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Abstract
The intracellular localization of photosensitizers can be studied by different methods. One method involves homogenization of the cells followed by differential ultracentrifugation which leads to fractions enriched in nuclear, mitochondrial, and microsomal material as well as a supernatant fraction. More detailed information can be obtained by electron microscopy of cells exposed to light in the presence of photosensitizers. This method is based on the assumption that damage is primarily induced at intracellular sites where the concentration of photosensitizer is high. By irradiating the cells at 6 degrees C, where biochemical reactions are slow, and then incubating them for different times at 37 degrees C, it is possible to follow the development of damage. The amount of photosensitized damage to enzymes or cell functions whose localization in the cells is known gives information about the intracellular localization of the sensitizer. Fluorescence microscopy is the most direct method and is widely applicable because most photosensitizers fluoresce. Lipophilic dyes generally localize in membrane structures. In future more attention should be paid to the localization of dyes in lysosomes, as suggested by early reports. Mitochondria, the endoplasmic reticulum and nuclear membrane are other important loci for intracellular localization of sensitizers.
... It is also possible for the photosensitizer electron to perform electron transfer reactions with oxygen, forming reaction oxygen species [24]. These excited oxygen Pharmaceutics 2023, 15, 1721 4 of 27 molecules then act by inflicting photodynamic damage on nearby cells [32]. Affected cells will die via three main pathways; apoptosis, necrosis, or autophagy-associated cell death. ...
... It is also possible for the photosensitizer electron to perform electron transfer reactions with oxygen, forming reaction oxygen species [24]. These excited oxygen molecules then act by inflicting photodynamic damage on nearby cells [32]. Affected cells will die via three main pathways; apoptosis, necrosis, or autophagy-associated cell death. ...
As a prevalent medical problem that burdens millions of patients across the world, chronic wounds pose a challenge to the healthcare system. These wounds, often existing as a comorbidity, are vulnerable to infections. Consequently, infections hinder the healing process and complicate clinical management and treatment. While antibiotic drugs remain a popular treatment for infected chronic wounds, the recent rise of antibiotic-resistant strains has hastened the need for alternative treatments. Future impacts of chronic wounds are likely to increase with aging populations and growing obesity rates. With the need for more effective novel treatments, promising research into various wound therapies has seen an increased demand. This review summarizes photodynamic therapy, probiotics, acetic acid, and essential oil studies as developing antibiotic-free treatments for chronic wounds infected with Pseudomonas aeruginosa. Clinicians may find this review informative by gaining a better understanding of the state of current research into various antibiotic-free treatments. Furthermore. this review provides clinical significance, as clinicians may seek to implement photodynamic therapy, probiotics, acetic acid, or essential oils into their own practice.
... The consequences of PDT are highly dependent on the location of the PS at the time of illumination. This is due to the short half-life of singlet oxygen molecules, decreasing the probability that it interacts with molecules further away from its site of origin [55,59,60,60,61]. For this reason, the location of a PS during illumination coincides with the type of cellular damage caused. ...
... For this reason, the location of a PS during illumination coincides with the type of cellular damage caused. Because PS displays preferences for different (sub)cellular locations [61], PDT can therefore facilitate the destruction of solid tumors in several ways. These can be further divided into direct consequences of photodynamic effect, including damage to tumor cells, to the tumor (micro)environment, and/or disruption of the tumor surrounding vasculature and indirect effects involving the induction of an innate (inflammatory) immune response ( Figure 3) [41,[62][63][64]. ...
Background:
Photodynamic therapy (PDT) is an established, minimally invasive treatment for specific types of cancer. During PDT, reactive oxygen species (ROS) are generated that ultimately induce cell death and disruption of the tumor area. Moreover, PDT can result in damage to the tumor vasculature and induce the release and/or exposure of damage-associated molecular patterns (DAMPs) that may initiate an antitumor immune response. However, there are currently several challenges of PDT that limit its widespread application for certain indications in the clinic.
Methods:
A literature study was conducted to comprehensively discuss these challenges and to identify opportunities for improvement.
Results:
The most notable challenges of PDT and opportunities to improve them have been identified and discussed.
Conclusions:
The recent efforts to improve the current challenges of PDT are promising, most notably those that focus on enhancing immune responses initiated by the treatment. The application of these improvements has the potential to enhance the antitumor efficacy of PDT, thereby broadening its potential application in the clinic.
... Cercosporin has been of significant interest as it was the first plant pathogen toxin to be shown to be a photosensitizer, a compound that is activated by light to produce reactive oxygen species toxic to living cells ( Figure 2) [57]. Photosensitizers are documented to damage many cellular components including lipids, proteins, and nucleic acids, with the site of damage dependent on where the photosensitizer molecule localizes in cells, such as membranes, the cytoplasm, or nucleus [58]. ...
... In this activated state, it may react directly with oxygen to produce highly reactive singlet oxygen ( 1 O 2 ). It may also react through a reducing substrate to produce a reduced sensitizer molecule that can damage cells by reacting directly with cellular molecules or by generating free-radical forms of oxygen such as superoxide ( • O 2 − ) or the hydroxyl radical ( • OH) [57,58,90,104]. Cercosporin has been shown to produce both 1 O 2 and • O 2 − , but its toxicity has been primarily attributed to the production of 1 O 2 due to its high quantum yield and the ability of 1 O 2 quenchers to protect against cercosporin toxicity [105][106][107]. ...
Phytopathogenic cercosporoid fungi have been investigated comprehensively due to their important role in causing plant diseases. A significant amount of research has been focused on the biology, morphology, systematics, and taxonomy of this group, with less of a focus on molecular or biochemical issues. Early and extensive research on these fungi focused on taxonomy and their classification based on in vivo features. Lately, investigations have mainly addressed a combination of characteristics such as morphological traits, host specificity, and molecular analyses initiated at the end of the 20th century. Some species that are important from an economic point of view have been more intensively investigated by means of genetic and biochemical methods to better understand the pathogenesis processes. Cercosporin, a photoactivated toxin playing an important role in Cercospora diseases, has been extensively studied. Understanding cercosporin toxicity in relation to reactive oxygen species (ROS) production facilitated the discovery and regulation of the cercosporin biosynthesis pathway, including the gene cluster encoding pathway enzymes. Furthermore, these fungi may be a source of other biotechnologically important compounds, e.g., industrially relevant enzymes. This paper reviews methods and important results of investigations of this group of fungi addressed at different levels over the years.
... Bacterial cells have been shown to take up dyes such as phthalocyanines mesotetra(hydroxyphenyl)porphine (THPP) and acriflavine. Moan et al. (1989a) demonstrated that the uptake is governed by a number of factors such as whether the sensitiser is lipophilic and whether it aggregates. Also, in the case of heterogeneous photosensitisers, the uptake depends on the components of the photosensitiser and whether there is an uptake mechanism for each component, which would also govern its distribution (Moan et al. 1989a). ...
... Moan et al. (1989a) demonstrated that the uptake is governed by a number of factors such as whether the sensitiser is lipophilic and whether it aggregates. Also, in the case of heterogeneous photosensitisers, the uptake depends on the components of the photosensitiser and whether there is an uptake mechanism for each component, which would also govern its distribution (Moan et al. 1989a). Ben-Hur (1989) stated that the uptake also depended on a number of other factors such as the amount of serum in the growth medium, temperature and the structure of the photosensitiser. ...
Porphyromonas gingivalis has been implicated in the development of periodontal diseases and the current treatments used are not entirely successful in eradicating periodontopathogens from the pocket. Photodynamic therapy has been successfully used in the treatment of a number of cancers and has been used in vitro to kill a variety of bacteria. Lethal photosensitisation of Por. gingivalis using toluidine blue O (TBO) with Helium/Neon (HeNe) laser light was compared to lethal photosensitisation using aluminium disulphonated phthalocyanine (AIPCS2) with gallium aluminium arsenide (GaAs) laser light. It was found that there were substantial reductions in viable counts using TBO/HeNe laser light and when using AIPcS2/GaAs laser light. Substantial kills were obtained when lethal photosensitisation was carried out under conditions most likely to be encountered in the periodontal pocket (presence of serum, increasing pH values, different Por. gingivalis strains and biofilm-grown bacteria). The involvement of singlet oxygen and hydroxyl radicals was investigated by carrying out lethal photosensitisation in the presence of the singlet oxygen enhancer, deuterium oxide. It was found that singlet oxygen and possibly hydroxyl radicals are involved in the killing of Por. gingivalis. Distribution studies using 3H-TBO showed that most of the 3H-TBO was bound to the outer membrane and SDS-PAGE analysis showed that there were alterations to the outer and plasma membrane proteins from cells sensitised with TBO and exposed to laser light. It was also found that there was DNA degradation as a result of lethal photosensitisation. The final part of the study involved conjugating antibody against surface components of Por. gingivalis with TBO and specifically to target Por gingivalis to light-induced killing when in the presence of commensal bacteria or host tissue. When sensitised with the antibody/TBO conjugate and exposed to laser light, there were no significant reductions in viable counts of Streptococcus sanguis or human gingival fibroblasts, whereas all of the Por. gingivalis present were killed. In conclusion, Por. gingivalis can be killed effectively (100%) when sensitised with 82 μM TBO at an energy dose of 4.4 J. Type II and possibly type I mechanisms are involved in the killing of Por gingivalis and the outer and plasma membrane proteins and DNA are adversely affected by lethal photosensitisation. Damage to oral commensal organisms and oral host tissue can be avoided by using laser light in combination with TBO conjugated to antibody against Por gingivalis surface components.
... Finally, particular attention has been paid to the question of nanocarrier internalization and interaction with membranes (both biomimetic and cellular), and the importance of intracellular targeting has been addressed. 180 Review ...
... Subcellular localization of the photosensitizer is largely governed by its concentration and its physicochemical properties (molecular weight, lipophilicity, amphiphilicity, ionic charge, and protein binding characteristics) [179]. On the one hand, lipophilic, anionic dyes generally localize in membrane structures (including plasma, mitochondrial, endoplasmic reticulum and nuclear membranes), while hydrophilic materials seem to accumulate in lysosomes [180]. On the other hand, cationic sensitizers such as rhodamines and cyanines preferentially accumulate in mitochondria [181] due to electrical potential gradients across the mitochondrial membrane, allowing a targeted approach for PDT [182,183]. ...
Photodynamic therapy is a technique already used in ophthalmology or oncology. It is based on the local production of reactive oxygen species through an energy transfer from an excited photosensitizer to oxygen present in the biological tissue. This review first presents an update, mainly covering the last five years, regarding the block copolymers used as nanovectors for the delivery of the photosensitizer. In particular, we describe the chemical nature and structure of the block copolymers showing a very large range of existing systems, spanning from natural polymers such as proteins or polysaccharides to synthetic ones such as polyesters or polyacrylates. A second part focuses on important parameters for their design and the improvement of their efficiency. Finally, particular attention has been paid to the question of nanocarrier internalization and interaction with membranes (both biomimetic and cellular), and the importance of intracellular targeting has been addressed.
