Antitumor efficacy of photodynamic therapy using novel nanoformulations of hypocrellin photosensitizer SL052

Recent preclinical and clinical testing of hypocrellin-based photosensitizer SL052 for use in photodynamic therapy (PDT) of cancer has shown encouraging results. Further optimization of its formulation for delivery could considerably extend the therapeutic efficiency of this drug. A nanoformulation encapsulating SL052 into biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) was developed using a single-emulsion solvent evaporation technique and characterized in terms of particle size and loading of the photosensitizing agent. This nanoformulation, SL052-PLGA-nanoparticles (NPs), was compared with recently created nanoformulation based on polyvinylpyrrolidone (SL052-PVP-NPs) and standard liposomal SL052 preparation in terms of efficacy when used for PDT treatment of squamous cell carcinomas SCCVII growing subcutaneously in syngeneic mice. The therapeutic effect of PDT using these three different SL052 formulations was tested for both 1 and 4 h intervals between drug injection and tumor light exposure. The longer time interval produced higher tumor cure rates with all SL052 preparations. With both drug-light intervals, PDT based on SL052-PLGA-NPs produced superior therapeutic benefit compared with the other two SL052 formulations.     

Photo-properties of a silicon naphthalocyanine: a potential photosensitizer for photodynamic therapy

Abstract— The photoproperties of derivatized silicon naphthalocyanine have been investigated in order to assess its potential as a photosensitizer for photo-dynamic therapy. Absorption, fluorescence, phosphorescence and triplet absorption spectra have been measured. Oxygen quenching of the triplet state formed singlet oxygen in significant yields.     

Water-soluble self-assembled butadiyne-bridged bisporphyrin: a potential two-photon-absorbing photosensitizer for photodynamic therapy

We have synthesized a novel, two-photon-absorbing photosensitizer for two-photon-absorption photodynamic therapy (2PA-PDT). The molecule is a butadiyne-bridged porphyrin dimer terminated with two water-soluble porphyrin monomers connected through Zn-imidazolyl self-assembly and covalently linked through olefin metathesis. It has an effective two-photon-absorption (2PA) cross-section value, sigma((2)), of 33 000+/-4600 GM with 5-ns pulses at 890 nm measured by using the open-aperture Z-scan technique. The compound was found to generate singlet oxygen, cytotoxic for tumor cells in photodynamic therapy (PDT), under 2PA conditions by conducting photobleaching experiments with anthracene-9,10-dipropionic acid sodium salt (ADPA).     

Pharmacokinetics, tissue distribution and excretion of a new photodynamic drug deuxemether

Deuxemether was a new photodynamic drug effective for many kinds of solid tumor therapy, which was mainly composed of 3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deutero-porphyrin IX (MHD) and 3,8-di(1-methoxyethyl)-deuteroporphyrin IX (DMD). The aims of this study were to elucidate its pharmacokinetic characteristics, tissue distribution, plasma protein binding and excretion properties and underlying mechanisms of deuxemether in rats based on the simultaneous determination of MHD and DMD. The pharmacokinetic profiles of both MHD and DMD in rats after intravenous doses were linear and best fitted to a two compartment model, characterized with a rapid distribution phase (MHD: t_1/2, 0.09–0.14 h; DMD: t_1/2, 0.07–0.11 h) and a relatively slow elimination phase (MHD: t_1/2β, 2.03–3.20 h; DMD: t_1/2β, 2.51–3.20 h). The tissue distributions of MHD and DMD in rats were rather limited as evidenced from their low distribution volume (0.75–1.70 L/kg) and the results of tissue distribution study. Protein binding of MHD and DMD were moderate (65.36–89.99% for MHD; 45.43–76.23% for DMD), independent of drug concentrations and similar between human and rat plasma over a concentration range of 0.50–50.0 μg/mL. Both MHD and DMD were predominantly (>74.1%) eliminated from rats as the parent drugs through the hepatobiliary systems and finally excreted into the feces. The multidrug resistance-associated proteins 2 (MRP2) inhibitors, bromosulfophthalein and probenecid, substantially inhibited the hepatobiliary elimination of MHD and DMD while the P-gp inhibitor digoxin had little effect, suggesting that MRP2 may contribute to the rapid and extensive hepatobiliary excretion of deuxemether. There were no significant differences between MHD and DMD for all pharmacokinetic characteristics studied. In conclusion, this study provided firstly the full pharmacokinetic characteristics and mechanisms of deuxemether, which would be helpful for its clinical regiment design.     

