Benzoporphyrin Derivative Monoacid Ring Research Articles

Self-aggregation of the proteolytic forms of Verteporfin: An in silico and in vitro study

Verteporfin is a benzoporphyrin derivative monoacid ring A that has been historically and successfully used as a photosensitizer in clinical applications and in clinical trials including for cancer phototherapy. However, the photo-efficiency of Verteporfin is limited by self-aggregation in water, in which the various proteolytic forms present distinct self-aggregation tendencies. In this work, spectroscopic and computational studies were used to elucidate the self-aggregation behavior of the three proteolytic forms of Verteporfin. Additionally, the intracellular self-aggregation of Verteporfin was evaluated in U87MG cells line, a type of brain cancer considered incurable. The results indicated that the self-aggregation of Verteporfin in water is most likely modulated by formal charge effects following the order: neutral > monoanionic > dicationic. UV–vis electronic spectroscopy and computational dynamics analyses suggested that J-type aggregates were primarily formed. Time-resolved fluorescence and fluorescence lifetime imaging microscopy (FLIM) confirmed that although such aggregates exist in solution and inside cells, there is no evidence of preferential self-aggregation in acidic organelles, such as lysosomes. The present approach allows a better understanding of the self-aggregation of other photosensitizers, particularly the influence of different proteolytic forms and organelles on the self-aggregation of compounds that can be protonated, and the heterogeneity of photosensitizers within intracellular compartments.

Photoproducts of the Photodynamic Therapy Agent Verteporfin Identified via Laser Interfaced Mass Spectrometry.

Verteporfin, a free base benzoporphyrin derivative monoacid ring A, is a photosensitizing drug for photodynamic therapy (PDT) used in the treatment of the wet form of macular degeneration and activated by red light of 689 nm. Here, we present the first direct study of its photofragmentation channels in the gas phase, conducted using a laser interfaced mass spectrometer across a broad photoexcitation range from 250 to 790 nm. The photofragmentation channels are compared with the collision-induced dissociation (CID) products revealing similar dissociation pathways characterized by the loss of the carboxyl and ester groups. Complementary solution-phase photolysis experiments indicate that photobleaching occurs in verteporfin in acetonitrile; a notable conclusion, as photoinduced activity in Verteporfin was not thought to occur in homogenous solvent conditions. These results provide unique new information on the thermal break-down products and photoproducts of this light-triggered drug.

Investigation on the chiral recognition mechanism between verteporfin and cholate salts by capillary electrophoresis.

In this article, capillary electrophoresis was applied to investigate the chiral recognition mechanism for the enantioseparation on a well-known second-generation photodynamic therapy drug of benzoporphyrin derivative monoacid ring A, that is, verteporfin. In our previous study, cholate salts have been studied as the chiral selectors, which can realize baseline separation of the four verteporfin isomers. Aiming to reveal the chiral recognition mechanism, the separation effect of several kinds of chiral selectors was discussed. According to the results and references, the chiral separation mechanism of this system was concluded: the analytes selectively combine with the chiral micelles, that is, dynamic H-bonds interactions occur between the hydroxyl groups on the outer side of the cholate micelles and the ester/carboxy groups of the four isomers. In addition, the role of dimethyl formamide as an organic modifier was also researched, including reducing the effective mobility of the analytes and mobility of electroosmotic flow, and preventing them from adsorbing to the capillary wall and self-aggregating of verteporfin, which are pretty beneficial for separation. The method used in this article provides a direct and reliable solution to study the mechanism of chiral separation.

Dextran-Benzoporphyrin Derivative (BPD) Coated Superparamagnetic Iron Oxide Nanoparticle (SPION) Micelles for T2-Weighted Magnetic Resonance Imaging and Photodynamic Therapy.

