In vivo resistance to photofrin-mediated photodynamic therapy in radiation-induced fibrosarcoma cells resistant to in vitro Photofrin-mediated photodynamic therapy.

The effects of Photofrin-mediated photodynamic therapy (PDT) on the in vitro cell survival and in vivo tumor growth of murine radiation-induced fibrosarcoma (RIF) cell tumors have been examined following in vivo PDT treatment of tumors. The response to in vivo PDT is examined in tumors derived from RIF-1 mouse fibrosarcoma cells and in tumors derived from RIF-8A cells, which show in vitro resistance to PDT. A significant reduction in tumor volume is observed over the first three days following in vivo PDT treatment of either 5 or 10 mg/ kg. The reduction in tumor volume is greater for a 10 compared to a 5 mg/ml dose and occurs to a similar extent for both RIF-1 and RIF-8A tumors. The re-growth is significantly delayed for RIF-1 compared to RIF-8A tumors, indicating a greater response for RIF-1 tumors compared to RIF-8A tumors following PDT. A reduced response of the RIF-8A compared to the RIF-1 tumor cells is also observed in the clonogenic survival of cells from tumors that were excised and explanted in vitro immediately following in vivo PDT treatment. These data indicate that the intrinsic cell sensitivity to PDT is an important component in the mechanism that leads to tumor response following in vivo photodynamic therapy.     

An in vitro study of the use of photodynamic therapy for the treatment of natural oral plaque biofilms formed in vivo.

Seven-day oral plaque biofilms have been formed on natural enamel surfaces in vivo using a previously reported in situ device. The devices are then incubated with a cationic Zn(II) phthalocyanine photosensitizer and irradiated with white light. Confocal scanning laser microscopy (CSLM) of the biofilms shows that the photosensitizer is taken up into the biomass of the biofilm and that significant cell death is caused by photodynamic therapy (PDT). In addition, the treated biofilms are much thinner than the control samples and demonstrate a different structure from the control samples, with little evidence of channels and a less dense biomass. Transmission electron microscopy (TEM) of the in vivo-formed plaque biofilms reveals considerable damage to bacteria in the biofilm, vacuolation of the cytoplasm and membrane damage being clearly visible after PDT. These results clearly demonstrate the potential value of PDT in the management of oral biofilms.     

Antimicrobial photodynamic therapy and photodynamic inactivation, or killing bugs with dyes and light--a symposium-in-print

In antimicrobial photodynamic therapy, the photosensitizer (PS) in its ground singlet state absorbs light to give the excited singlet state that can transition to the long-lived triplet state. This PS triplet may undergo energy transfer (Type 2) or electron transfer (Type 1) to oxygen to form reactive oxygen species (singlet oxygen and/or hydroxyl radicals) that can kill both Gram-positive and Gram-negative bacteria and fungi. Infections in animal models can also be treated.     

Antimicrobial photodynamic therapy as a strategy to arrest enamel demineralization: a short-term study on incipient caries in a rat model

In this study we developed a rat model of incipient caries to investigate the short-term effects of antimicrobial photodynamic therapy (aPDT) on oral microbiota regulation and demineralization arrestment. Twenty-nine male rats were submitted to caries induction. Early carious lesion was confirmed by optical coherence tomography (OCT) 5 days after experiment beginning in five animals. The remaining animals (n = 24) were randomly divided into two groups: control (n = 12), animals were untreated; and aPDT (n = 12), animals were treated with 100 μM of methylene blue for 5 min and irradiated by a light emitting diode at λ = 645 ± 30 nm, fluence rate of 480 mW cm(-2) and exposure time of 3 min. Bacterial burden was evaluated before, immediately after, 3, 7 and 10 days following treatment, and total number of microaerophilic bacteria was counted. OCT was also used to quantify teeth demineralization. A significant bacterial decrease of about 1.6 log was observed immediately after aPDT. Besides, bacterial load in aPDT group remained lower than control until 10 days post-treatment (P < 0.05) and variation of optical attenuation coefficient before and after aPDT was 15%, corroborating to caries arrestment. Put together, these findings suggest that aPDT was competent to reduce cariogenic bacteria and to avoid further mineral loss.     