Polymeric photosensitizer prodrugs for photodynamic therapy

A targeting strategy based on the selective enzyme-mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared "first-generation" PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly-(L)-lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T-24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable "second-generation" PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme-specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over-express a specific enzyme.     

Polycationic chitosan-conjugated photosensitizer for antibacterial photodynamic therapy

The complex nature of bacterial cell membrane and structure of biofilm has challenged the efficacy of antimicrobial photodynamic therapy. This study was aimed to synthesize a polycationic chitosan-conjugated rose bengal (CSRB) photosensitizer and test its antibiofilm efficacy on Enterococcus faecalis (gram positive) and Pseudomonas aeruginosa (gram negative) using photodynamic therapy. During experiments, CSRB was tested along with an anionic photosensitizer rose bengal (RB) and a cationic photosensitizer methylene blue (MB) for uptake and killing efficacy on 7-day-old E. faecalis and P. aeruginosa biofilms. Microbiological culture based analysis was used to analyze the cell viability, while laser scanning confocal microscopy (LSCM) was used to examine the structure of biofilm. The synthesized CSRB showed absorbance spectrum similar to the RB. The concentration of CSRB uptaken by both the bacterial biofilms was significantly higher than that of RB and MB (P < 0.05). Photoactivation resulted in significantly higher elimination of both bacterial biofilms sensitized with CSRB than RB and MB. The structure of biofilm under LSCM was found to be disrupted following CSRB treatment. The present study highlighted the importance of inherent cell membrane permeabilizing effect of chitosan and increased cell/biofilm uptake of conjugated photosensitizer to produce significant antibiofilm efficacy during photodynamic therapy.     

Assessment of photosensitizer dosimetry and tissue damage assay for photodynamic therapy in advanced-stage tumors

Photodynamic therapy (PDT) efficacy is a complex function of tissue sensitivity, photosensitizer (PS) uptake, tissue oxygen concentration, delivered light dose and some other parameters. To better understand the mechanisms and optimization of PDT treatment, we assessed two techniques for quantifying tissue PS concentration and two methods for quantifying pathological tumor damage. The two methods used to determine tissue PS concentration kinetic were in vivo fluorescence probe and ex vivo chemical extraction. Both methods show that the highest tumor to normal tissue PS uptake ratio appears 4 h after PS administration. Two different histopathologic techniques were used to quantify tumor and normal tissue damage. A planimetry assessment of regional tumor necrosis demonstrated a linear relationship with increasing light dose. However, in large murine tumors this finding was complicated by the presence of significant spontaneous necrosis. A second method (densitometry) assessed cell death by nuclear size and density. With some exceptions the densitometry method generally supported the planimetry results. Although the densitometry method is potentially more accurate, it has greater potential subjectivity. Finally, our research suggests that the tools or methods we are studying for quantifying PS levels and tissue damage are necessary for the understanding of PDT effect and therapeutic ratio in experimental in vivo tumor research.     

Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy.

The tissue photosensitizer protoporphyrin IX (PpIX) is an immediate precursor of heme in the biosynthetic pathway for heme. In certain types of cells and tissues, the rate of synthesis of PpIX is determined by the rate of synthesis of 5-aminolevulinic acid (ALA), which in turn is regulated via a feedback control mechanism governed by the concentration of free heme. The presence of exogenous ALA bypasses the feedback control, and thus may induce the intracellular accumulation of photosensitizing concentrations of PpIX. However, this occurs only in certain types of cells and tissues. The resulting tissue-specific photosensitization provides a basis for using ALA-induced PpIX for photodynamic therapy. The topical application of ALA to certain malignant and non-malignant lesions of the skin can induce a clinically useful degree of lesion-specific photosensitization. Superficial basal cell carcinomas showed a complete response rate of approximately 79% following a single exposure to light. Recent preclinical studies in experimental animals and human volunteers indicate that ALA can induce a localized tissue-specific photosensitization if administered by intradermal injection. A generalized but still quite tissue-specific photosensitization may be induced if ALA is administered by either subcutaneous or intraperitoneal injection or by mouth. This opens the possibility of using ALA-induced PpIX to treat tumors that are too thick or that lie too deep to be accessible to either topical or locally injected ALA.     