Photodynamic therapy (PDT) has attracted extensive attention in recent years as a noninvasive and locally targeted cancer treatment approach. Nanoparticles have been used to improve the solubility and pharmacokinetics of the photosensitizers required for PDT; however, nanoparticles also suffer from many shortcomings including uncontrolled drug release and low tumor accumulation. Herein, we describe a novel biodegradable nanoplatform for the delivery of the clinically used PDT photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA) to tumors. Specifically, the hydrophobic photosensitizer BPD was covalently conjugated to the amine groups of a dextran-b-oligo (amidoamine) (dOA) dendron copolymer, forming amphiphilic dextran-BPD conjugates that can self-assemble into nanometer-sized micelles in water. To impart additional imaging capabilities to these micelles, superparamagnetic iron oxide nanoparticles (SPIONs) were encapsulated within the hydrophobic core to serve as a magnetic resonance imaging (MRI) contrast agent. The use of a photosensitizer as a hydrophobic building block enabled facile and reproducible synthesis and high drug loading capacity (∼30%, w/w). Furthermore, covalent conjugation of BPD to dextran prevents the premature release of drug during systemic circulation. In vivo studies show that the intravenous administration of dextran-BPD coated SPION nanoparticles results in significant MR contrast enhancement within tumors 24 h postinjection and PDT led to a significant reduction in the tumor growth rate.

Activation of TAp73 and inhibition of TrxR by Verteporfin for improved cancer therapy in TP53 mutant pancreatic tumors.

Aim:TAp73 is a tumor suppressor, which compensates for p53 loss and induces apoptosis in tumors in response to genotoxic stress or small-molecule treatments. Pancreatic ductal adenocarcinoma has a late onset of the disease, responds poorly to the existing therapies and has a very low survival rates.Result:Here, using drug-repurposing approach, we found that protoporphyrin IX (PpIX) and benzoporphyrin derivative (BPD) monoacid ring A activate TAp73 and induce apoptosis in pancreatic cancer cells. PpIX and BPD induce reactive oxygen species and inhibit thioredoxin reductase 1.Conclusion:Thus, PpIX and BPD target cancer cells’ vulnerabilities namely activate TAp73 tumor suppressor and inhibit oncogenic Trx1. Our findings may contribute to faster repurposing of PpIX and BPD to treat pancreatic tumors.

Targeting of a Photosensitizer to the Mitochondrion Enhances the Potency of Photodynamic Therapy.

Photodynamic therapy (PDT) involves use of a photosensitizer, whose activation with light leads to the production of singlet oxygen (SOS), generation of reactive oxygen species (ROS), and initiation of associated cell toxicity. Because a cell’s mitochondria constitute sites where oxygen levels are high, ROS can be readily produced, and apoptosis is commonly initiated. Therefore, an ideal PDT agent might be a potent photosensitizer that could naturally accumulate in mitochondria. Although a number of mitochondria-targeting moieties, including triphenylphosphine, guanidinium, and bisguanidium, have been identified, a quantitative comparison of their efficacies in targeting mitochondria has not been performed. In this study, we have prepared triphenylphosphine, guanidinium, and bisguanidium derivatives of the FDA-approved PDT agent verteporfin (Visudyne, benzoporphyrin derivative-monoacid ring A: BPD-MA) and compared their abilities to induce the intracellular perturbations common to potent PDT agents. Cellular parameters examined included subcellular localization of the verteporfin, real-time monitoring of SOS production, quantitation of reactive oxygen species (ROS) generation, analysis of mitochondria and chromatin integrity, characterization of cytoskeletal disruption and evaluation of cytochrome C release as a measure of apoptosis. An analysis of these parameters demonstrates that the triphenylphosphine derivative (0323) has better mitochondria-targeting efficacy, SOS production, and mitochondria membrane toxicity than either unmodified verteporfin or its guanidinium derivatives. Consistent with this potency, 0323 also induced the most prominent mitochondria swelling, actin depolymerization, pyknosis, and cytochrome C release. We conclude that triphenylphosphine has a better mitochondria-targeting moiety than guanidinium or bis-guanidinium and those PDT photosensitizers with improved cytotoxicities can be prepared by conjugating a mitochondria-targeting moiety to the desired photosensitizer.

