazine/chemistry , Dacarbazine/metabolism , Light , Molecular Structure , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Rats , Temozolomide , Tumor Stem Cell AssayABSTRACT
Resistance to chemotherapy, molecular targeted therapy as well as radiation therapy is a major obstacle for cancer treatment. Cancer resistance may be exerted through multiple different mechanisms which may be orchestrated as observed in multidrug resistance (MDR). Cancer resistance may be intrinsic or acquired and often leaves patients without any treatment options. Strategies for alternative treatment modalities for resistant cancer are therefore highly warranted. Photochemical internalization (PCI) is a technology for cytosolic delivery of macromolecular therapeutics based on the principles of photodynamic therapy (PDT). The present report reviews the current knowledge of PCI of therapy-resistant cancers. In summary, PCI may be able to circumvent several of the major mechanisms associated with resistance towards chemotherapeutics including increased expression of drug efflux pumps, altered intracellular drug distribution and increased ROS scavenging. Current data also suggest PCI of targeted toxins as highly effective in cancers resistant to clinically available targeted therapy such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs). PCI may therefore, in general, represent a future treatment option for cancers resistant to other therapies.
Subject(s)
Drug Resistance, Neoplasm , Neoplasms/drug therapy , Photochemotherapy/methods , Photosensitizing Agents/administration & dosage , Humans , Neoplasms/physiopathologyABSTRACT
Despite progress in radio-, chemo- and photodynamic-therapy (PDT) of cancer, treatment resistance still remains a major problem for patients with aggressive tumours. Cancer stem cells (CSCs) or tumour-initiating cells are intrinsically and notoriously resistant to conventional cancer therapies and are proposed to be responsible for the recurrence of tumours after therapy. According to the CSC hypothesis, it is imperative to develop novel anticancer agents or therapeutic strategies that take into account the biology and role of CSCs. The present review outlines our recent study on photochemical internalisation (PCI) using the clinically relevant photosensitiser TPCS2a/Amphinex® as a rational, non-invasive strategy for the light-controlled endosomal escape of CSC-targeting drugs. PCI is an intracellular drug delivery method based on light-induced ROS-generation and a subsequent membrane-disruption of endocytic vesicles, leading to cytosolic release of the entrapped drugs of interest. In different proof-of-concept studies we have demonstrated that PCI of CSC-directed immunotoxins targeting CD133, CD44, CSPG4 and EpCAM is a highly specific and effective strategy for killing cancer cells and CSCs. CSCs overexpressing CD133 are PDT-resistant; however, this is circumvented by PCI of CD133-targeting immunotoxins. In view of the fact that TPCS2a is not a substrate of the efflux pumps ABCG2 and P-glycoprotein (ABCB1), the PCI-method is a promising anti-CSC therapeutic strategy. Due to a laser-controlled exposure, PCI of CSC-targeting drugs will be confined exclusively to the tumour tissue, suggesting that this drug delivery method has the potential to spare distant normal stem cells.
Subject(s)
Endosomes/drug effects , Endosomes/radiation effects , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/radiation effects , Photochemotherapy/methods , Animals , Drug Delivery Systems , Endosomes/physiology , Humans , Neoplastic Stem Cells/physiology , Photosensitizing Agents/administration & dosage , Reactive Oxygen Species/metabolismABSTRACT
Photochemical internalization (PCI) has shown great promise as a therapeutic alternative for targeted drug delivery by light-harnessed activation. However, it has only been applicable to therapeutic macromolecules or medium-sized molecules. Herein we describe the use of an amphiphilic, water-soluble porphyrin-ß-cyclodextrin conjugate (mTHPP-ßCD) as a "Trojan horse" to facilitate the endocytosis of CD-guest tamoxifens into breast-cancer cells. Upon irradiation, the porphyrin core of mTHPP-ßCD expedited endosomal membrane rupture and tamoxifen release into the cytosol, as documented by confocal microscopy. The sustained complexation of mTHPP-ßCD with tamoxifen was corroborated by 2D NMR spectroscopy and FRET studies. Following the application of PCI protocols with 4-hydroxytamoxifen (4-OHT), estrogen-receptor ß-positive (Erß+, but not ERß-) cell groups exhibited extensive cytotoxicity and/or growth suspension even at 72â h after irradiation.