... In addition, the lifetime of the generated singlet oxygen is very short, limiting its diffusion to only 10-55 nm (Dysart and Patterson, 2005). Therefore, the photodynamic damage is likely to occur only in close proximity to the location of the PS, thus, the ROS generation has to be induced at the target site, e.g., inside the biofilm (Moan et al., 1989). The higher the concentration of the PS inside the biofilm, the better the therapeutic performance (Bekmukhametova et al., 2020). ...
Bacterial biofilms can pose a serious health risk to humans and are less susceptible to antibiotics and disinfection than planktonic bacteria. Here, a novel method for biofilm eradication based on antimicrobial photodynamic therapy utilizing a nanoparticle in conjunction with a BODIPY derivative as photosensitizer was developed. Reactive oxygen species are generated upon illumination with visible light and lead to a strong, controllable and persistent eradication of both planktonic bacteria and biofilms. One of the biggest challenges in biofilm eradication is the penetration of the antimicrobial agent into the biofilm and its matrix. A biocompatible hydrophilic nanoparticle was utilized as a delivery system for the hydrophobic BODIPY dye and enabled its accumulation within the biofilm. This key feature of delivering the antimicrobial agent to the site of action where it is activated resulted in effective eradication of all tested biofilms. Here, 3 bacterial species that commonly form clinically relevant pathogenic biofilms were selected: Escherichia coli , Staphylococcus aureus and Streptococcus mutans . The development of this antimicrobial photodynamic therapy tool for biofilm eradication takes a promising step towards new methods for the much needed treatment of pathogenic biofilms.
... This Ce4 ring current induced shift reflects a direct interaction with cellular phospholipids suggesting Ce4 association with phospholipid membranes. This is in agreement with the assumption that cellular membranes are the preferential intracellular localization 15 sites of porphyrinic PSs [71,72]. Our previous model studies on externally added amphiphilic Ce6 derivatives to unilamellar phospholipid vesicles as model membranes have suggested a preferential association with the choline containing head group regions of the phospholipids [61,73,74]. ...
Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is af-fected by nanoparticles. In a systematic study, high resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of the PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP, but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline an important protective role of the polymeric carriers following cell internalization. Moreover, the current study allowed - to our knowledge for the first time - to trace the intracellular PS localization on an atomic level by NMR methods.
... This Ce4 ring current-induced shift reflects a direct interaction with cellular phospholipids, suggesting Ce4 association with phospholipid membranes. This is in agreement with the assumption that cellular membranes are the preferential intracellular localization sites of porphyrinic PSs [71,72]. Our previous model studies on externally added amphiphilic Ce6 derivatives to unilamellar phospholipid vesicles as model membranes have suggested a preferential association with the choline-containing head group regions of the phospholipids [61,73,74]. ...
Porphyrinic photosensitizers (PSs) and their nano-sized polymer-based carrier systems are required to exhibit low dark toxicity, avoid side effects, and ensure high in vivo tolerability. Yet, little is known about the intracellular fate of PSs during the dark incubation period and how it is affected by nanoparticles. In a systematic study, high-resolution magic angle spinning NMR spectroscopy combined with statistical analyses was used to study the metabolic profile of cultured HeLa cells treated with different concentrations of PS chlorin e4 (Ce4) alone or encapsulated in carrier systems. For the latter, either polyvinylpyrrolidone (PVP) or the micelle-forming polyethylene glycol (PEG)-polypropylene glycol triblock copolymer Kolliphor P188 (KP) were used. Diffusion-edited spectra indicated Ce4 membrane localization evidenced by Ce4 concentration-dependent chemical shift perturbation of the cellular phospholipid choline resonance. The effect was also visible in the presence of KP and PVP but less pronounced. The appearance of the PEG resonance in the cell spectra pointed towards cell internalization of KP, whereas no conclusion could be drawn for PVP that remained NMR-invisible. Multivariate statistical analyses of the cell spectra (PCA, PLS-DA, and oPLS) revealed a concentration-dependent metabolic response upon exposure to Ce4 that was attenuated by KP and even more by PVP. Significant Ce4-concentration-dependent alterations were mainly found for metabolites involved in the tricarboxylic acid cycle and the phosphatidylcholine metabolism. The data underline the important protective role of the polymeric carriers following cell internalization. Moreover, to our knowledge, for the first time, the current study allowed us to trace intracellular PS localization on an atomic level by NMR methods.
... It is well established that the success of PDT outcome is strictly related to the extent of PS uptake by tumor cells, while the subcellular localization significantly affects the cell death mechanism [49,50]. Therefore, the in vitro uptake of freeTPCS 2a , TPCS 2a @NPs, or MSC-TPCS 2a @NPs by breast cancer models such as MCF7 adenocarcinoma and MDA-MB-231 triple negative BC cells, and in the non-malignant mammary epithelial MCF10A cell line, was evaluated by flow cytometric studies. ...
Despite substantial improvements in breast cancer (BC) treatment there is still an urgent need to find alternative treatment options to improve the outcomes for patients with advanced-stage disease. Photodynamic therapy (PDT) is gaining a lot of attention as a BC therapeutic option because of its selectivity and low off-target effects. However, the hydrophobicity of photosensitizers (PSs) impairs their solubility and limits the circulation in the bloodstream, thus representing a major challenge. The use of polymeric nanoparticles (NPs) to encapsulate the PS may represent a valuable strategy to overcome these issues. Herein, we developed a novel biomimetic PDT nanoplatform (NPs) based on a polymeric core of poly(lactic-co-glycolic)acid (PLGA) loaded with the PS meso-tetraphenylchlorin disulfonate (TPCS2a). TPCS2a@NPs of 98.89 ± 18.56 nm with an encapsulation efficiency percentage (EE%) of 81.9 ± 7.92% were obtained and coated with mesenchymal stem cells-derived plasma membranes (mMSCs) (mMSC-TPCS2a@NPs, size of 139.31 ± 12.94 nm). The mMSC coating armed NPs with biomimetic features to impart long circulation times and tumor-homing capabilities. In vitro, biomimetic mMSC-TPCS2a@NPs showed a decrease in macrophage uptake of 54% to 70%, depending on the conditions applied, as compared to uncoated TPCS2a@NPs. Both NP formulations efficiently accumulated in MCF7 and MDA-MB-231 BC cells, while the uptake was significantly lower in normal breast epithelial MCF10A cells with respect to tumor cells. Moreover, encapsulation of TPCS2a in mMSC-TPCS2a@NPs effectively prevents its aggregation, ensuring efficient singlet oxygen (1O2) production after red light irradiation, which resulted in a considerable in vitro anticancer effect in both BC cell monolayers (IC50 < 0.15 µM) and three-dimensional spheroids.
... The 1 O 2 produced after PDT has a very short lifetime (about 10-320 nanoseconds), which makes its diffusion in cells only about 10 to 55 nanometers [31]. Therefore, photodynamic damage occurs where the PS is localized within the vicinity of the location [32]. PS localization is associated with cell death pathways. ...
Photodynamic therapy (PDT) has been used clinically to treat cancer for more than 40 years. Some solid tumors, including esophageal cancer, lung cancer, head and neck cancer, cholangiocarcinoma, and bladder cancer, have been approved for and managed with PDT in many countries globally. Notably, PDT for gastric cancer (GC) has been reported less and is not currently included in the clinical diagnosis and treatment guidelines. However, PDT is a potential new therapeutic modality used for the management of GC, and its outcomes and realization are more and more encouraging. PDT has a pernicious effect on tumors at the irradiation site and can play a role in rapid tumor shrinkage when GC is combined with cardiac and pyloric obstruction. Furthermore, because of its ability to activate the immune system, it still has a specific effect on systemic metastatic lesions, and the adverse reactions are mild. In this Review, we provide an overview of the current application progress of PDT for GC; systematically elaborate on its principle, mechanism, and the application of a new photosensitizer in GC; and focus on the efficacy of PDT in GC and the prospect of combined use with other therapeutic methods to provide a theoretical basis for clinical application.
... Meanwhile, due to the low uptake efficiency of cancer cells, it is difficult to accumulate in appropriate organelles, such as mitochondria. Therefore, ROS are only produced on the surface of cancer cells and are not sufficient to eliminate cancer cells [26][27][28][29] . These two disadvantages markedly limit the application of traditional photosensitizers. ...
Mitochondria are well-acknowledged as ideal targets for tumor therapy due to their important role in energy supply and cellular signal regulation. Mitochondria-specific photosensitizers have been reported to be critical for inducing cell apoptosis. Two-photon fluorescence imaging provides a new technique for delineating biological structures and activities in deep tissues. Herein, we developed a new aggregation-induced emission (AIE) active photosensitizer by attaching a pyridinium group for mitochondrial targeting. The rationally designed photosensitizer (TTTP) exhibited excellent photophysical properties, good biocompatibility, reactive oxygen species (ROS) stimulation ability, anticancer efficacy, and two-photon imaging properties. TTTP was highly taken up by cells and accumulated specifically in mitochondria but was selectively cytotoxic to cancer cells. Under light irradiation, the generation of ROS was significantly boosted, leading to actively induced apoptosis. The in vivo tumor photodynamic therapeutic efficacy of TTTP showed significant inhibition of tumor growth. Furthermore, the underlying mechanism of TTTP tumor suppression revealed that the apoptosis agonist Bax was markedly up-regulated while the antagonist Bcl-xL was down-regulated. This research provides a potential mitochondrial-targeted phototherapeutic agent for effective therapy and two-photon fluorescence imaging.
... If the wavelength is longer than 800 nm, the absorption of single photons does not provide enough energy to excite the oxygen at its singlet state, causing a poor production of reactive oxygen species (ROS) for some biological mechanisms, even in the activated light [5]. PS is expected to localize in different subcellular locations, such as mitochondria, lysosomes, endoplasmic reticulum, and plasma membrane for the photodynamic damage leading to cell apoptosis [6]. ...
The Article Abstract is not available.
... Phospholipid membranes belong to the preferential localization sites of porphyrinic PSs in living systems [126,127]. Therefore, interactions with membrane models have been of great interest in the context of PDT. Small unilamellar vesicles (SUVs) composed of phospholipids (PLs) have been used as suitable simplified membrane models providing access to PL-bilayers for solution NMR studies. ...