Improved Photodynamic Therapy Efficacy of Protoporphyrin IX-Loaded Polymeric Micelles Using Erlotinib Pretreatment.

Photodynamic therapy (PDT) has attracted widespread attention in recent years as a noninvasive and highly selective approach for cancer treatment. We have previously reported a significant increase in the 90-day complete response rate when tumor-bearing mice are treated with the epidermal growth factor receptor (EGFR) inhibitor erlotinib prior to PDT with the photosensitizer benzoporphyrin-derivative monoacid ring A (BPD-MA) compared to treatment with PDT alone. To further explore this strategy for anticancer therapy and clinical practice, we tested whether pretreatment with erlotinib also exhibited a synergistic therapeutic effect with a nanocarrier containing the clinically relevant photosensitizer protoporphyrin IX (PpIX). The PpIX was encapsulated within biodegradable polymeric micelles formed from the amphiphilic block copolymer poly(ethylene glycol)-polycaprolactone (PEG-PCL). The obtained micelles were characterized systematically in vitro. Further, an in vitro cytotoxicity study showed that PDT with PpIX loaded micelles did exhibit a synergistic effect when combined with erlotinib pretreatment. Considering the distinct advantages of polymeric nanocarriers in vivo, this study offers a promising new approach for the improved treatment of localized tumors. The strategy developed here has the potential to be extended to other photosensitizers currently used in the clinic for photodynamic therapy.

Evaluation of singlet oxygen explicit dosimetry for predicting treatment outcomes of benzoporphyrin derivative monoacid ring A-mediated photodynamic therapy.

Existing dosimetric quantities do not fully account for the dynamic interactions between the key components of photodynamic therapy (PDT) or the varying PDT oxygen consumption rates for different fluence rates. Using a macroscopic model, reacted singlet oxygen ( [ O 2 1 ] rx ) was calculated and evaluated for its effectiveness as a dosimetric metric for PDT outcome. Mice bearing radiation-induced fibrosarcoma tumors were treated with benzoporphyrin derivative monoacid ring A (BPD) at a drug-light interval of 3 h with various in-air fluences (30 to 350 ?? J / cm 2 ) and in-air fluence rates (50 to 150 ?? mW / cm 2 ). Explicit measurements of BPD concentration and tissue optical properties were performed and used to calculate [ O 2 1 ] rx , photobleaching ratio, and PDT dose. For four mice, in situ measurements of O 2

Hemoporfin-mediated photodynamic therapy on normal vasculature: implications for phototherapy of port-wine stain birthmarks.

Port-wine stain (PWS) birthmarks currently are treated using a pulsed dye laser (PDL) combined with transient cooling of the epidermis. PDL treatment protocols utilize short pulses of light (585 or 595 nm wavelength) to heat selectively the microvasculature due to absorption by intravascular hemoglobin. Although most patients respond to PDL therapy, few experience complete removal of the PWS. An alternate treatment option to PDL therapy of PWS is photodynamic therapy (PDT). Research groups have reported on various photosensitizers for PDT of PWS, including Hemoporfin, Benzoporphyrin Derivative monoacid ring A, and talaporfin sodium. Our aim was to evaluate, with an established preclinical in-vivo model, the efficacy of photodynamic therapy (PDT) with Hemoporfin to achieve persistent vascular shutdown. To monitor the microvasculature, a dorsal window chamber was surgically installed on 24 adult mice. The PDT excitation source emitted 150mW of 532nm light, with an irradiance of 100mW/cm2. A retroorbital injection of Hemoporfin (2 mg/kg) was performed to deliver the drug into the bloodstream. Laser irradiation was initiated immediately after injection. To monitor blood-flow dynamics in response to PDT, we used laser speckle imaging. We employed a dose-response experimental design to study the efficacy of Hemoporfin-mediated PDT to achieve persistent vascular shutdown observed on Day 7 after PDT. We observed four general hemodynamic responses to PDT: (1) At low radiant exposures, we did not observe any persistent vascular shutdown; (2) at intermediate radiant exposures, we observed delayed vascular shutdown effect with significant change to the vascular structure; (3) at intermediate radiant exposures, we observed an acute vascular shutdown effect with gradual restoration of blood flow and no significant changes to the vascular structure; and (4) at high radiant exposures, we observed acute vascular shutdown that persisted during the entire 7-day monitoring period, with no change in vascular structure. With light dose-response analysis, we estimated a characteristic radiant exposure of 359 J/cm2 that was required to achieve persistent vascular shutdown observed on Day 7 after PDT. The experimental data collectively suggest that Hemoporfin-mediated PDT can achieve persistent vascular shutdown of normal microvasculature. However, compared with our previous data using Talaporfin Sodium as photosensitizer, Hemoporfin-mediated PDT is less efficient and requires a considerably longer (~four times) irradiation time. Patients with PWS lesions may benefit from the advantages that PDT potentially offers over conventional PDL therapy. PDT potentially is safer for patients of all skin types and more effective at treatment of recalcitrant lesions. Although clinical data suggest that Hemoporfin-mediated PDT is a promising alternative to PDL therapy, our results suggest that additional study of other photosensitizers is warranted.