Subject(s)
Drug Carriers/chemistry , Nanoconjugates/chemistry , Tamoxifen/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Fluorescence Resonance Energy Transfer , Humans , Light , MCF-7 Cells , Magnetic Resonance Spectroscopy , Microscopy, Confocal , Porphyrins/chemistry , Tamoxifen/toxicity , beta-Cyclodextrins/chemistryABSTRACT
BACKGROUND: Tyrosin kinase inhibitors (TKIs) and monoclonal antibodies aimed to target epidermal growth factor receptor (EGFR) have shown limited effect as monotherapies and drug resistance is a major limitation for therapeutic success. Adjuvant therapies to EGFR targeting therapeutics are therefore of high clinical relevance. METHODS: Three EGFR targeting drugs, Cetuximab, Erlotinib and Tyrphostin AG1478 were used in combination with photodynamic therapy (PDT) in two EGFR positive cell lines, A-431 epidermoid skin carcinoma and WiDr colorectal adenocarcinoma cells. The amphiphilic meso-tetraphenylporphine with 2 sulphonate groups on adjacent phenyl rings (TPPS(2a)) was utilized as a photosensitizer for PDT. The cytotoxic outcome of the combined treatments was evaluated by cell counting and MTT. Cellular signalling was explored by Western blotting. RESULTS: PDT as neoadjuvant to Tyrphostin in A-431 cells as well as to Tyrphostin or Erlotinib in WiDr cells revealed synergistic cytotoxicity. In contrast, Erlotinib or Cetuximab combined with neoadjuvant PDT induced an antagonistic effect on cell survival of A-431 cells. Neoadjuvant PDT and EGFR targeting therapies induced a synergistic inhibition of ERK as well as synergistic cytotoxicity only when the EGFR targeting monotherapies caused a prolonged ERK inhibition. There were no correlation between EGFR inhibition by the EGFR targeting monotherapies or the combined therapies and the cytotoxic outcome combination-therapies. CONCLUSIONS: The results suggest that sustained ERK inhibition by EGFR targeting monotherapies is a predictive factor for synergistic cytotoxicity when combined with neoadjuvant PDT. GENERAL SIGNIFICANCE: The present study provides a rationale for selecting anticancer drugs which may benefit from PDT as adjuvant therapy.
Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/genetics , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Photosensitizing Agents/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Cell Count , Cell Line, Tumor/drug effects , Cell Survival/drug effects , Cetuximab , Drug Synergism , ErbB Receptors/metabolism , Erlotinib Hydrochloride , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/drug effects , Humans , Molecular Targeted Therapy , Neoadjuvant Therapy , Photochemotherapy , Porphyrins/pharmacology , Quinazolines/pharmacology , Signal Transduction/drug effects , Tyrphostins/pharmacologyABSTRACT
BACKGROUND: The 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. METHODS: We have used a sarcoma model, SW872 cells isolated from xenografts harboring CSCs within a ~2% CD133(high) 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. RESULTS: PCI 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. CONCLUSION: Here 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 SIGNIFICANCE: PCI 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.
Subject(s)
Glycoproteins/antagonists & inhibitors , Immunotoxins/administration & dosage , Neoplastic Stem Cells/drug effects , Peptides/antagonists & inhibitors , Photosensitizing Agents/administration & dosage , Sarcoma/drug therapy , AC133 Antigen , Animals , Antigens, CD/immunology , Cell Line, Tumor , Drug Delivery Systems , Glycoproteins/immunology , Humans , Mice , Mice, SCID , Peptides/immunology , Photochemistry , Sarcoma/pathology , Xenograft Model Antitumor AssaysABSTRACT
We have used the site specific and light-depended drug delivery method photochemical internalization (PCI) to release an immunotoxin (IT), targeting the CD44 receptor, into the cytosol of target cells. The IT consisted of a pan CD44 mAb (clone IM7) bound to the ribosome inactivating protein (RIP) saporin by a biotin-streptavidin linker named IM7-saporin. PCI is based upon photosensitizing compounds localized in the membrane of endosomes and lysosomes causing membrane rupture upon illumination followed by release of the IT into the cytosol. In this in vitro study, we have used 7 different human cancer cell lines of various origins to investigate the cytotoxic effect of PCI-based targeting of the cancer stem cell (CSC) marker CD44. Epi-fluorescence microscopy shows both specific binding and uptake of the IM7-Alexa488, after 30 min and 18 h of incubation, and colocalization with the PCI-photosensitizer TPCS2a prior to light-triggered cytosolic release of the CD44-targeting IT. PCI of IM7-saporin resulted in efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells. A higher level of reactive oxygen species (ROS) was found in untreated and photodynamic therapy (PDT)-treated LNCaP (CD44(neg)) compared to that of DU145 (CD44(pos)) prostate cancer (PC) cells. This may explain the PDT-resistance observed in the DU145 cells. PCI-based targeting of CD44-expressing cancer cells gives very potent and specific cytotoxic effects and may represent a rational strategy for achieving site-selective elimination of CSCs in aggressive androgen-independent and treatment-resistant PC cells preventing cytotoxic effects on distant normal stem cells.
