Photodynamic Therapy Efficacy Enhanced by Dynamics: The Role of Charge Transfer and Photostability in the Selection of Photosensitizers

Progress in the photodynamic therapy (PDT) of cancer should benefit from a rationale to predict the most efficient of a series of photosensitizers that strongly absorb light in the phototherapeutic window (650–800 nm) and efficiently generate reactive oxygen species (ROS=singlet oxygen and oxygen‐centered radicals). We show that the ratios between the triplet photosensitizer–O₂ interaction rate constant (k_D) and the photosensitizer decomposition rate constant (k_d), k_D/k_d, determine the relative photodynamic activities of photosensitizers against various cancer cells. The same efficacy trend is observed in vivo with DBA/2 mice bearing S91 melanoma tumors. The PDT efficacy intimately depends on the dynamics of photosensitizer–oxygen interactions: charge transfer to molecular oxygen with generation of both singlet oxygen and superoxide ion (high k_D) must be tempered by photostability (low k_d). These properties depend on the oxidation potential of the photosensitizer and are suitably combined in a new fluorinated sulfonamide bacteriochlorin, motivated by the rationale.     

First Report on Photodynamic Inactivation of Archaea Including a Novel Method for High-Throughput Reduction Measurement

Archaea are considered third, independent domain of living organisms besides eukaryotic and bacterial cells. To date, no report is available of photodynamic inactivation (PDI) of any archaeal cells. Two commercially available photosensitizers (SAPYR and TMPyP) were used to investigate photodynamic inactivation of Halobacterium salinarum. In addition, a novel high-throughput method was tested to evaluate microbial reduction in vitro. Due to the high salt content of the culture medium, the physical and chemical properties of photosensitizers were analyzed via spectroscopy and fluorescence-based DPBF assays. Attachment or uptake of photosensitizers to or in archaeal cells was investigated. The photodynamic inactivation of Halobacterium salinarum was evaluated via growth curve method allowing a high throughput of samples. The presented results indicate that the photodynamic mechanisms are working even in high salt environments. Either photosensitizer inactivated the archaeal cells with a reduction of 99.9% at least. The growth curves provided a fast and precise measurement of cell viability. The results show for the first time that PDI can kill not only bacterial cells but also robust archaea. The novel method for generating high-throughput growth curves provides benefits for future research regarding antimicrobial substances in general.     

A Possible Phenomenon of Persistence in Pseudomonas aeruginosa Treated with Methylene Blue and Red Light

Planktonic Pseudomonas aeruginosa cells harvested in stationary phase were exposed to red light in the presence of methylene blue to study the potential occurrence of persistence in bacterial populations submitted to photodynamic antimicrobial therapy. Survival curves revealed the existence of small subpopulations of cells exhibiting increased ability to tolerate the treatment. These subpopulations were detected even using high concentrations of photosensitizer, whether added in a single step or following a fractionated scheme, and when the irradiation medium was modified to delay the photodecomposition of methylene blue. When cells grown from survivors to the treatment were cultured and exposed to red light and dye, their responses were similar to that of the original strain. These results exclude exhaustion of the photosensitizer and selection of resistant mutants as explanations for the features of the survival curves. Cells able to tolerate the treatment were found even when radiation was imparted at a high‐dose rate. They exhibit a response typical of persisters, which tolerate antimicrobial agents due to transient and reversible changes in their phenotype, suggesting that persistence is a factor to consider upon evaluating the efficacy of photodynamic antimicrobial therapy.     

Poly(ethylene glycol) conjugated nano-graphene oxide for photodynamic therapy

A novel methoxy-poly(ethylene glycol) modified nano-graphene oxide (NGO-mPEG) was designed and synthesized as a photosensitizer (PS) carrier for photodynamic therapy of cancer. NGO with a size below 200 nm was prepared using a modified Hummers’ method. NGO was observed by AFM to exhibit a structure with single-layer graphene oxide sheets down to a few nanometers in height. Hydrophilic mPEG conjugation of NGO (NGO-mPEG) was found to enhance solubility in cell culture media. No apparent cytotoxicity of the NGO-mPEG was observed towards MCF-7 carcinoma cell line. Zinc phthalocyanine (ZnPc), a photosensitizer for photodynamic therapy, was loaded in the NGO-PEG through π-π stacking and hydrophobic interactions, with the drug loading efficiency up to 14 wt%. Hydrophobic ZnPc was internalized in MCF-7 cells, exhibiting a pronounced phototoxicity in the cells under Xe light irradiation. The results indicate a great potential of NGO-mPEG for photodynamic therapy of cancer.     

