Accumulation Of Photosensitizer Research Articles

CRGD Peptide-Conjugated Pyropheophorbide-a Photosensitizers for Tumor Targeting in Photodynamic Therapy.

Pyropheophorbide-a (Pyro) is a highly promising photosensitizer for tumor photodynamic therapy (PDT), although its very limited tumor-accumulation ability seriously restricts its clinical applications. A higher accumulation of photosensitizers is very important for the treatment of deeply seated and larger tumors. The conjugation of Pyro with tumor-homing peptide ligands could be a very useful strategy to optimize the physical properties of Pyro. Herein, we reported our studies on the conjugation of Pyro with a cyclic cRGDfK (cRGD) peptide, an integrin binding sequence, to develop highly tumor-specific photosensitizers for PDT application. To further reduce the nonspecific uptake and, thus, reduce the background distribution of the conjugates in normal tissues, we opted to add a highly hydrophilic polyethylene glycol (PEG) chain and an extra strongly hydrophilic carboxylic acid group as the linker to avoid the direct connection of the strongly hydrophobic Pyro macrocycle and cRGD ligand. We reported here the synthesis and characterization of these conjugates, and the influence of the hydrophilic modification on the biological function of the conjugates was carefully studied. The tumor-accumulation ability and photodynamic-induced cell-killing ability of these conjugates were evaluated through both in vitro cell-based experiment and in vivo distribution and tumor therapy experiments with tumor-bearing mice. Thus, the synthesized conjugate significantly improved the tumor enrichment and tumor selectivity of Pyro, as well as abolished the xenograft tumors in the murine model through a one-time PDT treatment.

Comparison of a new nanoform of the photosensitizer chlorin e6, based on plant phospholipids, with its free form.

Photodynamic therapy is an advanced method of treating cancer and various benign diseases, including infections. It uses light‐activated molecules [photosensitizers (PSs)] to generate reactive oxygen species (ROS) when irradiated with light of a specific wavelength. This study examined the photophysical and photosensitizing activity of the PS chlorin e6 incorporated in a delivery system based on plant phospholipids. This new nanoform of chlorin e6 comprised particles with a diameter of 18.4 ± 2.5 nm and zeta potential of −34.6 ± 3.0 mV. Incorporation of chlorin e6 in phospholipid nanoparticles was observed to cause a bathochromic shift of Q‐band absorption maximum by 14 nm without an absorption change in the range of the Soret band. Fluorescence intensity of chlorin e6 embedded in the phospholipid nanoparticles increased 1.7‐fold. Chlorin e6 in phospholipid nanoparticles, when irradiated, was able to generate ROS as shown by oxidation of polyunsaturated fatty acids of the phospholipid matrix of the delivery system and reduced l‐glutathione. In vivo it was demonstrated that the new nanoform of chlorin e6 provides more accumulation of PSs in tumor tissue than its free form. Moreover, its accumulation in the skin was lower and its elimination from the skin almost five times faster than when administered in free form. The observed differences of this new nanoform of chlorin e6 should lead to enhancement of antitumor efficacy and a decrease in phototoxicity.

Porphyrin-grafted Lipid Microbubbles for the Enhanced Efficacy of Photodynamic Therapy in Prostate Cancer through Ultrasound-controlled In Situ Accumulation.

Photodynamic therapy (PDT) holds promise for focal therapy of prostate cancer (PCa). However, the therapeutic efficacy needs improvement, and further development of PDT for PCa has challenges, including uncertainty of photosensitizers (PSs) accumulation at the tumor site and difficulty in visualizing lesions using conventional ultrasound (US) imaging. We have developed novel porphyrin-grafted lipid (PGL) microbubbles (MBs; PGL-MBs) and propose a strategy to integrate PGL-MBs with US imaging to address these limitations and enhance PDT efficacy.Methods: PGL-MBs have two functions: imaging guidance by contrast-enhanced ultrasound (CEUS) and targeted delivery of PSs by ultrasound targeted microbubble destruction (UTMD). PGL-MBs were prepared and characterized before and after low-frequency US (LFUS) exposure. Then, in vitro studies validated the efficacy of PDT with PGL-MBs in human prostate cancer PC3 cells. PC3-xenografted nude mice were used to validate CEUS imaging, accumulation at the tumor site, and in vivo PDT efficacy.Results: PGL-MBs showed good contrast enhancement for US imaging and were converted into nanoparticles upon LFUS exposure. The resulting uniquely structured nanoparticles avoided porphyrin fluorescence quenching and efficiently accumulated at the tumor site through the sonoporation effect created with the assistance of US to achieve excellent PDT efficacy.Conclusions: This is the first preclinical investigation of MBs applied in PDT for PCa. PGL-MBs possess favorable CEUS imaging effects to enhance the localization of tumors. PGL-MBs with LFUS control PS accumulation at the tumor site to achieve highly effective PDT of PCa. This strategy carries enormous clinical potential for PCa management.