Photostability of bacteriochlorophyll a and derivatives: potential sensitizers for photodynamic tumor therapy

The photostabilities of bacteriochlorophyll a and several of its derivatives, which are of interest as potential sensitizers in photodynamic tumor therapy, were investigated. The pigments were irradiated with light >630 nm in organic solvents (acetone, tetrahydrofuran, pyridine, methanol, ethanol, n-propanol, 2-propanol and toluene) and in aqueous detergent solutions (cetyl-trimethyl-ammonium bromide [CTAB], lauryldimethyl-aminoxide [LDAO] or sodium dodecyl-sulfate [SDS] and Triton X-100 [TX100]). Their stabilities in these different solvents were determined in the presence and absence of an external sensitizer (pyromethyl-pheophorbide a), oxygen, sodium ascorbate and inert gas (Ar) or vacuum. The photodegradation products of bacteriochlorophyll a in acetone solution were isolated, purified by HPLC and analyzed by their absorption spectra and mass spectroscopy. Besides the well-known dehydrogenation products, such as [3-acetyl]-chlorophyll a, which were obtained as by-products, the major products had low absorption in the visible-near infrared spectral range. The spectral signature of the major component of these products was characteristic of linear open-chain tetrapyrroles, but they lacked the characteristic protonation-deprotonation behavior and reactivity of bilins with Zn(++).     

Tetra-trifluoroethoxyl zinc phthalocyanine: potential photosensitizer for use in the photodynamic therapy of cancer.

Tetra(trifluoroethoxyl) zinc phthalocyanine, which could be dissolved in most organic solvents, was synthesized. The compound displays a good photocytotoxicity on myeloma cells and Chinese hamster ovary cells in vitro in the presence of the emulsifying agent F68.     

Photophysical parameters, photosensitizer retention and tissue optical properties completely account for the higher photodynamic efficacy of meso-tetra-hydroxyphenyl-chlorin vs Photofrin

Meso-tetra-hydroxyphenyl-chlorin (mTHPC) is one of the most potent photosensitizers currently available for clinical photodynamic therapy (PDT). However the reason or reasons for its high photodynamic efficacy remain(s) unresolved. To investigate the PDT efficacy of mTHPC vs Photofrin we use the knowledge of photophysical parameters extracted from the analysis of oxygen electrode measurements in spheroids to compute and compare their respective singlet oxygen (1O2) dose depositions. The electrode measurements indirectly report the bleaching kinetics of mTHPC and indicate that its photobleaching mechanism is consistent with 1O2-mediated reactions. mTHPC's photodegradation via 1O2 reactions is confirmed by a more direct evaluation of the spatially resolved fluorescence in confocal sections of intact spheroids during irradiation. The PDT efficacy comparisons establish that mTHPC's enhanced potency may be accounted for completely on the basis of its ability to sequester tightly in cells and its photophysical properties, in particular its higher extinction coefficient at a redshifted wavelength. We extend the efficacy comparison to include the influence of hemoglobin absorption of PDT treatment light and show that incorporating the influence of wavelength-dependent light attenuation in tissue further contributes to significantly higher efficacy for mTHPC- vs Photofrin-PDT.     

Metal phthalocyanine as photosensitizer for photodynamic therapy (PDT)

A series of sulfonated (S) phthalimidomethyl (P) zinc phthalocyanines (Pc) was synthesized in a reaction, in which both kinds of substituents were introduced to ZnPc simultaneously. The products were separated by HPLC. The five different fractions obtained were further purified by a membrane separation method, and then characterized by UV/Vis, IR, element analysis and the abilities to generate singlet oxygen upon irradiation by light as well as a preliminary determination ofin vitro antitumor activities. The results show that one of the five separating parts with formula of ZnPcS₂P₂ exhibited rather good PDT activity. The compound was further characterized by NMR, MS and thermal analysis. Studies onin vivo antitumor activities of ZnPcS₂P₂ as photosensitizer show that its inhibitory rate was up to 89.8% and 90.8% for S₁₈₀ and U₁₄ solid tumors transplanted in mice respectively when the dosage of drug was 2 mg/kg and the dosage of laser light with 670 nm wavelength was 72 J/cm². Several structural factors influenced on the PDT activities were discussed.     