A Phthalocyanine–Peptide Conjugate with High In Vitro Photodynamic Activity and Enhanced In Vivo Tumor‐Retention Property

A novel zinc(II) phthalocyanine conjugated with a short peptide with a nuclear localization sequence, Gly‐Gly‐Pro‐Lys‐Lys‐Lys‐Arg‐Lys‐Val, was synthesized by click chemistry and a standard Fmoc solid‐phase peptide synthesis protocol. The conjugate was purified by HPLC and characterized with UV/Vis and high‐resolution mass spectroscopic methods. Both this compound and its non‐peptide‐conjugated analogue are essentially non‐aggregated in N,N‐dimethylformamide and can generate singlet oxygen effectively with quantum yields (Φ_Δ) of 0.84 and 0.81, respectively, relative to unsubstituted zinc(II) phthalocyanine (Φ_Δ=0.56). Conjugation of the peptide sequence, however, can enhance the cellular uptake, efficiency in generating intracellular reactive oxygen species, and photocytotoxicity of the phthalocyanine‐based photosensitizer against HT29 human colorectal carcinoma cells. The IC₅₀ value of the conjugate is as low as 0.21 μM . In addition, the conjugate shows an enhanced tumor‐retention property in tumor‐bearing nude mice. After 72 h post‐injection, the dye concentration in the tumor was significantly higher than that in other organs. The results suggest that this phthalocyanine–peptide conjugate is a highly promising photosensitizer for photodynamic therapy.     

Chlorin e6 Derivative Radachlorin Mainly Accumulates in Mitochondria,Lysosome and Endoplasmic Reticulum and Shows High Affinity toward Tumors in Nude Mice in Photodynamic Therapy

The efficacy of photodynamic therapy (PDT) depends upon the amount of photosensitizer accumulated in the malignant tissues. Radachlorin is a popular photosensitizer used in photodynamic therapy to treat various types of cancer. In this study, we have studied the main organelles responsible for the accumulation of radachlorin in human anaplastic thyroid cancer in vitro and in vivo. The optimal time window for uptake and clearance of radachlorin also was studied. Confocal microscopic images confirmed that the radachlorin is mainly acquired by mitochondria and partially by lysosome and endoplasmic reticulum. Studies also showed that the maximum amount of radachlorin was accumulated within 3–6 h after the treatment. Radachlorin also showed a higher affinity toward malignant tumors compared to the other organs in mice xenograft model. Uptake of radachlorin reached an optimum amount within 6 h and most of the radachlorins were also cleared from the body in next 48 h. Therefore, detailed information regarding exact accumulation sites and a time window in which maximum amount of drug is accumulated and cleared were obtained by this study. Hence, not only the efficacy of the treatment can be increased but the phototoxicity after the treatment also can be controlled.     

介孔钛纳米复合团簇应用于光热-光动力疗法

采用水热法合成了一种新型的介孔二氧化钛/碳/亚甲蓝复合纳米团簇(TiO_2@C-MB),并应用于肿瘤细胞的光动力(PDT)和光热治疗(PTT)。系统中介孔二氧化钛作为有效的光敏剂,MB作为重要的光敏添加剂以改善二氧化钛纳米晶的光化学效应,并将其光响应区域拓宽至光动力学疗法的理想治疗窗(650~900 nm)。柠檬酸在水热条件下被还原成碳并裹覆在二氧化钛表面。碳层表现出良好的光热效果,也充当多功能的电子受体以加速生成单线态氧。该纳米团簇不仅可以保持肿瘤细胞内部高浓度的MB和二氧化钛以产生大量的单线态氧杀死肿瘤细胞,而且可以避免MB退化失活。     

Photodynamic Effects of Zinc(II) Phthalocyanine‐Loaded Polymeric Micelles in Human Nasopharynx KB Carcinoma Cells

A major difficulty in photodynamic therapy is the poor solubility of the photosensitizer (PS) under physiological conditions which correlates with low bioavailability. PS aggregation leads to a decrease in the photodynamic efficiency and a more limited activity in vitro and in vivo. To improve the aqueous solubility and reduce the aggregation of 2,9(10),16(17),23(24)‐tetrakis[(2‐dimethylamino)ethylsulfanyl]phthal‐ocyaninatozinc(II) (Pc9), the encapsulation into four poloxamine polymeric micelles (T304, T904, T1107 and T1307) displaying a broad spectrum of molecular weight and hydrophilic–lipophilic balance was investigated. The aqueous solubility of Pc9 was increased up to 30 times. Morphological evaluation showed the formation of Pc9‐loaded spherical micelles in the nanosize range. UV/Vis and fluorescence studies indicated that Pc9 is less aggregated upon encapsulation in comparison with Pc9 in water–DMSO 2% and remained photostable. Pc9‐loaded micelles generated singlet molecular oxygen in high yields. Photocytotoxicity assays using human nasopharynx KB carcinoma cells confirmed that the encapsulation of Pc9 in T1107 and T1307 increases its photocytotoxicity by 10 times in comparison with the free form in water–DMSO. In addition, Pc9 incorporated into cells was mainly localized in lysosomes.     