Erlotinib Pretreatment Improves Photodynamic Therapy of Non-Small Cell Lung Carcinoma Xenografts via Multiple Mechanisms.

Aberrant expression of the epidermal growth factor receptor (EGFR) is a common characteristic of many cancers, including non-small cell lung carcinoma (NSCLC), head and neck squamous cell carcinoma, and ovarian cancer. Although EGFR is currently a favorite molecular target for the treatment of these cancers, inhibition of the receptor with small-molecule inhibitors (i.e., erlotinib) or monoclonal antibodies (i.e., cetuximab) does not provide long-term therapeutic benefit as standalone treatment. Interestingly, we have found that addition of erlotinib to photodynamic therapy (PDT) can improve treatment response in typically erlotinib-resistant NSCLC tumor xenografts. Ninety-day complete response rates of 63% are achieved when erlotinib is administered in three doses before PDT of H460 human tumor xenografts, compared with 16% after PDT-alone. Similar benefit is found when erlotinib is added to PDT of A549 NCSLC xenografts. Improved response is accompanied by increased vascular shutdown, and erlotinib increases the in vitro cytotoxicity of PDT to endothelial cells. Tumor uptake of the photosensitizer (benzoporphyrin derivative monoacid ring A; BPD) is increased by the in vivo administration of erlotinib; nevertheless, this elevation of BPD levels only partially accounts for the benefit of erlotinib to PDT. Thus, pretreatment with erlotinib augments multiple mechanisms of PDT effect that collectively lead to large improvements in therapeutic efficacy. These data demonstrate that short-duration administration of erlotinib before PDT can greatly improve the responsiveness of even erlotinib-resistant tumors to treatment. Results will inform clinical investigation of EGFR-targeting therapeutics in conjunction with PDT.

SU‐C‐303‐05: Photosensitizer Determination for PDT Using Interstitial and Surface Measurements of Fluorescence

Purpose:Photosensitizer concentration during photodynamic therapy (PDT) is an important parameter for accurate dosimetry. Fluorescence signal can be used as a measure of photosensitizer concentration. Two methods of data acquisition were compared to an ex vivo study both for in vivo and phantom models.Methods:Fluorescence signal of commonly used photosensitizer benzoporphyrin derivative monoacid ring A (BPD) was obtained in phantoms and mouse tumors using an excitation light of 405 nm. Interstitial fluorescence signal was obtained using a side‐cut fiber inserted into the tumor tissue of interest. Using a previously developed multi‐fiber probe, tumor surface fluorescence measurements were also collected. Signals were calibrated according to optical phantoms with known sensitizer fluorescence. Optical properties for each sample were determined and the influence of different absorption and scattering properties on the fluorescence signals was investigated. Using single value decomposition of the spectra, the sensitizer concentration was determined using the two different measurement geometries. An ex vivo analysis was also performed for tumor samples to determine the sensitizer concentration.Results:The two fluorescence signals obtained from the surface multi‐fiber probe and the interstitial measurements were compared and were corresponding for both phantoms and mouse models. The values obtained were comparable to the ex vivo measurements as well. Despite the difference in geometry, the surface probe measurements can still be used as a metric for determining the presence of sensitizer in small volume tumors.Conclusion:The multi‐fiber contact probe can be used as a tool to measure fluorescence at the surface of the treatment area for PDT and predict sensitizer concentration throughout the tumor. This is advantageous in that the measurement does not damage any tissue. Future work will include investigating the dependence of these results on intratumor sensitizer distribution.