Porphyrinic compounds are widespread in nature and play key roles in biological processes such as oxygen transport in blood, enzymatic redox reactions or photosynthesis. In addition, both naturally derived as well as synthetic porphyrinic compounds are extensively explored for biomedical and technical applications such as photodynamic therapy (PDT) or photovoltaic systems, respectively. Their unique electronic structures and photophysical properties make this class of compounds so interesting for the multiple functions encountered. It is therefore not surprising that optical methods are typically the prevalent analytical tool applied in characterization and processes involving porphyrinic compounds. However, a wealth of complementary information can be obtained from NMR spectroscopic techniques. Based on the advantage of providing structural and dynamic information with atomic resolution simultaneously, NMR spectroscopy is a powerful method for studying molecular interactions between porphyrinic compounds and macromolecules. Such interactions are of special interest in medical applications of porphyrinic photosensitizers that are mostly combined with macromolecular carrier systems. The macromolecular surrounding typically stabilizes the encapsulated drug and may also modify its physical properties. Moreover, the interaction with macromolecular physiological components needs to be explored to understand and control mechanisms of action and therapeutic efficacy. This review focuses on such non-covalent interactions of porphyrinic drugs with synthetic polymers as well as with biomolecules such as phospholipids or proteins. A brief introduction into various NMR spectroscopic techniques is given including chemical shift perturbation methods, NOE enhancement spectroscopy, relaxation time measurements and diffusion-ordered spectroscopy. How these NMR tools are used to address porphyrin–macromolecule interactions with respect to their function in biomedical applications is the central point of the current review.
... 216 It is noteworthy that photodynamic damage is likely to occur very close to the intracellular location of the PS, which plays a crucial part in the apoptosis process, in conjunction with other factors such as the overall PDT dose (PS concentration ⨰ light fluence). 217 Interestingly, SARS-CoV-2 has a number of distinguishing features including a protein envelope and lipids that would make it susceptible to treatment with PDT, 218 as well as positive stranded RNA virus and belongs to the beta CoVs category. It has a round/elliptic and often pleomorphic form, and a diameter of approximately 60-140 nm. ...
Initially, the SARS-CoV-2 virus was considered as a pneumonia virus; however, a series of peer reviewed medical papers published in the last eight months suggest that this virus attacks the brain, heart, intestine, nervous and vascular systems, as well the blood stream. Although many facts remain unknown, an objective appraisal of the current scientific literature addressing the latest progress on COVID-19 is required. The aim of the present study was to conduct a critical review of the literature, focusing on the current molecular structure of SARS-CoV-2 and prospective treatment modalities of COVID-19. The main objectives were to collect, scrutinize and objectively evaluate the current scientific evidence-based information, as well to provide an updated overview of the topic that is ongoing. The authors underlined potential prospective therapies, including vaccine and phototherapy, as a monotherapy or combined with current treatment modalities. The authors concluded that this review has produced high quality evidence, which can be utilized by the clinical scientific community for future reference, as the knowledge and understanding of the SARS-CoV-2 virus are evolving, in terms of its epidemiological, pathogenicity, and clinical manifestations, which ultimately map the strategic path, towards an effective and safe treatment and production of a reliable and potent vaccine.
... [30][31][32] Due to the short half-life and di®usion distance of singlet oxygen ( 1 O 2 ) and most radicals, intracellular location of the PSs is crucial to induce photodynamic damage during PDT. 33,34 During preclinical research, researchers in our group explored the molecular mechanisms of Photocyanine on human hepatocellular carcinoma (HepG2) cells to guide further drug development. 35,36 The amphipathic Photocyanine was found to permeate through the Photocyanine: A novel and e®ective pc-based ps for cancer treatment cell membrane of HepG2, predominately locate to lysosome and mitochondria, and to a lesser extent, concentrate in cytoplasm and on nuclear membrane. ...
As one of the three key components of photodynamic therapy (PDT), photosensitizers (PSs) greatly influence the photodynamic efficiency in the treatment of tumors. Photosensitizers with tetrapyrrole structure, such as porphyrins, chlorins and phthalocyanines, have been extensively investigated for PDT and some of them have already received clinical approval. However, only a few of porphyrin-based photosensitizers are available for clinical applications, and PDT has not received wide recognition in clinical practice. In this regard, PSs remain a limiting factor. Our research focuses on the rational design of new PSs. Photocyanine, a Zinc (II) phthalocyanine (ZnPc) type photosensitizer with low dark toxicity and high single oxygen quantum yield, is one of the promising PSs candidates and currently being tested in clinical trials. Here, we present an overview on the development of Photocyanine, including its design, synthesis, purification, characterization and preclinical studies, wishing to contribute to the research of more promising PSs.
... The distance of photodynamic damage is limited to the immediate intracellular localization of the PS (Moan et al., 1989) due to the short lifetime 1 O2 (approximately 10-320 ns) and limited diffusion in biological media (up to 100 nm) (Dysart and Patterson, 2005). ...
Photodynamic therapy (PDT) is an alternative cancer treatment which offers a more targeted and less invasive treatment regimen compared to traditional modalities. Temoporfin (mTHPC, medicinal product name: Foscan®), is one of the most potent clinically approved PS. However, its poor solubility in aqueous medium caused several complications of its administration. The present study is aimed at the development of drug-in-cyclodextrin-in-liposome (DCL) nanoparticles by coupling two independent delivery systems: cyclodextrin/mTHPC inclusion complexes and liposomal vesicles to improve the transport and penetration of mTHPC to the target tissue. The formation of inclusion complexes between cyclodextrins and mTHPC was studied in detail. Based on these data, single and double loaded mTHPC-DCLs have been prepared, optimized and characterized. It was demonstrated that mTHPC-DCLs are stable and almost all mTHPC is bound to β-CDs in the inner aqueous liposome lumen. The influence of DCLs on mTHPC accumulation, distribution and photodynamic efficiency was studied in human adenocarcinoma HT29 cellular monolayer and spheroid models. Using 3D multicellular HT29 tumor spheroids we demonstrated that trimethyl-β-CD-based DCL provides homogenous accumulation of mTHPC across tumor spheroid volume thus supposing optimal mTHPC distribution.
... Taking the short lifetime of the ROS (about 10-320 ns) into consideration, the time extension of the synergistic interaction between diverse components in H. perforatum total extract and biofilm constituents such as EPS may be crucial for the aPDT-induced antimicrobial efficacy 17,21 . The highest antimicrobial effect can be achieved when the photosensitizer remains as close as possible to the targeted bacteria 43,88 . ...
Due to increasing antibiotic resistance, the application of antimicrobial photodynamic therapy (aPDT) is gaining increasing popularity in dentistry. The aim of this study was to investigate the antimicrobial effects of aPDT using visible light (VIS) and water-filtered infrared-A (wIRA) in combination with a Hypericum perforatum extract on in situ oral biofilms. The chemical composition of H. perforatum extract was analyzed using ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS). To obtain initial and mature oral biofilms in situ, intraoral devices with fixed bovine enamel slabs (BES) were carried by six healthy volunteers for two hours and three days, respectively. The ex situ exposure of biofilms to VIS + wIRA (200 mWcm−2) and H. perforatum (32 mg ml−1, non-rinsed or rinsed prior to aPDT after 2-min preincubation) lasted for five minutes. Biofilm treatment with 0.2% chlorhexidine gluconate solution (CHX) served as a positive control, while untreated biofilms served as a negative control. The colony-forming units (CFU) of the aPDT-treated biofilms were quantified, and the surviving microorganisms were identified using MALDI-TOF biochemical tests as well as 16 S rDNA-sequencing. We could show that the H. perforatum extract had significant photoactivation potential at a concentration of 32 mg ml−1. When aPDT was carried out in the presence of H. perforatum, all biofilms (100%) were completely eradicated (p = 0.0001). When H. perforatum was rinsed off prior to aPDT, more than 92% of the initial viable bacterial count and 13% of the mature oral biofilm were killed. Overall, the microbial composition in initial and mature biofilms was substantially altered after aPDT, inducing a shift in the synthesis of the microbial community. In conclusion, H. perforatum-mediated aPDT using VIS + wIRA interferes with oral biofilms, resulting in their elimination or the substantial alteration of microbial diversity and richness. The present results support the evaluation of H. perforatum-mediated aPDT for the adjunctive treatment of biofilm-associated oral diseases.
... The higher subcellular localization of sulfonamide bacteriochlorins in the ER and mitochondria is consistent with their higher photoxicities than F2BOH. It is worth to notice that the localization of a photosensitizer is important to determine the initial targets of PDT [18,55,56]. Furthermore, to confirm the colocalization of Cl2BHep (red signal) with each organelle-specific probe (green signal) more clearly, the spectral profiles for these components were defined. ...
: Photodynamic therapy (PDT) augments the host antitumor immune response, but the role of the PDT effect on the tumor microenvironment in dependence on the type of photosensitizer and/or therapeutic protocols has not been clearly elucidated. We employed three bacteriochlorins (F2BOH, F2BMet and Cl2BHep) of different polarity that absorb near-infrared light (NIR) and generated a large amount of reactive oxygen species (ROS) to compare the PDT efficacy after various drug-to-light intervals: 15 min. (V-PDT), 3h (E-PDT) and 72h (C-PDT). We also performed the analysis of the molecular mechanisms of PDT crucial for the generation of the long-lasting antitumor immune response. PDT-induced damage affected the integrity of the host tissue and developed acute (protocol-dependent) local inflammation, which in turn led to the infiltration of neutrophils and macrophages. In order to further confirm this hypothesis, a number of proteins in the plasma of PDT-treated mice were identified. Among a wide range of cytokines (IL-6, IL-10, IL-13, IL-15, TNF-α, GM-CSF), chemokines (KC, MCP-1, MIP1α, MIP1β, MIP2) and growth factors (VEGF) released after PDT, an important role was assigned to IL-6. PDT protocols optimized for studied bacteriochlorins led to a significant increase in the survival rate of BALB/c mice bearing CT26 tumors, but each photosensitizer (PS) was more or less potent, depending on the applied DLI (15 min, 3 h or 72 h). Hydrophilic (F2BOH) and amphiphilic (F2BMet) PSs were equally effective in V-PDT (>80 cure rate). F2BMet was the most efficient in E-PDT (DLI = 3h), leading to a cure of 65 % of the animals. Finally, the most powerful PS in the C-PDT (DLI = 72 h) regimen turned out to be the most hydrophobic compound (Cl2BHep), allowing 100 % of treated animals to be cured at a light dose of only 45 J/cm2.
... The lifetime of singlet oxygen greatly decreases in biological environment due to the presence of various quenchers, and is calculated to be about 10-330 ns [32]. This short lifetime allows the diffusion of singlet oxygen to a distance from 10 to 55 nm at the sub-cellular [H 33H , H34H], thus limiting the photodestructive effect to the immediate intracellular localization of the PS [35]. ...