Study of level and selectivity of liposomal form of photosensitiser hydroxyaluminium tetra-3-phenylthiophthalocyanine accumulation on transplantable mice tumor models at different ways of transplantation

Background. Current work is devoted to the study in vivo of concentration and selectivity of accumulation of infrared photosensitizer (PS) hydroxyaluminium tetra-3-phenylthiophthalocyanine in liposomal form in intramuscularly and subcutaneously transplanted mice tumor models in comparison to normal tissues. Objective: to study the level and selectivity of accumulation of hydroxyaluminium tetra-3-phenylthiophthalocyanine liposomal form on mice tumor models in order to optimize the transplantation approach and the starting of photodynamic treatment. Materials and methods. A range of transplantable mice tumors was used in the study: solid carcinoma Ehrlich (ELD) and solid sarcoma S-37, epidermoid Lewis lung carcinoma (LLC) and colon adenocarcinoma (AKATOL). For the assessment of concentration of PS in tissues was evaluated by fluorescence spectroscopy in vivo. Results. The optimum transplantation approaches were shown to be as follows. Solid carcinoma Ehrlich (ELD) provided the highest accumulation of PS when transplanted intramuscularly. Five hours after administration concentration of PS in tumor achieves more than 7 mg/kg, with selectivity in comparison to normal tissue 3 : 1. The maximum concentration of PS in sarcoma S-37 was observed with subcutaneous transplantation, achieving at 5 h after administration the value of 5.4 mg/kg with selectivity of accumulation 4.3: 1. Both LLC and AKATOL showed optimum results with intramuscular transplantation. Maximum concentration of PS in LLC was observed 5 h after administration, achieving 7.5 mg/kg with selectivity exceeding 4. Concentration of photosensitizer in AKATOL 7h post administration achieved 6.8 mg/kg with selectivity about 2. Conclusions. Liposomal form of PS with intravenous administration selectively accumulates in tumors. The obtained experimental data allows to recommend the method of listed tumors models transplantation for the studies of PS.

ABCG2-mediated suppression of chlorin e6 accumulation and photodynamic therapy efficiency in glioblastoma cell lines can be reversed by KO143

BackgroundPhotodynamic therapy (PDT) of malignant brain tumors is a promising adjunct to standard treatment, especially if tumor stem cells thought to be responsible for tumor progression and therapy resistance were also susceptible to this kind of treatment. However, some photosensitizers have been reported to be substrates of ABCG2, one of the membrane transporters mediating resistance to chemotherapy. Here we investigate, whether inhibition of ABCG2 can restore sensitivity to photosensitizer chlorin e6-mediated PDT. MethodsAccumulation of chlorin e6 in wild type U87 and doxycycline-inducible U251 glioblastoma cells with or without induction of ABCG2 expression or ABCG2 inhibition by KO143 was analyzed using flow cytometry. In U251 cells, ABCG2 was inducible by doxycycline after stable transfection with a tet-on expression plasmid. Tumor sphere cultivation under low attachment conditions was used to enrich for cells with stem cell-like properties. PDT was done on monolayer cell cultures by irradiation with laser light at 665nm. ResultsElevated levels of ABCG2 in U87 cells grown as tumor spheres or in U251 cells after ABCG2 induction led to a 6-fold lower accumulation of chlorin e6 and the light dose needed to reduce cell viability by 50% (LD50) was 2.5 to 4-fold higher. Both accumulation and PDT response can be restored by KO143, an efficient non-toxic inhibitor of ABCG2. ConclusionGlioblastoma stem cells might escape phototoxic destruction by ABCG2-mediated reduction of photosensitizer accumulation. Inhibition of ABCG2 during photosensitizer accumulation and irradiation promises to restore full susceptibility of this crucial tumor cell population to photodynamic treatment.