Macroscopic singlet oxygen model incorporating photobleaching as an input parameter.

A macroscopic singlet oxygen model for photodynamic therapy (PDT) has been used extensively to calculate the reacted singlet oxygen concentration for various photosensitizers. The four photophysical parameters (ξ, σ, β, δ) and threshold singlet oxygen dose ([1O2]r,sh) can be found for various drugs and drug-light intervals using a fitting algorithm. The input parameters for this model include the fluence, photosensitizer concentration, optical properties, and necrosis radius. An additional input variable of photobleaching was implemented in this study to optimize the results. Photobleaching was measured by using the pre-PDT and post-PDT sensitizer concentrations. Using the RIF model of murine fibrosarcoma, mice were treated with a linear source with fluence rates from 12 - 150 mW/cm and total fluences from 24 - 135 J/cm. The two main drugs investigated were benzoporphyrin derivative monoacid ring A (BPD) and 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH). Previously published photophysical parameters were fine-tuned and verified using photobleaching as the additional fitting parameter. Furthermore, photobleaching can be used as an indicator of the robustness of the model for the particular mouse experiment by comparing the experimental and model-calculated photobleaching ratio.

β-Cyclodextrin Nanosponges as Multifunctional Ingredient in Water-Containing Semisolid Formulations for Skin Delivery

A β-cyclodextrin nanosponge cross-linked with pyromellitic dianhydride (βNS-PYRO) is reported for the first time as multifunctional ingredient in semisolid formulations for drug delivery to the skin. The role of βNS-PYRO on solubilization and stabilization of the photosensitizer benzoporphyrin-derivative monoacid ring A (BPDMA) and all-trans retinoic acid (atRA) as well as its effect on skin permeation of diclofenac (DIC) was investigated. Aqueous solutions, gels, and cream-gels were prepared from mixtures of βNS-PYRO with a conventional gelling agent at specific ratios. The incorporation of BPDMA in βNS-PYRO water solutions prevented its aggregation and gave kinetically stable complexes with high photostability and singlet oxygen generation upon irradiation. atRA incorporated in the βNS-PYRO-containing gel demonstrated a remarkable stability as compared with the formulation without βNS-PYRO, resulting in an eightfold increase of its lifetime. Skin permeation studies highlighted that βNS-PYRO in gels and cream-gels containing DIC significantly decreased the amount of drug permeated through the skin while increasing its amount in stratum corneum and viable epidermis. Overall, swellable βNS-PYRO turns to be a multifunctional coingredient with potential in topical monophasic and biphasic formulations to stabilize light-sensitive drugs and to localize the action of highly penetrating drugs in the external layers of skin.

Parameter determination for BPD mediated vascularPDT.