Subject(s)
Hyaluronan Receptors/metabolism , Immunotoxins/chemistry , Neoplastic Stem Cells/drug effects , Ribosome Inactivating Proteins, Type 1/chemistry , Antibodies, Monoclonal/chemistry , Biotin/chemistry , Cell Line, Tumor , Cytosol/metabolism , Drug Carriers/chemistry , Endosomes/metabolism , Flow Cytometry , Humans , Light , Lysosomes/metabolism , Photochemotherapy/methods , Photosensitizing Agents/chemistry , Reactive Oxygen Species/metabolism , Saporins , Sensitivity and Specificity , Streptavidin/chemistry , Time FactorsABSTRACT
Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80% of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments.PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect.
Subject(s)
Antifungal Agents/pharmacology , Cytosine Deaminase/genetics , Cytosine Deaminase/metabolism , Flucytosine/pharmacology , Cell Line, Tumor/drug effects , Dose-Response Relationship, Drug , Glioma/pathology , Humans , Indoles/pharmacology , Organometallic Compounds/pharmacology , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Photochemical Processes/drug effects , Photosensitizing Agents/pharmacology , TransfectionABSTRACT
Many photosensitizers (PSs) for use in photodynamic therapy (PDT) are characterized by poor solubility and a tendency to aggregate in aqueous environments. Nanovehicles of Pluronics block copolymers may be used for drug delivery of antineoplastic agents and may also exert a separate effect in enhancing drug efficiency. The objective of this study was to determine the effects of selected Pluronics (F127, P123, L44 and F68) on the dark cytotoxicity, photocytotoxicity and localization of four model photosensitizers, tetraphenyl porphyrins 4-substituted on the phenyl groups with trimethylamine (TAPP), hydroxyl (THPP), sulfonate (TSPP) and carboxyl (TCPP) in cancer cells. The selected PSs showed a 3 log range in sensitivity to cellular photoinactivation. Pluronics were found to efficiently deaggregate the PSs and improve PS solubility as analyzed by fluorescence spectroscopy and dynamic light scattering. The Pluronics had moderate to profound effects on intracellular localization of the PSs and cellular sensitivity to photoinactivation. Confocal microscopy was used to determine the localization of PSs in colon adenocarcinoma cell line (WiDr), guided by co-staining with nuclear (Hoechst 33342) and endolysosomal (LysoTracker Green DND® 26 and Dextran Alexa Fluor® 488) markers. Of the most significant effects P123 and F127 strongly attenuated the uptake and photocytotoxicity of THPP and redirected the cellular uptake to endocytosis. P123 stimulated translocation of TAPP from endocytic vesicles to a cytosolic and nuclear localization followed by an enhanced phototoxicity. In the absence of Pluronics TCPP was found to localize partly in endocytic vesicles and partly in the cytosol and nucleus, while P123 and F127 lowered the fraction in endocytic vesicles followed by a reduced sensitivity to photoinactivation. F68 had only moderate effects on intracellular localization of the evaluated PSs with the exception of a higher endocytic accumulation of TCPP and lowered photocytotoxicity of TCPP and THPP. In conclusion, Pluronics are attractive solubilizers of porphyrin-based PSs which have in many cases substantial effects on intracellular localization and efficacy of the PSs.
Subject(s)
Cell Nucleus/drug effects , Drug Carriers/pharmacology , Endosomes/drug effects , Lysosomes/drug effects , Photosensitizing Agents/pharmacology , Poloxamer/pharmacology , Porphyrins/pharmacology , Amines , Benzimidazoles , Biological Transport , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival/drug effects , Dose-Response Relationship, Drug , Endosomes/metabolism , Female , Fluorescent Dyes , Humans , Light , Lysosomes/metabolism , Male , Microscopy, Confocal , Photochemical Processes , Photosensitizing Agents/chemical synthesis , Porphyrins/chemical synthesis , Spectrometry, FluorescenceABSTRACT
BACKGROUND: Photochemical internalization (PCI) is a modality for cytosolic release of drugs trapped in endocytic vesicles. The method is based upon photosensitizers localized in the membranes of endocytic vesicles which create membrane rupture upon light exposure by generating reactive oxygen species (ROS), predominantly singlet oxygen ((1)O(2)). METHODS: The human epidermal growth factor receptor 2 (HER2)-targeted immunotoxin (IT), trastuzumab-saporin, was evaluated in combination with PCI using TPCS(2a) (Amphinex®), a new photosensitizer approved for clinical use. RESULTS: PCI synergistically enhanced the cytotoxicity of trastuzumab-saporin on trastuzumab-resistant HER2(+) Zr-75-1 cells. The PCI effect was only observed when the IT was administered prior to the photochemical treatment ("light after" strategy), while administration of a non-targeted drug may equally well be performed after light exposure. Mechanistic studies showed reduced ligand-induced HER2 phosphorylation and receptor-mediated endocytosis after TPCS(2a)-PDT. Photochemical disruption of the cytoplasmic domain of HER2 was found to be induced by (1)O(2) generated both by photosensitizer located in the endocytic vesicles and in the outer leaflet of the plasma membrane. CONCLUSIONS: Administration of the HER2-targeted toxin prior to light exposure is a prerequisite for successful PCI-mediated delivery of HER2-targeted toxins. GENERAL SIGNIFICANCE: PCI of HER2-targeted toxins is demonstrated as a highly effective treatment modality which may overcome trastuzumab resistance. The mechanistic studies of the lack of PCI effect of the "light first" procedure is of outermost importance when designing a clinical PCI treatment protocol for delivery of HER2-targeted therapies.