ALA-PpIX mediated photodynamic therapy of malignant gliomas augmented by hypothermia.

BackgroundMalignant gliomas are highly invasive, difficult to treat, and account for 2% of cancer deaths worldwide. Glioblastoma Multiforme (GBM) comprises the most common and aggressive intracranial tumor. The study hypothesis is to investigate the modification of Photodynamic Therapy (PDT) efficacy by mild hypothermia leads to increased glioma cell kill while protecting normal neuronal structures.MethodsPhotosensitizer accumulation and PDT efficacy in vitro were quantified in various glioma cell lines, primary rat neurons, and astrocytes. In vivo studies were carried out in healthy brain and RG2 glioma of naïve Fischer rats. Hypothermia was induced at 1 hour pre- to 2 hours post-PDT, with ALA-PpIX accumulation and PDT treatments effects on tumor and normal brain PDT quantified using optical spectroscopy, histology, immunohistochemistry, MRI, and survival studies, respectively.FindingsIn vitro studies demonstrated significantly improved post-PDT survival in primary rat neuronal cells. Rat in vivo studies confirmed a neuroprotective effect to hypothermia following PpIX mediated PDT by T2 mapping at day 10, reflecting edema/inflammation volume reduction. Mild hypothermia increased PpIX fluorescence in tumors five-fold, and the median post-PDT rat survival time (8.5 days normothermia; 14 days hypothermia). Histology and immunohistochemistry show close to complete cellular protection in normal brain structures under hypothermia.ConclusionsThe benefits of hypothermia on both normal neuronal tissue as well as increased PpIX fluorescence and RG2 induced rat survival strongly suggest a role for hypothermia in photonics-based surgical techniques, and that a hypothermic intervention could lead to considerable patient outcome improvements.

Phototoxicity in a laryngeal cancer cell line enhanced by a targeting amphiphilic chlorin photosensitizer

BackgroundPhotodynamic therapy (PDT) has been established in several countries as an alternative therapy for the treatment of various malignancies. This therapy involves the incorporation of a photosensitizer (PS) that is activated by visible light and form reactive oxygen species leading to target cell death by apoptosis or necrosis. Previously, our group has demonstrated that CHL-T (semi-synthesized from chlorophyll a and containing a linked solubilizing group TRISMA®) presented a pronounced potential to induce death in HeLa cell line after PDT. In the present study, besides confirm the high cytotoxicity in another cell line, we have further investigated the cell death mechanisms caused by CHL-T as a photosensitizer in laryngeal carcinoma cells. MethodsCells were exposed to different concentrations of three photosensitizers, namely, hypericin (HY), unmodified chlorin (CHL) and a synthesized amphiphilic chlorin derivative (CHL-T). PSs accumulation and localization were accessed by fluorescence assays. Photosensitization was induced at 6Jcm−2 using red LEDs (630±10nm). Viability was assessed by mitochondrial function (MTT); whereas apoptosis/necrosis was evaluated by fluorescence microscopy and flow cytometry. Expression of pro-apoptotic p53 protein was studied by Western blot. Results and conclusionsAll PS showed similar localization profile in the HEp-2 cells. The use of CHL-T increased the percentage of apoptotic cells and also p53 expression in comparison with the use of HY and CHL as photosensitizers. This study shows a significant effect of CHLT associated with red light (630±10nm and 18mWcm−2) irradiation on a cancer cell line, indicating the potential of this amphiphilic chlorin in enhancing the therapeutic effectiveness of Photodynamic Therapy (PDT).