The cell killing mechanism of benzoporphyrin derivative monoacid ring A (BPD) is known to be predominantly apoptotic or vascular, depending on the drug-light interval (DLI). With a 3 hour DLI, necrosis develops secondary to tumor cell damage, while with a 15 minute DLI, necrosis results from treatment-created vascular damage. The purpose of this study is to examine if the different mechanisms of cell death will affect the photochemical parameters for the macroscopic singlet oxygen model. Using the RIF model of murine fibrosarcoma, we determined the four photochemical parameters (ξ, σ, β, γ) and the threshold singlet oxygen dose for BPD-mediated PDT through evaluation of the extent of tumor necrosis as a function of PDT fluence rate and total fluence. Mice were treated with a linear source at fluence rates from 12-150 mW/cm and total fluences from 24-135 J/cm. BPD was administered at 1mg/kg with a 15 minute DLI, followed by light delivery at 690nm. Tumors were excised at 24 hours after PDT and necrosis was analyzed via H&E staining. The in-vivo BPD drug concentration is determined to be in the range of 0.05-0.30 μM. The determination of these parameters specific for BPD and the 15 minute DLI provides necessary data for predicting treatment outcome in clinical BPD-mediated PDT. Photochemical parameters will be compared between 1mg/kg DLI 3 hours and 1mg/kg DLI 15 minutes.

Dye Sensitizers for Photodynamic Therapy.

Photofrin® was first approved in the 1990s as a sensitizer for use in treating cancer via photodynamic therapy (PDT). Since then a wide variety of dye sensitizers have been developed and a few have been approved for PDT treatment of skin and organ cancers and skin diseases such as acne vulgaris. Porphyrinoid derivatives and precursors have been the most successful in producing requisite singlet oxygen, with Photofrin® still remaining the most efficient sensitizer (quantum yield = 0.89) and having broad food and drug administration (FDA) approval for treatment of multiple cancer types. Other porphyrinoid compounds that have received approval from US FDA and regulatory authorities in other countries include benzoporphyrin derivative monoacid ring A (BPD-MA), meta-tetra(hydroxyphenyl)chlorin (m-THPC), N-aspartyl chlorin e6 (NPe6), and precursors to endogenous protoporphyrin IX (PpIX): 1,5-aminolevulinic acid (ALA), methyl aminolevulinate (MAL), hexaminolevulinate (HAL). Although no non-porphyrin sensitizer has been approved for PDT applications, a small number of anthraquinone, phenothiazine, xanthene, cyanine, and curcuminoid sensitizers are under consideration and some are being evaluated in clinical trials. This review focuses on the nature of PDT, dye sensitizers that have been approved for use in PDT, and compounds that have entered or completed clinical trials as PDT sensitizers.

Temporary regression of locally invasive polypoid rhinosinusitis in a dog after photodynamic therapy

A 5-year-old male cross-breed dog with chronic rhinosinusitis was presented. Computed tomography (CT) revealed opacities consistent with soft tissue or fluid in the left nasal cavity and frontal sinus. A diagnosis of chronic rhinosinusitis with myxomatous polyps was based on the histology of biopsy samples, so prednisolone treatment was administered for 1 month. Although the clinical signs slightly improved, no changes were observed on the CT images. At 8 months after the initial presentation, the dog was treated with antivascular photodynamic therapy (PDT) using benzoporphyrin derivative monoacid ring A and, 11 days later, CT revealed remarkably decreased soft tissue opacity; the facial swelling and sneezing also resolved. Three additional sessions of PDT were performed at 114, 210, and 303 days after the first PDT because of recurrence of clinical signs. The disease was well controlled for approximately 10 months by antivascular PDT.

Enhanced antitumor effects of BPD-MA-mediated photodynamic therapy combined with adriamycin on breast cancer in mice

Photodynamic therapy (PDT) is an emerging treatment used to eradicate premalignant and early-stage cancers and to reduce tumor size in end-stage cancers. In this study, we investigated the effects of a combination of benzoporphyrin derivative monoacid ring A (BPD-MA)-mediated PDT with adriamycin (ADM) on 4T1 breast carcinoma cells in vivo and the mechanisms underlying this effect. Normal BALA/c female mice bearing 4T1 breast carcinoma xenografts were tested. The animals were treated with PDT (BPD-MA 1 mg/kg, iv, plus single-dose laser irradiation) or ADM (5 mg/kg, iv) alone, or a combination of PDT with ADM. The tumor growth rate was determined by measuring the tumor weight. Cell apoptosis was measured with flow cytometry, and the expression of apoptosis-related molecules was assessed using Western blot. Microvessel density (MVD) was determined with immunohistochemical staining. Compared to PDT or ADM alone, PDT plus ADM produced a combined inhibition on the tumor growth, prolonged life span, and enhanced apoptosis in the mice bearing 4T1 subcutaneously xenografted tumors. The combination of PDT and ADM exerted additive effects on the upregulation of Bax and the downregulation of Bcl-2, and on the reduction of MVD in 4T1 xenografted tumors. Our results demonstrate that PDT plus ADM exerts enhanced in vivo antitumor effect on breast cancer, which is closely associated with the cooperative regulation of extrinsic apoptotic pathways and the inhibition of tumor angiogenesis. Thus, PDT plus ADM is a promising combined treatment strategy for breast carcinoma.