In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy.

In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)-induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 +/- 25 nm emission band relative to the fluorescence intensity integrated over the entire 400-600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.     

An artificial photosensitizer drug network for mitochondria-selective photodynamic therapy

We report a new class of photosensitizer drug networks that can home into mitochondria and provide geospatial phototoxicity for tumors. We take advantage of the cleavable chemical network between the photosensitizer drug and poly(ethylene glycol), and find a significant increase in the efficiency of the multimeric drug network in mitochondria uptake and tumor inhibition.     

An ultra-stable bio-inspired bacteriochlorin analogue for hypoxia-tolerant photodynamic therapy

Photodynamic therapy (PDT) greatly suffers from the weak NIR-absorption, oxygen dependence and poor stability of photosensitizers (PSs). Herein, inspired by natural bacteriochlorin, we develop a bacteriochlorin analogue, tetrafluorophenyl bacteriochlorin (FBC), by one-step reduction of tetrafluorophenyl porphyrin (TFPP). FBC can realize deep tissue penetration, benefitting from the strong NIR absorption. The reactive oxygen species (ROS) generation capacity of FBC can retain around 60% with a 1.0 cm-thick pork skin as the barrier. Besides, FBC could not only produce oxygen-dependent ¹O₂, but also generate less oxygen-dependent O₂⁻˙ and ˙OH to achieve excellent PDT even in hypoxic tumors. Moreover, FBC exhibits an ultra-high stability and it is almost unchanged even under visible light at room temperature for 15 months. Interestingly, the high reactivity of the fluorophenyl group makes it easy for FBC to produce FBC derivatives. A biocompatible FBC nanogel could be directly formed by blending FBC with SH–PEG–SH. The FBC nanogel displays excellent photodynamic efficacy in vitro and in vivo. Thus, FBC would be a promising PS for the clinical PDT of deep tumors.

A hypoxia-tolerant photosensitizer FBC-based nanoplatform with strong NIR absorbance and ultra-high stability was facilely prepared for PDT of deep tumors.     

Synthesis and in vivo activity of some porphyrindione derivatives with potential in photodynamic therapy

A number of synthetic bacteriochlorins were prepared from porphyrindiones. The potential of these compounds as sensitizers for photodynamic therapy was examined using the FANFT-induced urothelial cell carcinoma (AY-27) transplanted into rats. In a number of cases, tumor regression was significant following treatment with both drug and light.     

In vivo damage to chorioallantoic membrane blood vessels by porphycene-induced photodynamic therapy

Photodynamic therapy (PDT) was performed in the chick embryo chorioallantoic membrane (CAM) for the purpose of quantitative evaluation of several porphycenes as potential photosensitizers. Porphycenes are structural isomers of porphine possessing lower symmetry of the macrocycle and are characterized by 10-fold higher absorption at the therapeutic wavelengths for PDT (λ > 630 nm). PDT-induced damage to CAM blood vessels included vasoconstriction and blanching, as was monitored during irradiation and videotaped. Image analysis techniques enabled us to follow PDT-induced constriction of vessel diameter (to 50%), reduction of blood perfusion (to 40% lower optical density) and shrinkage of implanted tumours (to 10% of their original area). The observed PDT efficacy of functionalized porphycenes is positively correlated with the number of polar substituents.     