The role of photosensitizer molecular charge and structure on the efficacy of photodynamic therapy against Leishmania parasites

Photodynamic therapy (PDT) is emerging as a potential therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). In order to establish a rationale for effective PDT of CL, we investigated the impact of the molecular charge and structure of photosensitizers on the parasitic phototoxic response. Two photosensitizers from the benzophenoxazine family that bear an overall cationic charge and two anionic porphyrinoid molecules were evaluated. The photodynamic activity of the photosensitizers decreases in the following order: EtNBSe > EtNBS > BpD > PpIX. The studies suggest that compared to hydrophobic anionic photosensitizers, the hydrophilic cationic benzophenoxazine analogs provide high effectiveness of PDT possibly due to (1) their strong attraction to the negatively charged parasitic membrane, (2) their hydrophilicity, (3) their high singlet oxygen quantum yield, and (4) their efficacy in targeting intracellular organelles.     

Synthesis, characterization and photodynamic activity of amino-substituted hypocrellin derivatives

Three new hypocrellin derivatives, amino- or amino acid-substituted on the side ring of hypocrellin B (HB), were synthesized by the reactions of HB with 3-methoxypropylamine, 6-aminohexanoic acid and gamma-amino-n-butyric acid, respectively. The structures of these compounds were characterized with proton nuclear magnetic resonance spectra, infrared spectra and mass spectra. The UV-visible absorption spectra, singlet oxygen-generating quantum yield and amphiphilicities of hypocrellin derivatives were measured and compared with HB, the parent compound. These derivatives showed strong absorption in the domain of the phototherapeutic window (600-900 nm) and improved amphiphilicity. HB and the derivatives were preliminarily tested for their photodynamic effects on human oral cavity epithelial carcinoma KB cell line in vitro. Two amino acid-substituted hypocrellins showed phototoxicity to the KB cell line. At an inhibitory dosage of 50% killing only 0.51 mumol L-1 compound 3 (or 0.88 mumol L-1 compound 2) and 0.5 J cm-2 irradiation were required. The hypocrellins exhibited some dark toxicity to the KB cell line. HB and amino acid-substituted hypocrellins showed lower dark toxicity to the KB cell line than amino-substituted hypocrellins in the assessment of cell survival.     

Effect of photosensitizer dose on fluence rate responses to photodynamic therapy

Photodynamic therapy (PDT) regimens that conserve tumor oxygenation are typically more efficacious, but require longer treatment times. This makes them clinically unfavorable. In this report, the inverse pairing of fluence rate and photosensitizer dose is investigated as a means of controlling oxygen depletion and benefiting therapeutic response to PDT under conditions of constant treatment time. Studies were performed for Photofrin-PDT of radiation-induced fibrosarcoma tumors over fluence rate and drug dose ranges of 25-225 mW cm(-2) and 2.5-10 mg kg(-1), respectively, for 30 min of treatment. Tumor response was similar among all inverse regimens tested, and, in general, tumor hemoglobin oxygen saturation (SO2) was well conserved during PDT, although the highest fluence rate regimen (225 mWx2.5 mg) did lead to a modest but significant reduction in SO2. Regardless, significant direct tumor cell kill (>1 log) was detected during 225 mWx2.5 mg PDT, and minimal normal tissue toxicity was found. PDT effect on tumor oxygenation was highly associated with tumor response at 225 mWx2.5 mg, as well as in all other regimens tested. These data suggest that high fluence rate PDT can be carried out under oxygen-conserving, efficacious conditions at low photosensitizer dose. Clinical confirmation and application of these results will be possible through use of minimally invasive oxygen and photosensitizer monitoring technologies, which are currently under development.     

A highly water-soluble C60-NVP copolymer: a potential material for photodynamic therapy

Water-soluble C60 polymers, which have the highest solubility of fullerene reported to date (7.8 mM of C60 in water), were prepared via copolymerization, and showed significant visible light-induced O2*- generation and DNA cleavage, indicating the potential of these water-soluble fullerene derivatives as agents for photodynamic therapy.     