Synthesis of a Photostable Near‐Infrared‐Absorbing Photosensitizer for Selective Photodamage to Cancer Cells

A new class of near‐infrared (NIR)‐absorptive (>900 nm) photosensitizer based on a phenothiazinium scaffold is reported. The stable solid compound, o‐DAP, the oxidative form of 3,7‐bis(4‐methylaminophenyl)‐10H‐phenothiazine, can generate reactive oxygen species (ROS, singlet oxygen and superoxide) under appropriate irradiation conditions. After biologically evaluating the intracellular uptake, localization, and phototoxicity of this compound, it was concluded that o‐DAP is photostable and a potential selective photodynamic therapy (PDT) agent under either NIR or white light irradiation because its photodamage is more efficient in cancer cells than in normal cells and is without significant dark toxicity. This is very rare for photosensitizers in PDT applications.     

The Endogenous Tryptophan‐derived Photoproduct 6‐formylindolo[3,2‐b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo

UV‐chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6‐formylindolo[3,2‐b]carbazole (FICZ), a tryptophan‐derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol^?1 cm^?1), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC‐25, HaCaT‐ras II‐4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm^?2) and FICZ (≥10 nm ), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg‐sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 “high‐risk” mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV‐induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.     

c(RGDfK)- and ZnTriMPyP-Bound Polymeric Nanocarriers for Tumor-Targeted Photodynamic Therapy

Active targeting strategies are currently being extensively investigated in order to enhance the selectivity of photodynamic therapy. The aim of the present research was to evaluate whether the external decoration of nanopolymeric carriers with targeting peptides could add more value to a photosensitizer formulation and increase antitumor therapeutic efficacy and selectivity. To this end, we assessed PLGA-PLA-PEG nanoparticles (NPs) covalently attached to a hydrophilic photosensitizer 5-[4-azidophenyl]-10,15,20-tri-(N-methyl-4-pyridinium)porphyrinato zinc (II) trichloride (ZnTriMPyP) and also to c(RGDfK) peptides, in order to target α_vβ₃ integrin-expressing cells. In vitro phototoxicity investigations showed that the ZnTriMPyP-PLGA-PLA-PEG-c(RGDfK) nanosystem is effective at submicromolar concentrations, is devoid of dark toxicity, successfully targets α_vβ₃ integrin-expressing cells and is 10-fold more potent than related nanosystems where the PS is occluded instead of covalently bound.     

GRP78‐Targeted HPMA Copolymer‐Photosensitizer Conjugate for Hyperthermia‐Induced Enhanced Uptake and Cytotoxicity in MCF‐7 Breast Cancer Cells

Photodynamic therapy (PDT) is a promising cancer treatment approach. However, the photosensitizers (PS) used for PDT are often limited by their poor solubility and selectivity for tumors. The goal of this study is to improve water solubility and delivery of the photosensitizer 2‐[1‐hexyloxyethyl]‐2‐divinyl pyropheophorbide‐a (HPPH) to breast cancer cells. An N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymer–HPPH photosensitizer conjugate is synthesized with heat shock receptor glucose‐regulated protein 78 (GRP78), targeting to GRP78 receptors of MCF‐7 cells, which are upregulated under mild hyperthermia. It is found that the uptake of the GRP78 targeted pep‐HPMA‐HPPH copolymer conjugate in MCF‐7 cells is improved through heat induction. Under mild hyperthermia the targeted copolymers are more effective compared to free HPPH. These results show potential for the utility of mild hyperthermia and copolymer delivery vehicles to enhance the efficacy of photodynamic therapy.     

A protein-targeted photosensitizer for highly efficient cancer therapy

Photodynamic therapy typically employs photo-triggered photosensitizers to generate reactive oxygen species to destroy cancer cells. However, the therapeutic effect of photodynamic therapy is often limited owing to the ultrashort diffusion distance of reactive oxygen species and easy efflux of photosensitizers. Herein, we design and synthesize a protein-targeted molecular photosensitizer for highly efficient photodynamic therapy. The designed photosensitizer can covalently bind with the sulfhydryl groups of intracellular proteins to achieve the protein targeting. Under irradiated with near infrared laser, the photosensitizer was locally activated, and the produced reactive oxygen species directly destroy intracellular bioactive proteins, causing cell dysfunction and ultimately inducing cell apoptosis. Significantly, the leakage of molecular photosensitizer is effectually avoided due to the protein targeting. In vivo experimental results indicated that the effect of treatment was efficiently enhanced with the protein-targeted strategy. This work can offer new insights for designing protein-based therapeutic drugs.     

In vitro induction of PDT resistance in HT29, HT1376 and SK-N-MC cells by various photosensitizers.