BODIPYs in antitumoral and antimicrobial photodynamic therapy: An integrating review

Nowadays, both cancer and infections caused by antibiotic resistant microorganisms are problems that affect the entire planet. Phototherapy (namely photodynamic therapy (PDT) and photodynamic inactivation (PDI) of microorganisms) are an alternative method for the treatment of these diseases. That requires adequate photosensitizers and, in this sense, boron-dipyrromethenes (BODIPYs) have interesting properties to act as phototherapeutic agents. In the present review, first, we describe the different strategies used to increase reactive oxygen species production. Then, we explain different architectures developed aiming to enhance the solubility of BODIPYs in biological media in order to optimize their targeting and delivery into the cells to be treated. Finally, we discuss the design of BODIPYs that are activated by specific stimuli present in the target tissues, allowing increasing the selectivity of the treatment. The data presented and discussed here show that BODIPYs are outstanding photosensitizers for the treatment of tumors and infections in the presence of oxygen and light.     

The history of photodetection and photodynamic therapy.

Light has been employed in the treatment of disease since antiquity. Many ancient civilizations utilized phototherapy, but it was not until early last century that this form of therapy reappeared. Following the scientific discoveries by early pioneers such as Finsen, Raab and Von Tappeiner, the combination of light and drug administration led to the emergence of photochemotherapy as a therapeutic tool. The isolation of porphyrins and the subsequent discovery of their tumor-localizing properties and phototoxic effects on tumor tissue led to the development of modern photodetection (PD) and photodynamic therapy (PDT). This review traces the origins and development of PD and PDT from antiquity to the present day.     

Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. In this study we report on the first use of a photochemical approach to destroy bacteria infecting a wound in an animal model. Following topical application, a targeted polycationic photosensitizer conjugate between poly-L-lysine and chlorin(e6) penetrated the gram (-) outer bacterial membrane, and subsequent activation with 660 nm laser light rapidly killed Escherichia coli infecting excisional wounds in mice. To facilitate real-time monitoring of infection, we used bacteria that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low-light imaging system. There was a light-dose dependent loss of luminescence in the wound treated with conjugate and light, not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the photodynamic treatment did not damage the host tissue. Our study points to the possible use of this methodology in the rapid control of wounds and other localized infections.     

Highly efficient drug delivery with gold nanoparticle vectors for in vivo photodynamic therapy of cancer

A highly efficient drug vector for photodynamic therapy (PDT) drug delivery was developed by synthesizing PEGylated gold nanoparticle conjugates, which act as a water-soluble and biocompatible "cage" that allows delivery of a hydrophobic drug to its site of PDT action. The dynamics of drug release in vitro in a two-phase solution system and in vivo in cancer-bearing mice indicates that the process of drug delivery is highly efficient, and passive targeting prefers the tumor site. With the Au NP-Pc 4 conjugates, the drug delivery time required for PDT has been greatly reduced to less than 2 h, compared to 2 days for the free drug.     

Experimental tests of the feasibility of singlet oxygen luminescence monitoring in vivo during photodynamic therapy

Singlet oxygen (1O2) is thought to be the cytotoxic agent in photodynamic therapy (PDT) with current photosensitizers. Direct monitoring of 1O2 concentration in vivo would be a valuable tool in studying biological response. Attempts were made to measure 1O2 IR luminescence during PDT of cell suspensions and two murine tumour models using the photosensitizers Photofrin II and aluminium chlorosulphonated phthalocyanine. Instrumentation was virtually identical to that devised by Parker in the one positive report of in vivo luminescence detection in the literature. Despite the fact that our treatments caused cell killing and tissue necrosis, we were unable to observe 1O2 emission under any conditions. We attribute this negative result to a reduction in 1O2 lifetime in the cellular environment. Quantitative calibration of our system allowed us to estimate that the singlet oxygen lifetime in tissue is less than 0.5 microsecond. Some technical improvements are suggested which would improve detector performance and perhaps make such measurements feasible.     