The alteration of temoporfin distribution in multicellular tumor spheroids by β-cyclodextrins

To be effective anticancer drugs must penetrate tissue efficiently, reaching all target population of cancer cells in a concentration sufficient to exert a therapeutic effect. This study aimed to investigate the ability of methyl-β-cyclodextrin (Me-β-CD) and 2-hydroxypropyl-β-cyclodextrin (Hp-β-CD) to alter the penetration and diffusion of temoporfin (mTHPC) in HT29 multicellular tumor spheroids. mTHPC had а nonhomogenous distribution only on the periphery of spheroids. The presence of β-CDs significantly altered the distribution of mTHPC consisting in the increase of both the depth of photosensitizer penetration and accumulation in HT29 spheroids. We suggest that this improvement is related to the nanoshuttle mechanism of β-CD action, when β-CDs facilitate mTHPC transportation to the cells in the inner layers of spheroids. As a result of mTHPC distribution improvement, β-CDs enhance mTHPC photosensitizing activity towards HT29 multicellular tumor spheroids. The observed effects strongly depend on the type of β-CD. Thus, varying the type of β-CD we can finely tune the possibility of using mTHPC for diagnostic (delimitation of tumor margins) or therapeutic purposes.

Determination of oxygen saturation and photosensitizer accumulation in the tumor with the help of LEDand laser diode-based irradiation sources and fiber-optics probes

Determination of oxygen saturation and photosensitizer accumulation in the tumor with the help of LEDand laser diode-based irradiation sources and fiber-optics probes

Endobronchial photodynamic therapy under fluorescence control: Photodynamic theranostics

BackgroundPhotodynamic therapy (PDT) has several advantages. However, one of the disadvantages is its inability to be individualized according to biological characteristics of malignant tumors. The objective of this study was to investigate a strategy for individualized endobronchial PDT in the treatment of centrally located non-small cell lung cancer. MethodsNew approach suggests taking fluorescence-based measurements of chlorine E6 photosensitizer (PS) accumulation in the malignant tumor tissue, and assess PS consumption rate during PDT. Two randomized groups of 45 patients took part in the comparative study of standard PDT procedure, 662nm, pulse-periodic mode, therapeutic light (reference group – RG) versus the investigated individualized approach under fluorescence control after irradiation with violet light, 408nm, diagnostic light (study group – SG). The PDT-treatment parameters and results of follow-up bronchoscopy were compared between the groups. Results43 (96%) of 45 patients in SG demonstrated intense fluorescence in the area of the tracheal/bronchial tumor stenosis. 4 (9%) of 45 patients (SG) demonstrated fluorescence of mucosa areas distant from the main tumor lesion after violet light irradiation. Mean fluence during the whole PDT procedure was 95±20J/cm2 (range 60–130J/cm2), which was significantly lower than in RG (p=0.01). Total exposure time was significantly lower in SG (365±65s), compared with RG (690±65s), P=0.001. According to the follow-up bronchoscopy the difference in the PDT-treatment results between the groups is statistically insignificant. ConclusionsThe investigated strategy suggests using fluorescence control of the efficacy of PDT-treatment (photodynamic theranostics) to optimize and individualize the PDT procedure.

Minimizing the Heat Effect of Photodynamic Therapy Based on Inorganic Nanocomposites Mediated by 808 nm Near-Infrared Light.

Photodynamic therapy (PDT) based on photosensitizers (PSs) constructed with nanomaterials has become popular in cancer treatment, especially oral carcinoma cell. This therapy is characterized by improved PS accumulation in tumor regions and generation of reactive oxygen species (ROS) for PDT under specific excitation. In the selection of near-infrared (NIR) window, 808 nm NIR light because it can avoid the absorption of water is particularly suitable for the application in PDT. Hence, multiband emissions under a single 808 nm near-infrared excitation of Nd3+ -sensitized upconversion nanoparticles (808 nm UCNPs) have been applied for the PDT effect. 808 nm UCNPs serve as light converter to emit UV light to excite inorganic PS, graphitic carbon nitride quantum dots (CNQDs), thereby generating ROS. In this study, a nanocomposite consisting UCNPs conjugated with poly-l-lysine (PLL) to improve binding with CNQDs is fabricated. According to the research results, NIR-triggered nanocomposites of 808 nm UCNP-PLL@CNs have been verified by significant improvement in ROS generation. Consequently, 808 nm UCNP-PLL@CNs exhibit high capability for ROS production and efficient PDT in vitro and in vivo. Moreover, the mechanism of PDT treatment by 808 nm UCNP-PLL@CNs is evaluated using the cell apoptosis pathway.