Epidermal growth factor receptor-targeted photosensitizer selectively inhibits EGFR signaling and induces targeted phototoxicity in ovarian cancer cells

Targeted photosensitizer delivery to EGFR-expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyrin derivative monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody.

Calculation of Singlet Oxygen Dose Using Explicit and Implicit Dose Metrics During Benzoporphyrin Derivative Monoacid Ring A (BPD‐MA)‐PDT In Vitro and Correlation with MLL Cell Survival

Photodynamic therapy (PDT) oxygen consumption, clonogenic cell survival, fluorescence photobleaching and photoproduct formation were investigated during benzoporphyrin derivative monoacid (BPD-MA)-PDT of MAT-LyLu cells in vitro. Cells were incubated with BPD-MA concentrations of 0.1, 0.5 or 2.5 μg mL(-1) for 2 h and then treated with 405 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment, and photobleaching and photoproduct generation were quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony-forming assay. The amount of oxygen consumed by PDT per photon absorbed decreased with BPD-MA intracellular concentration. Survival was correlated with the total amount of oxygen consumed by PDT per unit volume, which is assumed to be equivalent to the amount of singlet oxygen that reacted. A photobleaching-based singlet oxygen dose metric was also found to predict survival independent of intracellular BPD-MA concentration. The BPD-MA photoproduct was bleached during the treatment. Two singlet oxygen dose metrics based on photoproduct kinetics could not be correlated with cell survival over the full range of intracellular BPD-MA concentrations used.

Photodynamic therapy and the treatment of head and neck malignancies

The impact of the immunomodulatory photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) and visible light on the survival and surface receptor pattern of resting and activated murine T cells was evaluated. T cells treated for 48 h with immobilized anti-CD3 monoclonal antibody upregulated expression of the interleukin-2 receptor α-chain (CD25), transferrin receptor (CD71), the apoptosis-regulating Fas receptor (CD95), contained a greater level of the anti-apoptotic protein Bcl-2 and accumulated significantly more BPD-MA than their unactivated counterparts. Activated T cells displayed a modestly greater susceptibility to the photodynamic induction of DNA fragmentation than resting T cells. Resting T cells treated with sub-lethal levels of BPD-MA and light did not exhibit changes in surface levels of CD3, CD4, CD8, CD28, CD45 or T cell receptor (TCR) β-chain structures. However, levels of major histocompatibility complex (MHC) class I antigens were decreased while the density of Thy-1.2 (CD90) increased on these cells. Photodynamically treated T cells failed to express optimal CD25 levels when exposed to the mitogenic anti-CD3 antibody. Activated T cells treated with sub-lethal levels of BPD-MA and light exhibited lower CD25 levels, a temporary block in cell cycle transition, but unaltered expression of MHC Class I, CD3, CD4, CD8, CD45, CD54, CD71, CD122 (IL-2R β-chain) or TCR β-chain antigens 24 h afterward. Resting and activated T lymphocytes differ in susceptibility to PDT-mediated apoptosis but both types are sensitive to anti-proliferative effects the treatment exerts at sub-lethal photosensitizer levels. The marked sensitivity of activated T cells to photodynamic inactivation likely contributes to the immunomodulatory action of BPD-MA.