Photodynamic therapy (PDT) is a photochemical-based modality of cancer treatment that uses a combination of a photosensitizer, light and molecular oxygen. Application of liposomal nanocarriers to deliver photosensitizers to tumor targets has become a major direction of PDT research. The present study investigates conventional and sterically stabilized liposomal formulations of the photosensitizer mTHPC, Foslip® and Fospeg®, with a view to determine the parameters for optimizing liposomal PDT. The characterization of in vitro behaviour of liposomal mTHPC was conducted, with an emphasis on drug localization, aggregation state and photophysical properties of the compounds in liposomes. We demonstrated the monomeric state of mTHPC in lipid vesicles and a partial localisation of mTHPC in Fospeg® in a PEG shell, while the main part was bound to the lipid bilayer. We further studied the drug release kinetics and binding pattern to serum proteins and the destruction of liposomes in serum. With this aim, a fluorescence-based methodology of estimating mTHPC release both in vitro and in vivo was developed, as well as an in vitro assay to characterize liposome destruction. The release of mTHPC from PEGylated liposomes was delayed compared with conventional liposomes along with greatly diminished liposome destruction. Knowledge of these parameters allows to better predict the drug release rate, pharmacological parameters and in vivo tumoricidal effect. The PDT treatment could be more advantageous with Fospeg® compared to mTHPC embedded in conventional liposomes
... Pour des concentrations intracellulaires identiques, l'oxygène singulet, qui est l'espèce cytotoxique majoritairement responsable de l'efficacité PDT, présente un temps de vie compris entre 10-330 ns dans un milieu biologique [63]. Ce court temps de vie limite la diffusion de l'oxygène singulet à une distance de 10 à 55 nm dans la cellule [66], [263], qui réduit les effets phototoxiques à la localisation immédiate du PS [264]. C'est la raison pour laquelle une localisation lysosomale semble être moins favorable en terme de phototoxicité qu'une localisation dans le réticulum endoplasmique ou la mitochondrie [262]. ...
La thérapie photodynamique (PDT) est une modalité de traitement des cancers prometteuse, mettant en jeu une action combinée de l’oxygène moléculaire, de la lumière et d’un photosensibilisateur (PS). Néanmoins, les PSs utilisés souffrent d’une faible solubilité dans les milieux aqueux ainsi que d’un tumorotropisme limité qui sont des barrières à la réussite du traitement. Ainsi, actuellement, une attention particulière est portée au développement de nanoparticules (NPs) capables de pallier les défauts des PSs. Notre travail a consisté à étudier des dendrimères poly(amidoamine) (PAMAM), macromolécules polymériques tridimensionnelles, conjugués via une liaison covalente au PS, la Chlorine e6 (Ce6). Cette construction nous a permis de vectoriser 32 molécules de Ce6 par dendrimère. La production d’oxygène singulet et l’émission de fluorescence ont été modérément affectées par le greffage covalent de la Ce6 aux NPs. In vitro, les dendrimères PAMAM ont permis d’accroitre l’efficacité PDT de la Ce6 en potentialisant son internalisation cellulaire via un mécanisme actif d’endocytose. Néanmoins, l’efficacité PDT des NPs est limitée par la concentration locale élevée en Ce6 en périphérie des dendrimères qui réduit son rendement quantique en oxygène singulet moléculaire, espèce cytotoxique. Une libération de la Ce6 permettrait ainsi de potentialiser l’efficacité PDT des NPs en restaurant notamment les propriétés photophysiques de la Ce6. La suite de ce travail a été de concevoir une NP capable de libérer la Ce6 sous l’action d’estérases retrouvées dans les cellules. Leur caractérisation a permis de démontrer en solution que les propriétés photophysiques de la Ce6 étaient rétablies à la suite de son relargage des NPs. Cette dernière construction clivable est prometteuse pour de futures applications en PDT
... Lifetime of singlet oxygen varies from about 4 µs in water to 25-100 µs in non-polar organic solutions and greatly decreases (about 10-330 ns) in biological systems due to the presence of various quenchers (Baker & Kanofsky, 1992). This short lifetime allows the diffusion of singlet oxygen to a distance from 10 to 55 nm at the sub-cellular level (Dysart & Patterson, 2005;Moan & Berg, 1991), thus limiting the PDT effect to the immediate intracellular localization of the PS (Moan et al., 1989). ...
Photodynamic therapy (PDT) is a minimally invasive photochemical treatment with a promising clinical track record for oncological and some other disesases. Most PDT-drugs (photosensitizers) including a second-generation PS meta-tetra(hydroxyphenyl)chorin (mTHPC) are highly hydrophobic and require delivery systems. To improve mTHPC solubility and pharmacokinetic properties, β-cyclodextrins (β-CDs) derivatives were proposed.The present study investigates the effect of β-CDs on mTHPC behavior at various stages of its distribution in vitro and in vivo. Interaction of mTHPC with 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 and cellular cultures in vitro. In vivo study confirms the fact that the use of β-CDs allows modifying 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. In conclusion, application of β-CD derivatives can open up new possibilities to modify and control biodistribution and pharmacokinetics of mTHPC in the course of PDT.
... The lifetime of singlet oxygen ( 1 O 2 ) is very short (∼10-320 ns), limiting its diffusion to only approximately 10-55 nm in cells [12]. Thus, photodynamic damage is likely to occur very close to the intracellular location of the PS [13]. PDT can kill cells via the three main morphologies of cell death: apoptotic, necrotic and autophagy-associated cell death ( Figure 2). ...
Photodynamic therapy (PDT)was discoveredmore than 100 years ago, and has since become a well-studied therapy for cancer and various non-malignant diseases including infections. PDT uses photosensitizers (PSs, non-Toxic dyes) that are activated by absorption of visible light to initially form the excited singlet state, followed by transition to the long-lived excited triplet state. This triplet state can undergo photochemical reactions in the presence of oxygen to form reactive oxygen species (including singlet oxygen) that can destroy cancer cells, pathogenic microbes and unwanted tissue. The dual-specificity of PDT relies on accumulation of the PS in diseased tissue and also on localized light delivery. Tetrapyrrole structures such as porphyrins, chlorins, bacteriochlorins and phthalocyanines with appropriate functionalization have been widely investigated in PDT, and several compounds have received clinical approval. Other molecular structures including the synthetic dyes classes as phenothiazinium, squaraine and BODIPY (boron-dipyrromethene), transition metal complexes, and natural products such as hypericin, riboflavin and curcumin have been investigated. Targeted PDT uses PSs conjugated to antibodies, peptides, proteins and other ligands with specific cellular receptors. Nanotechnology has made a significant contribution to PDT, giving rise to approaches such as nanoparticle delivery, fullerene-based PSs, titania photocatalysis, and the use of upconverting nanoparticles to increase light penetration into tissue. Future directions include photochemical internalization, genetically encoded protein PSs, theranostics, two-photon absorption PDT, and sonodynamic therapy using ultrasound.
... 26 Thus, the mechanism of cell death induced by PDT depends on the intracellular localization of the photosensitizer. 27,28 Due to the limitation of light penetration and the targeting of the PS, novel strategies in PDT have been developed over the past several decades. With the development of nanotechnology, many drugdelivery platforms have been applied to PDT, including liposomes, nanoemulsions, micelles, polymer nanoparticles, and silica nanoparticles. ...
Photonics immunotherapy is a novel cancer treatment strategy that combines local phototherapy and immunotherapy. Phototherapy is a noninvasive or minimally invasive therapeutic strategy for local treatment of cancer, which can destroy tumor cells and release tumor antigens, inducing an in situ antitumor immune response. Immunotherapy, including the use of antibodies, vaccines, immunoadjuvants and cytokines, when combined with phototherapy, could bring a synergistic effect to stimulate a host immune response that effectuates a long-term antitumor immunity. This review will focus on the development of photonics immunotherapy and its systemic antitumor immunological effects.
... However, the high-energy radical in the system has the potential to induce oxidative damage to the encapsulated cells. Free radicals can cause damage to cells membrane, nucleic acids and proteins (Atsumi T et al., 1998;Moan J et al., 1989 caused minimal toxicity (cell death) over a broad range of mammalian cell types and species. The cytotoxicity of photoinitiators has been thought to be due in part to the hydrophobicity since permeability through phospholipid bilayers of cellular membranes increases with the hydrophobicity of a compound. ...
... This is extremely important, since it indicates that fluorescence microscopy can be used to identify these targets. In this way the following targets have been identified: all membranes, mitochondria, lysosomes, endoplasmic Reticulum, and Golgi complex [50,53,54]. Furthermore, since practically no PDT sensitizers localize in the nucleus (being negatively charged at pH~7), PDT causes little DNA damage and few chromosome changes, indicating a low carcinogenic potential. ...
The sections in this article areIntroductionThe Role of Oxygen in Photodynamic Therapy: 1O2 GenerationDependence of the Photosensitizing Effect on O2 ConcentrationThe Oxygenation Status in Tumors and Normal TissuesPDT-Induced Reduction of Tumor OxygenationO2 Consumption (Primary Reduction)Vascular Damage (Secondary Reactions)Photosensitizer Photobleaching (Secondary Reactions)Methods to Reduce Tumor Deoxygenation During PDTLow Fluence RatesFractionated Light ExposureOther Methods
Changes of Quantum Yields Related to Photosensitizer RelocalizationChanges of Optical Penetration Caused by Changes in O2 ConcentrationConclusion
Keywords:photodynamic therapy;oxygen;photosensitization;oxygenation;tumor deoxygenation;cancer
Photodynamic therapy is an effective method for treating superficial forms of malignant neoplasms, characterized by a minimal risk of damage to normal tissues. In this study, we presented our experience of treating cancer of the oral mucosa using photodynamic therapy, and analyzed the immediate and long-term results of treatment. 38 patients with squamous cell carcinoma of oral cavity mucosa, with a depth of invasion no more than 7 mm, were included in the study. All patients underwent photodynamic therapy with chlorine e6 based photosensitizer. Photosensitizers were administered intravenously 3 hours before irradiation, at a dosage of 1 mg/kg of the patient’s weight. Photodynamic therapy was performed with the following parameters: P – 1.0 W, Ps – 0.31 W/cm2, E – 300 J/cm2. The area of one irradiation field ranged 1.0-2.0 cm2. Treatment effect was evaluated by RECIST 1.1. Overall survival, cancer-specific survival, and disease-free survival were calculated using Kaplan-Meier curves. Evaluation of adverse events was made by .TCAE 5.0 criteria. At 35 (92.1%) out of 38 cases, complete regression was observed after photodynamic therapy. Among them in 3 out of 35 patients relapse was diagnosed in 11.5 to 43.2 months. The total number of patients who didn’t respond to treatment was 6 (15.8%). Follow-up period was 4.2-87.3 months. (mean 42.9). 34 (89.5%) out of 38 patients are alive, 1 (2.6%) died from progression, and three died from other causes. The 5-year overall survival rate was 82.1%, cancer-specific survival rate was 97.0%, and disease-free survival rate was 81.1%. Among the factors significantly (p < 0.05) influencing relapse-free survival: depth of invasion < 5 mm (p – 0.013) and the presence of leukoplakia (p – 0.007). When assessing cancer-specific survival, factors worsening the prognosis were: age >70 years (p – 0.034) and the presence of leukoplakia (p – 0.007). Photodynamic therapy is an alternative treatment method of oral cancer superficial lesions, in case of proper assessment of primary lesion and in case of possibility of full irradiation of the tumor. Moreover, after using photodynamic therapy, the underlying connective-muscular structures are preserved, which promotes rapid healing with minimal scarring, the functions of the affected organ remain intact, and cosmetic defects do not form.