Activity of phosphatase-sensitive 5-aminolevulinic acid prodrugs in cancer cell lines

5-aminolevulinc acid (5-ALA)-based photodynamic therapy (PDT) and photodiagnosis (PD) present many advantages over treatments with conventional photosensitizers (PS). It offers great tumor specificity, reduced photosensitivity reactions caused by PS accumulation in non-targeted tissues and also inherent PS metabolism into endogenous non-fluorescent heme. However, chemical instability, low bioavailability and poor pharmacokinetic profile limit systemic efficacy of 5-ALA. Here, we present a comprehensive in vitro evaluation of novel phosphatase-sensitive prodrugs of 5-ALA. These prodrugs are designed to be activated by ubiquitously expressed phosphatases with much improved chemical stability and reduced acute toxicity profile. PpIX kinetic measurements and flow cytometry show accumulation of PpIX upon incubation with phosphatase-sensitive prodrugs in PC3 human prostate cell cancer, MCF7 breast adenocarcinoma, U87MG glioblastoma, T24 bladder cancer and A549 lung carcinoma cells. They revealed a different fluorescence kinetics and dose-response curves for the different types of 5-ALA prodrugs. These experiments have allowed us to identify the most promising cancer cell types for phospho- 5-ALA prodrugs. Confocal fluorescence microscopy provided further evidence of fluorescent protoporphyrin IX accumulation and sub-cellular localisation. These findings, together with the low toxicity profile of phosphatase-sensitive prodrugs of 5-ALA and good response to PDT provide solid basis for future translational development in PC3, MCF7 and U87MG cancer types.

Effect of photodynamic therapy with the bacteriochlorophyll a derivative on growth and functional morphology of rat sarcoma M-1

Background : In recent years, the method of photodynamic therapy (PDT) has been increasingly used in clinical oncology. Three non-toxic components play a key role in realization of the anti- tumor effect of PDT: a photosensitizer (PS), local irradiation of the tumor with light/laser of a certain wavelength corresponding to the sensitizer absorption peak, and oxygen. The highly reactive biological oxidants formed during photochemical reactions exert a destructive effect on cells and tumor vasculature with subsequent activation of the immune response. Efficiency of PDT is determined by the level of PS accumulation in tumors, as well as by its photophysical parameters and photochemical activity. To create a new PS, natural chlorophylls and their derivatives with intense absorption in the long-wave region of the spectrum are of particular interest. Aim : To study the efficacy and mechanism of PDT action (using a conjugate containing two molecules of dipropoxy-bacteriopurpurinimide with a cystamine residue as a PS) on the growth and functional morphology of transplanted solid connective tissue tumor. Materials and methods : The study was carried out in female white outbred rats with subcutaneously implanted sarcoma M-1. PS was injected to rats of the experimental group intraperitoneally. PDT was performed during the maximal index of tumor/ healthy tissue contrast after the administration of the PS. The criterion of PDT efficacy was a change in the tumor growth/regression coefficient. The research methods included immunohistochemistry for PCNA and CD31, evaluation of mitotic activity and apoptosis of tumor cells, as well as computer analysis of microscopic images. Results : After PDT with the new PS, a 16-fold decrease of growth coefficient of tumor nodes was registered. The photo-induced antitumor effect was shown to be due to destruction of sarcoma M-1 vascular bed, rapid inhibition of proliferative activity and devitalization of tumor cells. At early stages after PDT, destruction of the microvasculature and photo-cytostatic shock of tumor cells with subsequent development of necrosis appears to be caused by direct influence of the light flux on sensitized cellular elements in tumor parenchyma and stroma. Conclusion : The final result of PDT is determined by the sequence of destructive and inflammatory changes in the tumor parenchyma and surrounding tissues, as well as by a proliferative potential of malignant cells surviving after treatment. The surviving tumor cells, which determine the recurrent growth of neoplasms after PDT, are still present, maybe due to an insufficient concentration of the sensitizer in certain weakly vascularized areas of sarcoma M-1.