Our approach to examine the mechanism(s) of action for photodynamic therapy (PDT) has been via the generation of PDT-resistant cell lines. In this study we used three human cell lines, namely, human colon adenocarcinoma (HT29), human bladder carcinoma and human neuroblastoma. The three photosensitizers used were Photofrin, Nile Blue A and aluminum phthalocyanine tetrasulfonate. The protocol for inducing resistance consisted of repeated in vitro photodynamic treatments with a photosensitizer to the 1-10%-survival level followed by regrowth of single surviving colonies. Varying degrees of resistance were observed. The three induced variants of the HT29 cell line were the most extensively studied. Their ratios of increased survival at the LD90 level range between 1.5- and 2.62-fold more resistant.     

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON ESCHERICHIA COLI MEMBRANES AND DNA IN VIVO

Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in rec A, uvr A, or pol A functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was provided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-ß-D-galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.     

Intracellular Modulation of Excited‐State Dynamics in a Chromophore Dyad: Differential Enhancement of Photocytotoxicity Targeting Cancer Cells

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O₂(a¹Δ_g)], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer‐related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.     

Damage Threshold of Normal Rat Brain in Photodynamic Therapy

Normal brain tissue response to photodynamic therapy (PDT) must be quantified in order to implement PDT as a treatment of brain neoplasm. We therefore calculated the threshold for PDT-induced tissue necrosis in normal brain using Photofrin (porfimer sodium, Quadralogic Technologies Inc., Vancouver, BC) as the photosensitizer. The absolute light fluence-rate distribution for superficial irradiation and effective attenuation depth were measured in vivo using an invasive optical probe. Photosensitizer uptake in cerebral cortex was measured with chemical extraction and fluorometric analysis. Photodynamic therapy-induced lesion depths at various drug dose levels were measured as a biological end point. The PDT threshold for normal brain necrosis was calculated as in the magnitude of 10¹⁶ photons/cm³. Thus normal rat brain is extremely vulnerable to PDT damage. This suggests that extra precautions must be exercised when PDT is used in brain.     

Development of a Novel Covalent Folate‐Albumin‐Photosensitizer Conjugate

There is considerable interest in the development of novel and more efficient delivery systems for improving the efficacy of photodynamic therapy (PDT). The authors in this highlighted issue describe the synthesis and the photobiological characterizations of two photosensitizer (PS) conjugates based on β‐carboline derivatives covalently conjugated to folic acid (FA) coupled to bovine serum albumin (BSA) as a carrier system specifically targeting cancer cells overexpressing FA receptor alpha (FRα). Accordingly, only the FA–BSA–β‐carboline conjugates are internalized specifically in FRα‐positive cells and are proved to be phototoxic. On the other hand, albumin–β‐carboline conjugates without FA or β‐carboline derivatives alone are not internalized and nontoxic. This conjugate is among the first to produce a conjugate composed of a PS and FA molecules that are directly conjugated to BSA. In addition, the in vitro studies are the first evidence that directly conjugated FA‐BSA can be used as carriers to selectively enhance cytotoxicity by PDT relative to unmodified PS or nontargeted BSA‐PS. This strategy is a positive step forward for the covalent design and construction of a photodynamic nanomedicine for FR‐positive tumors.     

Cell-penetrating riboflavin conjugate for antitumor photodynamic therapy

Riboflavin (RF, vitamin B2) is an essential vitamin and has been considered as a promising natural photosensitizer for photodynamic therapy (PDT). However, further exploration of RF in antitumor application was limited by its poor cellular uptake. In this study, using cell-penetrating peptides Arg₈, (Cha-Arg)₃ and small molecule triphenylphosphine (TPP) as delivery compounds, three RF conjugates were prepared to increase the accumulation of RF in cells, termed as Arg₈-RF, (Cha-Arg)₃-RF and TPP-RF, respectively. Compared with TPP-RF and Arg₈-RF, (Cha-Arg)₃-RF exhibited better cell internalization and stronger cytotoxicity against HeLa cells upon exposure to blue light. Further researches proved that (Cha-Arg)₃-RF generated reactive oxygen species (ROS) under irradiation, which could indiscriminately destroy endogenous proteins and mitochondria, ultimately inducing cell death. This work provides a new approach to explore RF as a natural photosensitizer for antitumor photodynamic therapy.     

[1,2,3,4-Tetrakis(α/β-d-galactopyranos-6-yl)phthalocyaninato]zinc(II): a water-soluble phthalocyanine

A novel water-soluble asymmetrical sugar-phthalocyanine was prepared via a statistical cross-condensation of tetrakis(1,2:3,4-di-O-isopropylidene-α-d-galactopyranos-6-yl)phthalonitrile with phthalonitrile. The new compound, with amphiphilic character, can be useful as a selective photosensitizer in photodynamic therapy, as well as for constructing phthalocyanine-based supramolecular systems.