Photodynamic therapy (PDT) has been established as a substitute modality for managing nonneoplastic and neoplastic diseases. It is a non-invasive treatment comprising three non-toxic components, i.e., photosensitizer (PS), light, and molecular oxygen. The simultaneous interaction of three components generates highly toxic reactive oxygen species (ROS). These ROS are subsequently involved in the demolition of unwanted cells at specific sites. The discovery of biocompatible photosensitizers with optimized properties is critical for accomplishing the therapeutic effect. This book chapter describes the uses of various photosensitizers, their PDT applications in antibacterial and anticancer treatment, and the mechanisms of cell death. This chapter also included the principle and mechanism of PDT and a list of clinically approved photosensitizers for PDT application.
Subject. The tendency of growing antibiotic resistance causes scientific medical community to develop new antimicrobial treatment protocols. Recently, the increased interest in photodynamic therapy has been noted. Photodynamic therapy (PDT) is a non-surgical method of treatment patients with inflammatory diseases and neoplasms in the maxillofacial region, based on the interaction between special light-sensitive chemical compounds — photosensitizers and light radiation. The literature reports the existence of synthetic and natural photosensitizers. Despite the higher stability present by the synthetic photosensitizers, they are more prone to collateral effects. Recently, a growing body of evidence shows the promising applications of curcumin against different diseases, including the pathologies in maxillofacial region. Curcumin is a bioactive compound isolated from the roots of Curcuma longa that has antibacterial, antiviral, anti-inflammatory, and antioxidant properties. The disadvantage of curcumin is that it is unstable at physiological pH, has low water solubility and is rapidly metabolized by the body. The objective of this work is to review current research aimed at improving curcumin as a photosensitizer used for photodynamic therapy. Methodology. The analysis of scientific articles from databases of medical and biological publications — scientific electronic library (Elibrary), PubMed and Web of Science, dedicated to the use of curcumin in photodynamic therapy. Results and conclusion. The results of modern research in the field of laser technologies presented in this review indicate that photodynamic therapy with curcumin, as a photosensitizer is a promising treatment option in many fields of medicine. The aforementioned scientific studies give the understanding that the study and improvement of delivery systems for curcumin photosensitizer by combining it with nanoparticles is a scientific interest.
Sugar beet (Beta vulgaris L.) is commercially cultivated in the northern temperature zone, i.e. between 30° and 60° latitudes north. Sugar beet is the second source of sugar (sucrose) after sugarcane; it is covering about 40% of the world’s sugar demands. On the contrary with sugarcane, sugar beet stores sugar in the roots not the stalks as in sugarcane. Like other crops, the plant density in the unit area is one of the most important factors affecting sugar beet production. Various fungal pathogens can decrease the number of cultivated plants and cause substantial economic losses at all plant stages, especially damping-off diseases in the seedling stage and root rot diseases during growth. On the other hand, foliar diseases (leaf spots, rust, powdery mildew, and viral diseases) also affect sugar production as well as the quality of roots. In this chapter, we will discuss the economic root diseases and foliar diseases that affect sugar beet and sucrose production quantitatively and qualitatively. This chapter will discuss foliar and root diseases and the etiology, epidemiology, and management of each disease.KeywordsDisease controlEpidemiologyPlant pathogensSugar beet
The development of improved photosensitizers is a key aspect in the establishment of photodynamic therapy (PDT) as a reliable treatment modality. In this chapter, we discuss how molecular design can lead to photosensitizers with higher selectivity and better efficiency, with focus on the importance of specific intracellular targeting in determining the cell death mechanism and, consequently, the PDT outcome.Key wordsPhotosensitizerActivatable photosensitizersPhotobleachingDrug deliveryOrganelle targetingCell death
Photodynamic therapy has great potential to treat diverse types of cancer and infections. PDT involves the use of a photoactive drug called photosensitizer and visible light of the appropriate wavelength. The excited photosensitizer generates reactive oxygen species, which kill the cancer cells and microorganisms and destroy the tumor. PDT also generates immune responses by generating acute inflammation that activates the innate immune response. This is followed by priming of tumor-specific T lymphocytes that have the potential to destroy distant untreated tumor cells, besides developing a long-term memory “shield” that is effective in fighting possible recurrence of the cancer. Additionally, PDT can play a role in overcoming the escape mechanism used by progressing tumors trying to escape immune attack. Moreover, in cases of infections, PDT can have a beneficial effect by attracting and accumulating neutrophils into the infected area that can directly kill the bacterial cells.
Photodynamic therapy (PDT) produces localised destruction of tissue with light after prior administration of a photosensitising agent. Connective tissue is relatively unaffected but attempts to treat the entire urothelium in the bladder using the photosensitiser Photofrin have led to detrusor muscle scarring and irritable, contracted bladders and obstructive uropathy. This thesis studied PDT on the normal rat bladder using the photosensitising agent, 5-aminolaevulinic acid (ALA). ALA solution was given intravesically, and the kinetics of the active derivative, protoporphyrin IX (PpIX) followed with fluorescence microscopy. Peak urothelial levels were seen at 5 hours, when treatment with 50J red light gave uniform urothelial necrosis without muscle damage which healed by regeneration. The PDT effect was enhanced by giving the iron chelator, CP94. Further studies looked at PDT on normal canine prostate using 3 photosensitisers: ALA, meso-tetra-(m-hydroxyphenyl)chlorin (mTHPC) and aluminium disulphonated phthalocyanine (A1S2Pc). Kinetic studies were undertaken on serial biopsies after sensitisation. Light was delivered interstitially and the animals killed up to 90 days later, revealing glandular necrosis (2.5cm diam. mTHPC, 1.2cm A1S2PC, 0.2cm ALA) but little effect on connective tissues, and no change in the gland size and shape. Urethral damage sometimes caused urinary retention which resolved in a week. Deliberate treatment of the sphincter in rats caused incontinence in 25%. Neoplastic areas of both prostate and bladder take up at least as much photosensitiser as adjacent normal tissue. Thus PDT using intravesical ALA is promising for carcinoma in situ of the bladder and preventing bladder tumour recurrence and PDT with mTHPC for prostate cancers localised to the gland, care being taken to avoid the sphincter. Both techniques are now ready for preliminary clinical trials.
La thérapie photodynamique (PDT) est basée sur l'action d'un photosensibilisateur qui en présence de la lumière et de l'oxygène induit la synthèse d'espèces réactives de l'oxygène, toxiques pour les cellules. La PDT a été utilisée pour le traitement et la prévention du cancer de la vessie. Les études ont été réalisées in vivo. La PDT a été développé en utilisant l'Hexvix® précurseur de la protoporphyrine IX (PpIX). Les paramètres biologiques liés à la PpIX et qui régissent l'efficacité du traitement photodynamique des tumeurs vésicales ont été déterminés en fonction de deux concentrations d'Hexvix® (8 et 16 mM) induisant des effets photodynamiques diamétralement opposés. L'efficacité du traitement photodynamique obtenue pour 8 mM d'Hexvix® est liée à la localisation mitochondriale de la PpIX induisant des dommages pro-apoptotiques. La PDT entraîne la diminution significative du pourcentage de l'implantation tumorale après la TUR et permet ainsi de prévenir les récidives du cancer de la vessie.
La thérapie pbotodynamique (PDT est une modalité de traitement des petites tumeurs localisées accessibles à la lumière. Son principe repose sur l'action conjuguée d'un photo sensibilisant, de la lumière et de l'oxygène. Une dosimétrie correcte est nécessaire pour assurer le traitement complet et des résultats reproductibles. Un élément clé de la dosimétrie à prendre en compte est le photoblanchiment, étant donné qu'il diminue la concentration du photosensibilisant au cours du traitement. Il est donc indispensabte d'appréhender les mécanismes du photoblanchiment pour maîtriser au mieux le traitement. Le rendement quantique de photoblanchiment de la m-THPBC en solution avec des protéines est de 5.7 X 10-4, les formes agrégées de photosensibilisants photoblanchissent plus lentement que les formes monomérisées. La m-THPBC se transforme sous l'effet de la lumière en m-THPC, ce rendement de phototransformation est influencé par l'état d'agrégation de la molécule. D'autres photoproduits tels que la m-THPBC di-bydroxylée et la m-THPC dibydroxylée ainsi que des dérivés dipyrines ont été observés. L'espèce responsable du photoblanchiment a été identifiée comme étant l'oxygène singulet. ln vitro, la LD50 de la mTHPBC est 0.7 J cm-2. Le photoblanchiment est 3 à 5 fois plus sensible que celui de la m-THPC. L'étude de la localisation intra-cellulaire des photosensibilisants a montré une différence, la m-THPC s'accumulant dans le réticulum endoplasmique alors que la m-THPBC se localise dans les mitochondries. ln vivo, la m-THPBC possède un photoblanchiment 4 à 10 fois plus élevé que la m-THPC, ainsi qu'une bonne accumulation tumorale. Ces résultats in vitro et in vivo sont particulièrement intéressants en terme de ratio thérapeutique, de sélectivité du traitement et de photosensibilité cutanée.
Photodynamic therapy (PDT) is a useful tool against cancer and various other diseases. PDT is capable to induce different cell death mechanisms, due to the PDT evoked reactive oxygen species (ROS) production and is dose dependent. It is known that cytoskeleton is responsible for numerous cell functions, including cell division, maintenance of cell shape, their adhesion ability and movement. PDT initiated redistribution and subsequent disintegration of cytoskeletal components that precedes cell death. Here was present our results in HeLa and G361 cells subjected to sublethal PDT treatments using α,β,χ,δ porphyrin-Tetrakis (1-methylpyridinium-4-yl) p-Toluenesulfonate porphyrin (TMPyP). The photosensitizer (PS) induced transient increasing of mitotic index (MI) observable early after PDT, cell cycle arrest, microtubule (MTs) disorganization of interphase cells, aberrant mitosis and formation of rounded cells with partial loss of adherence. Some cells were partly resistant to PDT induced MTs disorganization. The differences between both cell lines to PDT response were described. This is the first evidence of TMPyP - PDT induced microtubule disorganization and the cell death mechanisms known as mitotic catastrophe and the first detail analysis of microtubule aberrations of mitotic and interphase cells in HeLa and G361 cell lines. New modification of techniques of protein immunolabeling was developed.