MnO2 Gatekeeper: An Intelligent and O2‐Evolving Shell for Preventing Premature Release of High Cargo Payload Core, Overcoming Tumor Hypoxia, and Acidic H2O2‐Sensitive MRI

Premature leakage of photosensitizer (PS) from nanocarriers significantly reduces the accumulation of PS within a tumor, thereby enhancing nonspecific accumulation in normal tissues, which inevitably leads to a limited efficacy for photodynamic therapy (PDT) and the enhanced systematic phototoxicity. Moreover, local hypoxia of the tumor tissue also seriously hinders the PDT. To overcome these limitations, an acidic H2O2‐responsive and O2‐evolving core–shell PDT nanoplatform is developed by using MnO2 shell as a switchable shield to prevent the premature release of loaded PS in core and elevate the O2 concentration within tumor tissue. The inner core SiO2‐methylene blue obtained by co‐condensation has a high PS payload and the outer MnO2 shell shields PS from leaking into blood after intravenous injection until reaching tumor tissue. Moreover, the shell MnO2 simultaneously endows the theranostic nanocomposite with redox activity toward H2O2 in the acidic microenvironment of tumor tissue to generate O2 and thus overcomes the hypoxia of cancer cells. More importantly, the Mn(ΙΙ) ion reduced from Mn(ΙV) is capable of in vivo magnetic resonance imaging selectively in response to overexpressed acidic H2O2. The facile incorporation of the switchable MnO2 shell into one multifunctional diagnostic and therapeutic nanoplatform has great potential for future clinical application.

Plasmon-Enhanced Photodynamic Cancer Therapy by Upconversion Nanoparticles Conjugated with Au Nanorods.

Photodynamic therapy (PDT) based on photosensitizers (PSs) constructed with nanomaterials has been widely applied to treat cancer. This therapy is characterized by an improved PS accumulation in tumor regions. However, challenges, such as short penetration depth of light and low extinction coefficient of PSs, limit PDT applications. In this study, a nanocomposite consisting of NaYF4:Yb/Er upconversion nanoparticles (UCPs) conjugated with gold nanorods (Au NRs) was developed to improve the therapeutic efficiency of PDT. Methylene blue (MB) was embedded in a silica shell for plasmon-enhanced PDT. UCPs served as a light converter from near-infrared (NIR) to visible light to excite MB to generate reactive oxygen species (ROS). Au NRs could effectively enhance upconversion efficiency and ROS content through a localized surface plasmon resonance (SPR) effect. Silica shell thickness was adjusted to investigate the optimized MB loading amount, ROS production capability, and efficient distance for plasmon-enhanced ROS production. The mechanism of plasmon-enhanced PDT was verified by enhancing UC luminescence intensity through the plasmonic field and by increasing the light-harvesting capability and absorption cross section of the system. This process improved the ROS generation by comparing the exchange of Au NRs to Au nanoparticles with different SPR bands. NIR-triggered nanocomposites of UCP@SiO2:MB-NRs were significantly confirmed by improving ROS generation and further modifying folic acid (FA) to develop an active component targeting OECM-1 oral cancer cells. Consequently, UCP@SiO2:MB-NRs-FA could highly produce ROS and undergo efficient PDT in vitro and in vivo. The mechanism of PDT treatment by UCP@SiO2:MB-NRs-FA was evaluated via the cell apoptosis pathway. The proposed process is a promising strategy to enhance ROS production through plasmonic field enhancement and thus achieve high PDT therapeutic efficacy.