新型光敏剂32-(4-甲氧基苯基)-152-天冬氨酸-二氢卟吩e6的药代动力学及组织分布特征

二氢卟吩类衍生物3²-(4-甲氧基苯基)-15²-天冬氨酸-二氢卟吩e6(DYSP-C34)是从程海湖螺旋藻中提取并合成的新型光敏剂。研究DYSP-C34在生物体内的药代动力学及组织分布过程对光动力疗法(PDT)的有效性和安全性至关重要。该文运用高效液相色谱-紫外(HPLC-UV)检测技术,建立了大鼠血浆中DYSP-C34的检测方法。采用沉淀蛋白-液液萃取法处理血浆和组织样品,采用Unitary C₁₈色谱柱(250 mm×4.6 mm, 5 μm)分离,流动相为甲醇-5 mmol/L四丁基磷酸氢铵缓冲盐溶液(70∶30, v/v),流速为1.0 mL/min,进样量为20 μL,检测波长为400 nm,柱温为40 ℃。实验结果表明,大鼠血浆药物质量浓度在1~200 μg/mL范围内线性良好,判定系数(r²)为0.9941。在低、中、高(8、40、120 μg/mL)3个添加水平下的提取回收率分别为74.39%、69.71%和65.89%,日内和日间相对标准偏差(RSD)均在5%以内。运用此方法测定静脉注射DYSP-C34(16 mg/kg)后大鼠血浆中以及荷瘤小鼠组织中的药物浓度,采用DAS 2.0计算出药物半衰期t_1/2z为6.98 h,药-时曲线下面积AUC_(0-∞)为1025.01 h·mg/L,平均驻留时间MRT_(0-∞)为9.19 h。DYSP-C34在荷瘤小鼠体内的分布结果显示,DYSP-C34可以在肿瘤组织中蓄积,并具有一定的滞留作用。综上,该文建立了大鼠血浆中DYSP-C34的HPLC-UV测定方法,并进行了方法学验证,此方法简便、快速,结果准确。阐明了DYSP-C34在静脉给药方式下大鼠体内药代动力学和荷瘤小鼠组织中的分布特征,对临床合理用药和药效学研究具有重要意义。     

Curcumin as a photosensitizer: From molecular structure to recent advances in antimicrobial photodynamic therapy

Nowadays multi-drug resistant microorganisms is a serious public health problem worldwide. To overcome it, new antimicrobial strategies have been developed. Among them, antimicrobial photodynamic therapy is an efficient tool against various micro-organisms in different medical and healthcare fields. The antimicrobial photodynamic protocol is based on the interaction of a photosensitizer, molecular oxygen, and an appropriate light source. Herein, we described the main physical and chemical proprieties of curcumin, an useful natural photosensitizer, including its degradation pathways, analytical methods for quantification, extraction method, synthetic methodologies, and pharmaceutical formulations used. Moreover, a comprehensive review of the past 10 years (2010−2019) concerning the application of curcumin as photosensitizer against microorganisms is described and discussed.     

Spectroscopic studies and photodynamic actions of hypocrellin B in liposomes

Hypocrellin B (HB), a lipid-soluble natural pigment of perylenequinone derivative, is considered as potential photosensitizer for photodynamic therapy. Liposomes loaded with HB can constitute a simple model system, appropriate for better understanding the photodynamic action of HB in vivo. The steady-state absorption and emission spectra, quantum yield and lifetime of fluorescence of HB incorporated into egg L-a-phosphatidyl-choline (EPC) liposome were examined. The photochemical properties (Type I and/or Type II) of HB have also been studied in aqueous dispersions of small unilamellar liposomes of EPC using electron paramagnetic resonance and spectrophotometric methods, respectively. The quantum yield of 1O2 generated by HB is ca 0.76 in chloroform solution and it did not change upon the incorporation of HB into liposomes of EPC. The superoxide anion radical was generated by the electron transfer from the anion radical of HB (HB.-) to oxygen. The disproportionation of O2.- can generate H2O2 and ultimately the highly reactive .OH via the Fenton reaction. It could be that the disproportionation proceeded too fast, so we could not detect O2.- directly in aqueous dispersions of liposome EPC. Moreover, the self-sensitized photooxygenation of HB embedded in liposomes was studied, and almost fully (87%) inhibiting this reaction of HB by p-benzoquinone (as the quencher of O2.-) in aqueous dispersion of liposome EPC indicated that the radical mechanism (Type I) might be mainly involved in this oxygenation. All these findings suggested that the photodynamic action of HB proceeded via both Type-I and -II mechanisms, but Type-I mechanism might play a more important role in the aqueous dispersion.