Most nonviral gene transfection vectors deliver transfecting DNA into cells through the endocytic pathway (1,2). Poor escape from endocytic vesicles in many cases constitutes a major barrier for delivery of a functional gene, since the endocytosed transfecting DNA is unable to reach the cytosol and be further transported to the nucleus, but rather is trapped in endocytic vesicles and finally degraded in lysosomes (3). Therefore, the development of endosome-disruptive strategies is of great importance for the further progress of gene transfection. We have developed a new technology, termed photochemical internalization (PCI), to achieve light-inducible permeabilization of endocytic vesicles (4–8). The technology is based on photochemical reactions initiated by photosensitizers localized in endocytic vesicles and inducing rupture of these vesicles upon light exposure (4). This leads to the release of endocytosed macromolecules such as transfecting DNA from endocytic vesicles into the cytosol (Fig. 1). As a light-dependent treatment, PCI-mediated transfection (photochemical transfection) allows the possibility of directing the gene delivery to a desired site, e.g., achieving tumor-specific expression of a therapeutic gene in gene therapy in vivo. Fig. 1. Principle of photochemical internalization (PCI). I, endocytosis of the pho-tosensitizer (S) and the transfecting gene (G); II, localization of the photosensitizer and the transgene in the same endocytic vesicles; III, rupture of endosomal membrane upon light exposure and subsequent release of the transfecting gene into the cytosol.
Photodynamic therapy is a novel treatment for solid tumors based on the selective induction of cell death by the generation of cytotoxic reactive oxygen species within neoplastic tissues. Oxygen photosensitization is promoted as a consequence of the activation (using light) of a photosensitizer, which must reach the desired tissue by cellular transport. Hydrophobicity (expressed as the logarithm of octanol/water partition coefficient, logP), becomes a key factor in these processes. Unfortunately, there is no computational method to unambiguously predict the logP value for high hydrophobic photosensitizers. In this study, a total of 12 computational methods have been tested for predicting the logP value of tetrapyrrolic derivatives. Furthermore, in the attempt to correlate logP with experimental HPLC measurements (log(k')), validation of the results leads to the proposal of a sigmoidal regression for the two parameters (log(k') and logP).
The investigation of the influence of mTHPC distribution in tumor, plasma and leukocytes on PDT response shows that photosensitizer accumulation in leukocytes exhibits a good correlation with PDT efficacy. This result suggests that leukocytes could play an important role in the mechanism of PDT-induced vascular damage.The study of mTHPC monomerisation in the course of interactions with plasma proteins demonstrates slow rate of disaggregation kinetics. The fraction of aggregated mTHPC at equilibrium and sensitizer disaggregation rates strongly depends on protein content and incubation temperature. The data obtained show that in aqueous media mTHPC forms free large-scale aggregates with strong interaction between sensitizer molecules.Kinetic analysis demonstrates that mTHPC is characterized by very slow redistribution rates from the complexes with plasma proteins. Low redistribution rates as compared to other sensitizers are consistent with the unique binding properties of mTHPC. The existence of both collisional and aqueous mediated transfer of mTHPC was supposed. Confocal microscopy study reveals diffuse mTHPC localization pattern at 3h and formation of highly fluorescent spots of sensitizer at 24h incubation in MCF-7 cells. The parameters of sensitizer absorption, fluorescence lifetime and photobleaching have shown that at 24h incubation mTHPC is much more aggregated compared to 3h. The yield of cellular photoinactivation is about 2 times higher for 3h time point. Such difference is attributed to different sensitizer aggregation states and interactions with cellular components. Theoretical and spectroscopic study of mTHPC, mTHPP and mTHPBC allowed us to define their aggregates structure in aqueous media. For this purpose new quantum mechanic semi-empirical method was developed on the basis of which the spectral shifts in different solvents were calculated. mTHPC and mTHPP form linear dimers in aqueous media, whereas mTHPBC form zigzag dimers.
Photodynamic therapy (PDT) is a photochemical-based modality of cancer treatment that uses a combination of a photosensitizer, light and molecular oxygen. Application of liposomal nanocarriers to deliver photosensitizers to tumor targets has become a major direction of PDT research. The present study investigates conventional and sterically stabilized liposomal formulations of the photosensitizer mTHPC, Foslip® and Fospeg®, with a view to determine the parameters for optimizing liposomal PDT. The characterization of in vitro behaviour of liposomal mTHPC was conducted, with an emphasis on drug localization, aggregation state and photophysical properties of the compounds in liposomes. We demonstrated the monomeric state of mTHPC in lipid vesicles and a partial localisation of mTHPC in Fospeg® in a PEG shell, while the main part was bound to the lipid bilayer. We further studied the drug release kinetics and binding pattern to serum proteins and the destruction of liposomes in serum. With this aim, a fluorescence-based methodology of estimating mTHPC release both in vitro and in vivo was developed, as well as an in vitro assay to characterize liposome destruction. The release of mTHPC from PEGylated liposomes was delayed compared with conventional liposomes along with greatly diminished liposome destruction. Knowledge of these parameters allows to better predict the drug release rate, pharmacological parameters and in vivo tumoricidal effect. The PDT treatment could be more advantageous with Fospeg® compared to mTHPC embedded in conventional liposomes.
Photodynamic therapy (PDT) is a therapeutic strategy for the treatment of small localized tumors accessible to the visible light irradiation. It is based on the combined action of photosensitizer (PS), light and molecular oxygen. Tumoricidal effect of PDT is triggered by direct damage of malignant cells and indirect vascular damage followed by an activation of the immune system. The present study investigates the relationship between photoinduced apoptosis in each compartment of interest (vascular versus neoplastic) and mTHPC-PDT treatment efficiency in function of the intratumoral distribution of mTHPC. The latter was defined by the drug-light intervals. In the first part, we demonstrated the importance of the intratumoral distribution of mTHPC to optimize photodynamic parameters. The fractionation of the PS administration permitted to obtain a tumor cure rate of 100% correlated to a massive apoptosis of pathological tissues. Moreover, this treatment strategy induced only limited skin damages and few inflammation which could be an advantage in clinical context. In the second part, we evidenced the mTHPC redistribution from liposomal formulations (Foslip®, Fospeg®) in vivo in the chick chorioallantoic membrane model (CAM) and its influence on photoinduced vascular damage.
Photodynamic therapy (PDT) is the combination of photosensitizers and visible light used as a treatment for cancer and infections. As a direct consequence of PDT, reactive oxygen species and other “danger signals” activate the innate immune system and by doing so generate acute inflammation. This cascade of effects is followed by priming of tumor-specific T lymphocytes that have the potential to destroy distant untreated tumor cells, in addition to developing an innate memory “shield” that can be effective in combating recurrence of the cancer. Moreover, PDT can be successfully applied for overcoming the escaping mechanisms employed by progressing tumors attempting to evade immune attack. Furthermore, PDT can assert a beneficial effect on cases of bacterial infection through attracting and accumulating neutrophils into the infected regions rather than by directly killing the bacterial cells.
This review article briefly describes: (a) the advantages in developing multifunctional nanoparticles for cancer-imaging and therapy, (b) the advantages and limitations of most of the porphyrin-based compounds in fluorescence imaging and photodynamic therapy (PDT), (c) problems associated with current Food and Drug Administration (FDA) approved photosensitizers, (d) challenges in developing in vivo target-specific PDT agents, (e) development of porphyrin-based nuclear-imaging agents (PET, SPECT) with an option of PDT, (f) the importance of light dosimetry in PDT, (g) the role of whole body or local hyperthermia in enhancing tumor-uptake, tumor-imaging and phototherapy and finally, (h) the advantages of photosensitizer-gold nanocages (Ps-Au NC) in photoacoustic and PDT.
The new peripherally and non-peripherally tetra-2-[2-(dimethylamino)ethoxy]ethoxy substituted zinc (II) phthalocyanine complexes (2 and 4) and their quaternized amphiphilic derivatives (2a and 4a) have been synthesized and characterized for the first time. The quaternized complexes show excellent sol-ubility in both organic and aqueous solutions, which makes them potential photosensitizer for use in photodynamic therapy (PDT) of cancer. Photophysical (fluorescence quantum yields and lifetimes) and photochemical (singlet oxygen generation and photodegradation under light irradiation) properties of these novel phthalocyanines are investigated in dimethylsulfoxide (DMSO) for non-quaternized complexes and in DMSO, phosphate buffered solution (PBS) or PBS + triton X-100 (TX) for quaternized complexes. In this study, the effects of the aggregation of the molecules, quaternization and position (peripherally or non-peripherally) of the substituents and nature of the solvents (DMSO, PBS or PBS + triton X-100) on the photophysical and photochemical parameters of the zinc (II) phthalocya-nines are also reported. A spectroscopic investigation of the binding of the quaternized cationic zinc (II) phthalocyanine complexes to bovine serum albumin (BSA) is also presented in this work.
Singlet oxygen, created in photosensitization, peroxidizes unsaturated fatty-acids of the membrane's lipids. This generates alcoholic or aldehyde groups at double bonds' breakage points. In a previous study we examined the leakage of a K(+) -induced cross-membrane electric potential of liposomes that undergo photosensitization. The question remains to what extent peroxidized lipids can compromise the stability of the membrane. In this work, we studied the effect of the oxidatively modified lipids PGPC and ALDOPC in the membrane on its stability, by monitoring the membrane electric potential with the potentiometric dye DiSC2 (5). As the content of the modified lipids increases the membrane becomes less stable, and even at just 2% of the modified lipids the membrane's integrity is affected, in respect to the leakage of ions through it. When the liposomes that contain the modified lipids undergo photosensitization by hematoporphyrin, the lipid bilayer becomes even more unstable and passage of ions is accelerated. We conclude that the existence of lipids with a shortened fatty acid that is terminated by a carboxylic acid or an aldehyde and more so when photosensitized damage occurs to unsaturated fatty acids in lecithin, add up to a critical alteration of the membrane, which becomes leaky to ions. This article is protected by copyright. All rights reserved.
Two novel Cu(2+) sensors, and , bearing naphthalimide and a DPA moiety were synthesized to study copper accumulation in organelles by selective Cu(2+) sensing. The ER-selective Cu(2+) sensor that we developed serves as a valuable tool for understanding the subcellular compartmentalization and roles of copper ions in physiology and pathophysiology.
The cytochrome P-450 in hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative was shown to be rapidly destroyed in the presence of long-wave ultraviolet light. The photocatalytic destruction of the heme-protein was dependent on both the dose of ultraviolet light and of hematoporphyrin derivative administered to the animals. The destructive reaction was accompanied by increased formation of cytochrome P-420, loss of microsomal heme content, and diminished catalytic activity of cytochrome P-450-dependent monooxygenases such as aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The specificity of the effect on cytochrome P-450 was confirmed by the observation that other heme-containing moieties such as myoglobin and cytochrome c were not susceptible to photocatalytic destruction. The destruction of cytochrome P-450 was a photodynamic process requiring oxygen since quenchers of singlet oxygen, including 2,5-dimethylfuran, histidine, and beta-carotene, each substantially diminished the reaction. Scavengers of superoxide anion such as superoxide dismutase and of H2O2 such as catalase did not protect against photodestruction of cytochrome P-450, whereas inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethyl alcohol, did afford protection. These results indicate that lipid-rich microsomal membranes and the heme-protein cytochrome P-450 embedded therein are potential targets of injury in cells exposed to hematoporphyrin derivative photosensitization.