THE WAYS TO OPTIMIZE CLINICAL OUTCOMES OF PHOTODYNAMIC THERAPY BY OPTICAL IMAGING TECHNIQUES

Background: Photodynamic therapy (PDT) is a modern minimally invasive technique for treatment of a wide range of diseases, including malignancies. One of directions for PDT development is the individualization of exposure modes that can be achieved with effective treatment monitoring. There are a number of approaches employing imaging techniques, the most promising of them being optical ones. Aim: To analyze factors affecting clinical outcomes of PDT in non-melanoma skin tumors, and to evaluate the prospects of optical imaging techniques for PDT planning and monitoring. Materials and methods: We retrospectively analyzed various aspects of the results PDT obtained in 855 patients with non-melanoma skin tumors. PDT was performed with systemic chlorine photosensitizers. As a source of irradiation, the laser at a wavelength of 662 nm was used following exposure modes: mean power density 0.3 W/cm², the laser irradiation dose of 200 J/cm² for basal cancer and 300 J/cm² for squamous cell carcinoma. Clinical evaluation was performed based on tumor response according to RECIST criteria, by the presence or absence of recurrence during long term follow up and by the presence or absence of cosmetic defects. Fluorescence imaging and optical coherence tomography were used as non-invasive imaging techniques. Results: It was found that clinical predictors of treatment failure included tumor recurrence, squamous type of tumor, and advanced exophytic or infiltrative component. Fluorescence imaging showed an association between clinical outcomes of PDT and fluorescence characteristics of the photosensitizer. The best clinical outcomes were achieved in 147 patients with a combination of high contrast fluorescence (FC>1.2) and a high degree of photobleaching of the agent (ΔIt/IN >25%): the number of complete tumor responses was 94% (138 of 147), with recurrence seen in 3 (2%) patients only with the follow up from 6 to 53 month duration. Clinical predictors of PDT cosmetic failures are tumor recurrence and tumor stage above T2. The most vulnerable zones are the outer ear and nose wings; this fact is related to an involvement of the cartilage located directly beneath the thin skin in the photodynamic reaction. This was demonstrated by optical coherence tomography. Conclusion: Presence of clinical predictors of PDT failure justifies correction of light exposure modes that can be optimally implemented with techniques for objective evaluation of the tumor borders, photosensitizer accumulation and photobleaching. Dynamic non-invasive monitoring of PDT procedure with fluorescence imaging and optical coherence tomography seems promising for implementation of an individual approach resulting in optimal oncological and functional outcomes.

Anti- and Pro-apoptotic Bcl2 Proteins Distribution and Metabolic Profile in Human Coronary Aorta Endothelial Cells Before and After HypPDT.

Understanding apoptosis regulatory mechanisms in endothelial cells (ECs) has great importance for the development of novel therapy strategies for cancer and cardiovascular pathologies. An oxidative stress with the generation of reactive oxygen species (ROS) is a common mechanism causing ECs' dysfunction and apoptosis. The generation of ROS can be triggered by various stimuli including photodynamic therapy (PDT). In most PDT treatments, photosensitizer (PS) is administered systemically, and thus, possibility of high exposure to PS in the ECs remains high. PS accumulation in ECs may be clinically relevant even without PDT, if PS molecules affect the pro-apoptotic cascade without illumination. In the present work, we focused on Hypericin (Hyp) and HypPDT effects on the cell viability, oxidative stress, and the distribution of Bcl2 family members in human coronary artery endothelial (HCAEC) cells. Our findings show that the presence of Hyp itself has an effect on cell viability, oxidative stress, and the distribution of Bcl2 family members, without affecting the mitochondria function. In contrast, HypPDT resulted in mitochondria dysfunction, further increase of oxidative stress and effect on the distribution of Bcl2 family members, and in primarily necrotic type of death in HCAEC cells.

Inclusion complexation with β-cyclodextrin derivatives alters photodynamic activity and biodistribution of meta-tetra(hydroxyphenyl)chlorin