A first comparative study of purpurinimide-based fluorinated vs. nonfluorinated photosensitizers for photodynamic therapy

A first report on the synthesis and comparative in vitro-in vivo photosensitizing efficacy of various fluorinated and the corresponding nonfluorinated, purpurinimide-based photosensitizers is discussed. In preliminary in vivo screening, compared with the nonfluorinated analogs, purpurinimides bearing trifluoromethyl substituents showed enhanced photosensitizing efficacy. Among compounds (isomers) with similar lipophilicity, the position of the substituents was found to play a decisive role in biological efficacy.     

Synthesis,and in vitro and in vivo evaluation of a diphenylchlorin sensitizer for photodynamic therapy

This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13017 M(-1) cm(-1) for SIM01 and 22718 M(-1) cm(-1) for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 microg ml(-1)). However, phototoxicities were nearly identical for HT29 cells (0.45 microg ml(-1) for 5 h incubation followed by 300 mW, 20 J cm(-2)). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg(-1) dye dose and laser irradiation at 300 J cm(-2) (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg(-1) injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.     

Changes in tumor interstitial pressure induced by photodynamic therapy.

This study has examined the changes in tumor interstitial pressure exhibited during and after photodynamic therapy (PDT). The kinetics of these changes are marked by an initial decrease, followed by a rapid rise in tumor interstitial pressure. We have also employed two inhibitory agents to evaluate the different components of the pressure curve. Specially designed pressure chambers were seeded with chondrosarcoma and implanted subcutaneously in rats. Animals were injected with 0-50 mg/kg Photofrin II (i.v.) 7 days post-implantation and tumors were exposed to 0-540 J/cm2 630 nm 24 h later. Interstitial pressure was monitored via a transducer connected to the implanted chamber. Additional groups of animals were injected with either indomethacin (an inhibitor of thromboxane synthesis) or Ketanserin (a serotonin antagonist) before light treatment. Porphyrin doses of 10 mg/kg and above (135 J/cm2), or light doses of 135 J/cm2 and above (25 mg/kg Photofrin II) were effective in modifying interstitial pressure. Porphyrin doses greater than 25 mg/kg, or light doses greater than 270 J/cm2 produced no further increases in interstitial pressure. Animals given indomethacin (10 mg/kg i.p.) exhibited the initial decrease in pressure during light treatment, but showed no increase past baseline levels. Animals given Ketanserin (10 mg/kg i.p.) demonstrated no decrease in pressure during PDT, but showed the same elevations in pressure as controls. This suggests that two independent mechanisms account for the different components of the pressure curve, and that serotonin release may occur during PDT.     

Long-term effects of photodynamic therapy on fluorescence spectroscopy in the human esophagus.

Clinical interest in laser-induced fluorescence (LIF) spectroscopy and photodynamic therapy (PDT) is growing rapidly and may ultimately lead to close parallel use of these techniques. However, variations in LIF due to photosensitizer retention as well as tissue damage and healing processes may interfere with autofluorescence-based diagnostic methods. We have investigated the compatibility of these two techniques by quantifying PDT-induced changes in LIF in the human esophagus. Fluorescence spectra were collected endoscopically at excitation wavelengths (lambda ex) of 337, 400 and 410 nm in 32 patients. Measurements were performed immediately before and after PDT treatment with porfimer sodium and during follow-up procedures. In the months following PDT regions of reepithelialized squamous showed reduced autofluorescence in comparison with untreated squamous regions (P = 0.0007). Photosensitizer fluorescence was undetectable with lambda ex = 337 nm during follow-up procedures, whereas for lambda ex = 400 and 410 nm porfimer sodium fluorescence was noted for nearly a year after treatment. Therefore, residual photosensitizer fluorescence is likely to affect certain LIF-based diagnostic techniques during a period when patients are at high risk for tumor recurrence. Modification of LIF systems and/or the use of alternative photosensitizers may be required to optimize the detection of lesions in the post-PDT patient. Given the potential of LIF as a method for surveillance following cancer therapy, further investigation of the compatibility of specific LIF approaches with cancer pharmaceuticals may be warranted.