Subject(s)
Breast Neoplasms/drug therapy , Endocytosis/drug effects , Immunotoxins/pharmacology , Photochemotherapy , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Ribosome Inactivating Proteins, Type 1/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Blotting, Western , Breast Neoplasms/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Female , Humans , Light , Phosphorylation/drug effects , Receptor, ErbB-2/metabolism , Saporins , Trastuzumab , Tumor Cells, CulturedABSTRACT
In order to apply curcumin as a photosensitizer in photodynamic therapy (PDT) one needs a formulation that can solubilize and stabilize the compound. Pluronics® (Pluronic) are reported to both solubilize and stabilize curcumin against hydrolytic degradation. The aim of the present work was therefore to investigate the influence of Pluronic formulation on the photocytotoxicity of curcumin. Interactions between curcumin and Pluronics were investigated by fluorescence emission and absorption spectroscopy. Cell survival was measured with the MTT assay. The location of curcumin in the cells was investigated with fluorescence microscopy, and the cellular uptake was measured with fluorescence emission spectroscopy. Pluronics P123 and F127 in contrast to Pluronic P85 and PEG 400 may solubilize curcumin under non-cytotoxic conditions. An inverse relationship between the concentration of Pluronic and the photocytotoxicity of curcumin was observed. Curcumin could rapidly translocate across the cell membrane by passive diffusion. The fluorescence from curcumin in the cells (in the cytoplasm) after 1 hour of incubation was lowered by the presence of Pluronics in the formulation. However, the absolute amount of cell-bound curcumin after 1 hour of incubation was independent of the presence of Pluronics. Curcumin was bound more strongly to cells when incubated with formulations without Pluronics compared to cells incubated with curcumin formulations with Pluronics. Incubation of WiDr cells with curcumin for 6 hours resulted in lysosomal accumulation of curcumin independent of the presence of Pluronics. Lysosomally located curcumin could not be observed in HT1080 cells after 6 hours of incubation. The Pluronics P123 and F127 were found to be suitable for solubilizing and stabilizing curcumin, but inhibited photocytotoxic effects of curcumin unless the Pluronic concentration during treatment of the cells was less than 5-10× above the critical micellar concentration.
Subject(s)
Curcumin/metabolism , Curcumin/pharmacology , Darkness/adverse effects , Photosensitizing Agents/metabolism , Photosensitizing Agents/pharmacology , Poloxamer/chemistry , Biological Transport , Cell Line, Tumor , Chemistry, Pharmaceutical , Curcumin/chemistry , Curcumin/toxicity , Drug Stability , Humans , Intracellular Space/metabolism , Photosensitizing Agents/chemistry , Photosensitizing Agents/toxicity , Polyethylene Glycols/chemistry , Solvents/chemistry , Spectrometry, FluorescenceABSTRACT
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.
Subject(s)
Photochemical Processes , Photosensitizing Agents/chemistry , Photosensitizing Agents/metabolism , Porphyrins/chemistry , Porphyrins/metabolism , Biological Transport , Cell Line, Tumor , Head and Neck Neoplasms/pathology , Humans , Ribosome Inactivating Proteins, Type 1/metabolism , Saporins , Singlet Oxygen/chemistry , Spectrometry, FluorescenceABSTRACT
Kohler [Einführung in die Phonetik des Deutschen (Erich Schmidt Verlag, Berlin, Germany, 1995)] stated that German [É] and [a] in unstressed syllables are merging. The present study tested this hypothesis. The contrast was found intact word-internally and word-finally. Neighborhood density enhanced its phonetic characteristics, but no effects of frequency and conditional probability were found.