Application of meta-tetra(hydroxyphenyl)chorin (mTHPC) one of the most effective photosensitizer (PS) in photodynamic therapy of solid tumors encounters several complications resulting from its insolubility in aqueous medium. To improve its solubility and pharmacokinetic properties, two modified β-cyclodextrins (β-CDs) methyl-β-cyclodextrin (M-β-CD) and 2-hydroxypropyl-β-cyclodextrin (Hp-β-CD) were proposed. The aim of this work was to evaluate the effect of β-CDs on mTHPC behavior at various stages of its distribution in vitro and in vivo. For this purpose, we have studied the influence of the β-CDs on mTHPC binding to the serum proteins, its accumulation, distribution and photodynamic efficiency in HT29 cells. In addition, the processes of mTHPC biodistribution in HT29 tumor bearing mice after intravenous injection of PS alone or with the β-CDs were compared. Interaction of mTHPC with studied β-CDs leads to the formation of inclusion complexes that completely abolishes its aggregation after introduction into serum. It was demonstrated that the β-CDs have a concentration-dependent effect on the process of mTHPC distribution in blood serum. At high concentrations, β-CDs can form inclusion complexes with mTHPC in the blood that can have a significant impact on PS distribution out of the vascular system in solid tissues. Besides, the β-CDs increase diffusion movement of mTHPC molecules that can significantly accelerate the delivery of PS to the targets cells and tissues. In vivo study confirms the fact that the use of β-CDs allows to modify mTHPC distribution processes in tumor bearing animals that is reflected in the decreased level of PS accumulation in skin and muscles, as well as in the increased PS accumulation in tumor. Further studies are underway to verify the optimal protocols of mTHPC/β-CD formulation for photodynamic therapy.

F8-SIP mediated targeted photodynamic therapy leads to microvascular dysfunction and reduced glioma growth

The extra domain A (ED A) of fibronectin has been identified as a tumor vessel specific neovascular marker in glioma. Antibody based vascular targeting against ED A of fibronectin allows precise accumulation of photosensitizer in glioma microvasculature and thereby promises to overcome drawbacks of current photodynamic therapy (PDT) for glioma treatment. Our aim was to characterize microcirculatory consequences of F8-small immunoprotein (SIP) mediated PDT by intravital microscopy (IVM) and to analyze the effects on glioma growth. For IVM SF126 glioma cells were implanted into dorsal skinfold-chamber of nude mice. PDT was performed after intravenous injection of photosensitizer (PS)-coupled F8-SIP or PBS (n=4). IVM was performed before and after PDT for 4days. Analysis included total and functional (TVD, FVD) vessel densities, perfusion index (PI), microvascular permeability and blood flow rate (Q). To assess tumor growth SF126 glioma cells were implanted subcutaneously. PDT was performed as a single and repetitive treatment after PS-F8-SIP injection (n=5). Subcutaneous tumors were treated after uncoupled F8-SIP injection as control group (n=5). PDT induced microvascular stasis and thrombosis with reduced FVD (24h: 115.98±0.7 vs. 200.8±61.9cm/cm(2)) and PI (39±11 vs. 70±10%), whereas TVD was not altered (298±39.2 vs. 278.2±51cm/cm(2)). Microvascular dysfunction recovered 4days after treatment. Microvascular dysfunction led to a temporary reduction of glioma growth in the first 48h after treatment with complete recovery 5days after treatment. Repetitive PDT resulted in sustained reduction of tumor growth. F8-SIP mediatedPDT leads to microvascular dysfunction and reduced glioma growth in a preclinical glioma model with recovery of microcirculation 4days after treatment. Repetitive application of PDT overcomes microvascular recovery and leads to prolonged antiglioma effects.

The in vitro cytotoxicity of metal-complexes of porphyrin sensitizer intended for photodynamic therapy

The sulphonated derivatives of porphyrins (e.g. TPPS4) are hydrophilic photosensitizers and have certain advantages like fully known structures and the possibility of synthetic production.The aim of this work was to study in vitro cytotoxicity and to compare the new photosensitizer MgTPPS4 with TPPS4 and its other metal-complexes (ZnTPPS4, PdTPPS4) on human skin melanom and mouse fibroblast cell lines. A photodynamic treatment was induced by light emitting diodes with three different total doses (1, 5 and 10J/cm2).For proper analysis and understanding of cell behavior after the administration of sensitizers, a complex battery of in vitro tests including the production of reactive oxygen species, the MTT viability test, a comet assay, a cell cycle and a type of cell death determination were used.We discovered that the most suitable photosensitizer is ZnTPPS4 because it had the biggest lethal influence on melanoma cells and the lowest lethal influence on fibroblast cells. The second most effective photosensitizer seemed to be MgTPPS4. On the basis of our results we can also assume that there is a higher accumulation of photosensitizer in a tumorous cell line. The higher concentration of photosensitizer and light dose resulted in more reactive oxygen species production and found more cells undergoing necrosis.