The photosensitizing activity of hematoporphyrin derivative (HPD) was investigated by studying selected enzymes localized to mitochondria and cytosol of R3230AC mammary adenocarcinomas. Experiments in vitro demonstrated that mitochondrial succinate dehydrogenase was inhibited in a drug dose- and light exposure time-related manner; at 7.0 micrograms of HPD per ml or higher, enzyme activity was inhibited greater than 50% after 15 min of photoradiation. The three cytosol enzymes studied under the same conditions in vitro demonstrated different photosensitivities. Pyruvate kinase activity was significantly inhibited in a dose- and time-related fashion, whereas lactate dehydrogenase was inhibited to a lesser extent, and glucose phosphate isomerase activity was inhibited only at the highest dose (70 micrograms of HPD per ml) used. The time-course of these responses was examined with an in vivo-in vitro protocol, consisting of photoradiation of mitochondria and cytosol prepared from tumors obtained at various times (up to 1 week) after a single injection of HPD (80 mg/kg). Pyruvate kinase activity was markedly inhibited at early times returning to initial levels by 48 hr; neither lactate dehydrogenase nor glucose phosphate isomerase was inhibited by this treatment. Mitochondrial succinate dehydrogenase and cytochrome c oxidase activities displayed significant photoradiation-induced inhibitions, with greatest inhibition occurring between 24 and 96 hr after injection of HPD; at 1 week, succinate dehydrogenase activity had returned to its initial level, but cytochrome c oxidase activity remained significantly inhibited. These data suggest that HPD-induced photosensitization of mitochondria may be an important site of action contributing to tumor cell cytotoxicity and regression as a result of photoradiation therapy.
NHIK 3025 cells derived from a carcinoma in situ were exposed to hematoporphyrin derivative (Hpd) and light and examined by light microscopy, freeze-etching, scanning, and transmission electron microscopy. The first morphologic changes observed were shrinkage of mitochondria and formation of vesicles on the cell membrane. Furthermore, increased membrane permeability led to accumulation of Hpd and cellular swelling, with a concomitant reduction in the number and size of the microvilli. Some of the vesicles seemed to originate from microvilli. The freeze-etching appearance of the membranes of the majority of the cells was unaltered by treatment with Hpd and light. However, in some cases clustering of membrane particles was observed. At low doses membrane vesiculation and cell swelling were reversed within a few hours after treatment, indicating that repair processes were operative.
In mice, feeding with griseofulvin leads to the rapid accumulation of protoporphyrin in liver mitochondria. When liver mitochondria from mice fed with griseofulvin for 2 days are exposed to irradiation (320-400 nm), uncoupling of oxidative phosphorylation followed by inhibition of respiration occurs at light doses above 3-5 kJ/m2. When combined preparations of mitochondria and lysosomes are irradiated, inactivation of enzymes occurs in the following order: succinate dehydrogenase greater than glutamate dehydrogenase greater than acid phosphatase greater than beta-glucuronidase. Qualitatively, the photodamaging effect of endogenously produced protoporphyrin is indistinguishable from that of externally added protoporphyrin. Quantitatively, however, when protoporphyrin is added externally, more protoporphyrin is taken up by lysosomes, and photoinactivation of the lysosomal enzymes is correspondingly more severe. The results are further evidence that porphyrin-induced photodamage is largely determined by the solubility properties of the porphyrins and the target structures [Sandberg & Romslo (1980) Biochim. Biophys. Acta 593, 187-195], and also that protoporphyrin-induced photodamage is essentially similar whether protoporphyrin is generated endogenously or added exogenously.
The combination of mesoporphyrin IX and light is cytotoxic to leukemia L1210 cells, resulting in membrane damage and loss of viability. In this study, mesoporphyrin transport and the cellular environment of accumulated drug were examined. The latter was characterized by measurements of absorbance and fluorescence spectra and of effects of irradiation on subsequent capacity of cells for transport of the nonmetabolized amino acid cycloleucine. We observed a rapid accumulation of drug at a relatively hydrophilic cellular environment (dielectric constant, 20) from which light-catalyzed inhibition of cycloleucine transport was clearly demonstrable. Washing at 37 degrees rapidly depleted this cellular region of drug. Longer incubations resulted in accumulation of porphyrin at more hydrophobic locci (dielectric constant, approximately 10) from which the drug was not readily washed and from which the efficiency of light-catalyzed damage to membrane transport was relatively low. These findings are consistent with the hydrophobic nature of mesoporphyrin (octanol:water partition ratio, 10).
In rats feeding with hexachlorobenzene leads to the accumulation of uro- and heptaporphyrins in hte liver, with the highest concentration in the lysosomes. Irradiation of post-nuclear supernatants of livers from hexachlorobenzene-fed rats results in a marked inactivation of β-glucuronidase, a slight inactivation of succinate dehydrogenase, and no change in the activity of lactate dehydrogenase and urate oxidase. When combined preparations of mitochondria and lysosomes are irradiated, there is release of lysosomal enzymes before there is uncoupling of oxidative phosphorylation. This is in contrast to the findings of Sandberg and Romslo (1981, Biochem. J. 198, 67-74) when preparations of lysosomes and mitochondria from livers of protoporphyric mice are irradiated. In that case protoporphyrin accumulates mainly in the mitochondria and, upon irradiation of combined preparations of mitochondria and lysosomes, the mitochondria are uncoupled before there is any leakage of lysosomal enzymes. The photodamage is dependent on oxygen, and it is enhanced when the experiments are run in D2O. The results are further evidence that porphyrin-induced photodamage is mediated by singlet oxygen and determined largely by the subcellular localization of the porphyrins.
The mitochondria of tumor cells are structurally and functionally different from those in normal cells. Because of a yet undetermined flaw in the mitochondria of most neoplasms, lipophilic cationic compounds tend to accumulate within the interior of these structures. By selecting a cationic lipophilic compound that tends to preferentially bind to tumor over normal mitochondria and which is photoactivated to a cytocidal species in the presence of penetrating near-infrared light, we and others hope to find a drug that is clinically effective for the photochemotherapeutic treatment of solid neoplasms.
Haematoporphyrin derivative photosensitization has been studied in single heart cells in tissue culture by laser micro-irradiation ( u =632·8 nm ). Changes of beating rate as well as cell death depend on the localization of the microbeam on the various parts of the cell. The results show that the targets for photodamage are the plasma membrane followed by the mitochondria.
Leukemia L1210 cells were incubated in vitro with the tumor-localizing product HPD (hem-atoporphyrin derivative) for 0.5. 4 and 18 h. Effects of subsequent irradiation on viability, membrane transport and integrity, DNA synthesis and intracellular ATP concentration were assessed. Intracellular porphyrin pools were analyzed by HPLC. A 30 min incubation led to concentration of a readily-exchangeable pool of monomeric HPD components at plasma membrane loci; irradiation resulted in photodamage to membrane transport and a loss in capacity for dye exclusion. In contrast, increasing the incubation time led to a corresponding increase in the size of a non-exchangeable intracellular pool of other HPD components. Subsequent irradiation led to depletion of intracellular ATP and loss of capacity for biosynthesis of DNA, but little plasma membrane damage.
10-n-Alkyl-acridinium-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl=methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K
–1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K=K
0at the standard temperature T=298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m+1 (m=1–9) (Table 2). In addition the standard dissociation enthalpies (energies) H
0 and dissociation entropies S
0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K
0, H
0, S
0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M
1=20.000 cm–1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm–1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D
1=18.700 and D
2=21.200 cm–1 with very different intensities. D
1 has very low intensity and is forbitten, D
2 is allowed. D
1 shows fluorescence. Phosphorescence has not been observed. D
1, D2and also M
1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition intensities agree with quantum mechanical model calculations for dimers with parallel or antiparallel molecular orientation. Hydrophobic interaction needs parallel orientation in the dimers of nonyl-AO. In the dimers of AO and of dyes with very short alkyl chains antiparallel orientation may occur.
When tumours and cells containing photofrin II (PII) are exposed to light at wavelengths and exposures relevant for photodynamic
therapy (PDT), changes in the shape of the prophyrin fluorescence spectra, as well as a decay of the fluorescence intensity,
can be observed. These changes are due to (a) photodestruction of porphyrin macrocycles resulting in loss of absorbance and
fluorescence; (b) a photoinduced chemical modification of the porphyrins, leaving the porphyrin macrocycle intact; or (c)
a photoinduced displacement of the porphyrins to different locations in the cells. The two processes (b) and (c) may lead
to changes in the shape of the fluorescence spectra as observed. In this study survival curves were determined for cells grown
in vitro, exposed to various concentrations of PII and irradiated with light. It was concluded that the quantum yield for
photoinactivation of the cells increased during light exposure under the conditions where changes in the spectral shape of
the fluorescence was seen. However, the photodegradation of PII resulted in the need for larger light fluences for cell inactivation
at low PII concentrations than could be expected on the basis of a reciprocal dependence between light exposure and applied
drug dose.
Human NHIK 3025 cells derived from a carcinoma in situ were incubated with tetrasodium-meso-tetra-(4-sulphonatophenyl)porphine (TPPS4) or HPD and exposed to light. Two different incubation conditions were applied: 0.5h in pure phosphate-buffered saline and
22h in a medium containing 10% newborn calf serum. The cell survival curves revealed different inactivation kinetics for TPPS4 as compared with HPD. Electron microscopy revealed qualitatively the same types of photoinduced damage to the cell nuclei
for both sensitizers when the cells were examined at the same survival level. The damage was manifested as nuclear swelling
and segmentation, swelling of the perinuclear cistern, simultaneous chromatin dilution and condensation, and nucleolar segregation
with cap formation. For HPD, mitotic arrest with disappearance of microtubules was also observed.
The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY-R, deficient in the excision repair of UV-induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY-S, deficienl in the repair of DNA double-strand breaks, was more sensitive than strain LY-R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY-S and LY-R sub-strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY-SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 10⁶ surviving cells. The induced frequency in strain LY-S1 was much higher than in either tk+l-heterozygous or ik+10 hemizygous strains of LY-R. The rate and extent of incorporation of CAPC by the LY-R strains was somewhat greater than for strain LY-S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.
Human cervix carcinoma cells of the line NHIK 3025 were exposed to light after 18 h incubation with Photofrin II. After this photodynamic treatment cells in the interphase were retarded with respect to entry into mitosis for a period which increased with increasing light dose. Following the prolonged interphase, an increase in the mitotic index was observed, giving rise to a 3-fold higher level of mitotic cells compared to the control level. Staining of methanol-fixed cells with the DNA-specific dye mithramycin indicated that the increase in mitotic index was due to a prolongation of the metaphase. For all the light doses studied most of the metaphase cells could be characterized as three-group metaphases or c-metaphase-like structures for the first 8 h after treatment. An approximately 10-fold increase above the control level in the number of tripolar mitoses was also observed. A 2h incubation in a Photofrin II-free medium after the 18 h incubation with Photofrin II and before light exposure reduced the fluorescence of the cells by 30 per cent. However, this wash-out period had no effect on the increase in mitotic index after light exposure. A light dose corresponding to 80 per cent survival (as assayed on asynchronous cells) was given to cells in mitosis after Photofrin II incubation. This treatment delayed more than 90 per cent of the metaphase cells from entering the anaphase for at least 1 h. Cells photodynamically treated in the anaphase and telophase entered the interphase at a similar rate as control cells. These observations indicate a temporary block in the initiation of the anaphase and a prolongation of the metaphase. A microscopic study of cells immunologically stained for beta-tubulin 1 h after photodynamic treatment indicated that the organization of the spindle apparatus was disturbed by the photodynamic treatment. Such perturbations are suggested to be the cause of the observed accumulation of cells in mitosis.
— Water soluble chloro aluminum phthalocyanines sulfonated to different degrees are studied for phototoxicity and cellular distribution inV–79 Chinese hamster cells. The more hydrophobic disulfonated dyes, with sulfonate substituents on adjacent benzyl groups of the phthalocyanine ring structure, exhibited the best cell penetrating properties and the highest phototoxicity. Fluorescence microscopy revealed that the dye was uniformly distributed in the cytoplasm but absent in the nucleus. The greater cell membrane penetrating properties of the lower as compared to the higher sulfonated dyes are attributed to the amphiphilic nature of the former.
Several parameters of the following dyes, all relevant as sensitizers for photochemotherapy of cancer, have been studied: Photofrin II (PII), hematoporphyrin (HP)-di-hexyl-ether, HP-di-ethyl-ether, tetra (3-hydroxyphenyl) porphyrin, (3THPP), tetraphenyl porphine tetrasulphonate (TPPS4) aluminium phthalocyanine tetrasulfonate (A1PCTS), aluminium phthalocyanine (A1PC), chlorin e, (Chi e6) and merocyanine 540 (MC 540). The following parameters and features of these dyes were studied: (1) Tumor uptake in C3H mouse mammary carcinomas. (2) Skin/tumor concentration ratio in the same animal system. (3) Triton X-114/H20 partition coefficients at different pH-values. (4) Uptake of the dyes by human cells of the line NHIK 3025. (5) Relative fluorescence quantum yields of the dyes bound to cells. (6) Absorption-, fluorescence-excitation- and fluorescence-emission spectra of the cell-bound dyes. (7) Relative quantum yields for photoinactivation of cells after 18 h incubation with the dyes. (8) Relative quantum yields of photodegradation of the singlet oxygen trap 1,3-diphenylisobenzofuran (DPBF) in cells after 18 h incubation with the dyes.
Chloroaluminum phthalocyanine (CAPC) was recently shown to photosensitize cell killing in culture and tumor destruction in vivo. Because this compound is potentially useful in the photodynamic therapy of cancer, its properties as a genotoxic agent were evaluated. Applying the technique of alkaline elution to study DNA integrity, it was found that CAPC could produce single-strand breaks in the DNA of Chinese hamster cells after exposure to white fluorescent light. At equicytotoxic doses, the number of DNA strand breaks produced by CAPC photosensitization was about three times lower than that induced by X-irradiation. During incubation in growth medium after exposure to CAPC-plus-fluorescent light, cells rejoined DNA strand breaks at a rate similar to that observed after X-irradiation. Resistance to 6-thioguanine (6-TG') or to ouabain (OUA') were used as end points of mutagenic potential. Following a treatment that caused -90% cell killing, there was a slight mutagenic effect, i.e. the frequencies were increased by -40% above the background or spontaneous mutations. However, this enhancement was not statistically significant. Taken together, the foregoing, plus an earlier observation that there is no variation in the sensitivity of cells to CAPC + light through the cell cycle, lead to the inferences that DNA damage does not play a major role in cell killing and that the mutagenic potential of this treatment is small.
Normal erythroid cells and both uninduced and induced erythroleukemia cells were stained with the leukemia-specific fluorescent probe merocyanine 540 and its analogs. The external membranes of normal intact cells bound the dye, but this general low-affinity binding was completely abolished by the addition of competing serum. In contrast, erythroleukemia cells bound the dye even in the presence of serum; binding was not affected by reversing the sign of the charge carried by MC540, but was abolished upon removal of certain hydrophobic side chains. When the erythroleukemia cells were induced to differentiate, the distribution of dye-binding regions was altered by the cell such that staining became localized to one region of the membrane. Concomitantly, conconavalin A binding sites were redistributed and became localized in the same region of the membrane as the merocyanine binding sites. Merocyanine 540 is thus shown to bind to a hematopoietic surface feature whose topological distribution is subject to cellular control during differentiation. This leukemia-specific marker may be one of several eliminated during enucleation of mammalian erythroid cells.
Several in vitro cell systems were exposed to hematoporphyrin derivative (HPD): established lines of rat kangaroo epithelial kidney; normal mouse embryonic fibroblasts; and differentiated neonatal rat myocardial cells. The uptake of HPD (25 to 100 micrograms/ml) by individual cells occurred rapidly over a 2-hr period and leveled off by 24 hr. HPD was excreted from cells by 48 hr after exposure. However, a low level of HPD (above background) was maintained in cells for up to 4 days following cessation of exposure. Intracellular binding of HPD was to mitochondria as demonstrated by fluorescence microscopy. HPD was also shown to have a growth-inhibiting effect on rat kangaroo cells without added light. The growth effects on mouse cells were less marked.
Several effects of hematoporphyrin derivative (HpD) and light on NHIK 3025 cells in vitro were studied. The treatment resulted in a partly repairable reduction of the rate of thymidine incorporation into DNA, a division delay, a reduced rate of protein synthesis, a reduced rate of active cellular uptake of α-aminoisobutyrate, a reduction in the colony-forming ability and an increased permeability of the cell membrane to chromate. Thymidine incorporation was by far the most sensitive parameter studied. However, comparison of the photodynamic effects after 1 and 18 h incubation with HpD prior to irradiation indicated that neither the reduced rate of DNA synthesis nor any of the other observed effects was the main primary cause of cell inactivation under all conditions. Several of the effects, such as increased permeability of the cell membrane to chromate, reduction in the rate of protein synthesis and reduction in the rate of repair of the damage to the mechanism of DNA synthesis, were clearly of secondary nature. When seen in relation to cellular survival, membrane damage was more important after short incubation times with HpD than after long incubation times.
— The photodynamically-induced liberation of lysosomal enzymes using ß-galactosidase as marker for the lysosomal enzymes has been studied by microspectrofluorometry on mouse L cells. Similar studies have been carried out using N-acetyl-ß-D-glucosaminidase as marker for the lysosomal enzymes of human fibroblasts. The high sensitivity of the fluorescence detection makes it possible to use 4-methylumbelliferyl substrates for the enzymes contained in a single cell. Methylene blue and hematoporphyrin readily incorporate into both cells and upon excitation, sensitize lysosomal membrane damages, leading to enzyme release accompanying strong morphological changes.
Carcinoma cells and normal epithelial cells differ in the mitochondrial retention of a permeant cationic compound, rhodamine
123. The possibility of utilizing this difference in carcinoma chemotherapy was investigated. Rhodamine 123 exhibited anticarcinoma
activity in mice, and this activity was potentiated by 2-deoxyglucose.
The porphyrin content of cells labelled with hematoporhyrin derivative (Hpd) and tumours of mice injected with Hpd was analysed by means of high pressure liquid chromatography (HPLC). The components of Hpd may be classified in 3 groups: (A) Components with a high fluorescence quantum yield and with sharp peaks in the HPLC chromatogram. These are monomers. (B) Components with a lower fluorescence quantum yield and with sharp peaks in the HPLC chromatogram. These are probably dimers or oligomers. (C) Components with a low fluorescence quantum yield, with a short retention time on a P-10 column and with a broad and unresolved peak in the HPLC chromatogram. These are probably large aggregates. Components of group A are rapidly accumulated by cells but are easily removed by washing the cells with medium containing serum. Porphyrins of group B are significantly more concentrated by cells in vitro than porphyrins of group A and B and accumulate over a time interval of about 18 h. Porphyrins of group B gradually migrate to sites in the cells where they are more strongly retained. Tumors in mice behave differently from tumor cells in vitro since they mainly contain porphyrins of group C after an i.p. injection of Hpd in the mice.
Commercial hematoporphyrin (Hp) and the tumor-localizing and photosensitizing agent hematoporphyrin derivative (Hpd) were analysed by means of high pressure lipid chromatography (HPLC). Furthermore, their efficiencies in sensitizing the photoinactivation of human cells in vitro were compared. The comparison showed that the least polar components of Hpd played the major role in this sensitization. In Hpd solutions used for injection in photochemotherapeutic treatment of cancer, these active components seem to be present as aggregates.
Different doses (0.5-20 mg/kg) of hematoporphyrin (HP) have been injected intraperitoneally into normal rats and rats affected by Yoshida ascites hepatoma. About 80% of HP reaching the liver was recovered in the extracellular compartment after liver perfusion, the ratio of extra- to intracellular HP being essentially independent of the administered dose. Similar data were obtained at different times after injection of 20 mg/kg HP. Intracellular HP largely accumulates in the mitochondria and in the membrane components of the nuclear fraction of isolated hepatocytes. Kinetic studies suggest that the cell receptors of highest affinity for HP are present in the external membrane. The latter result obtains for ascites hepatoma cells in an even more evident way, although the latter cells exhibit secondary HP binding sites probably constituted by cytoplasmatic proteins. Moreover, the clearance of intracellular HP from malignant cells occurs at a remarkably lower rate as compared with HP clearance from liver cells.
The intracellular localization of porphyrin taken up by a transplanted rat tumor has been studied. Quantitative determination of the porphyrin concentrations in the subcellular fractions and direct observation by fluorescence microscopy indicate that porphyrin accumulates in the soluble fraction and not in the cell wall, nucleus, mitochondrial, or microsomal particles. There is suggestive evidence that selective uptake by the soluble fraction is due to a specific constituent to which the porphyrin is bound.
Evaluation of in vivo tissue localization properties and in vitro photosensitization reactions of hematoporphyrin derivative
- C J Gomer
Biological activities of phthalocyanines. VIII
- Paquette
Porphyrinsensitized photoinactivation of human cells in vitro
- Moan J