Synthesis and biological activities of chlorin e6-based conjugate of fluorouracil as dual-mode antitumor photosensitizer / 药学实践杂志

Objective To design and synthesize the conjugate (compound 1) of chlorin e6 (compound 3) with fluorouracil (5-Fu) as novel pH-responsive dual-mode antitumor photosensitizer by acyl hydrazone bond coupling, based on literature reports that combination of 5-Fu and photosensitizer possess synergistic anti-tumor effect, and investigate its photodynamic antitumor activity and mechanism. Methods Lead compound 3 was obtained by alkali degradation with 25% KOH-CH3OH on pheophorbide a (compound 4) which was prepared through acid hydrolysis of chlorophyll a in crude chlorophyll extracts from silkworm excrement. Reflux reaction of 5-Fu with P2S5 in pyridine formed crude 4-thio-5-fluorouracil which was followed to react with hydrazine hydrate (N2H4·H2O) in CH3OH to give 5-fluorouracil-4-hydrazone (compound 2). Then, treatment of compound 3 i.e. acid alkali degradation product of chlorophyll a in silkworm excrement with EDC·HCl generated its 171- and 152 cyclic anhydride which was followed to directly react with intermediate compound 2 to successfully get title compound 1. In addition, its pH-responsive 5-Fu release and photodynamic antitumor activity and their mechanisms in vitro were investigated. Results Compound 1 could responsively release 5-Fu at pH 5.0, with a cumulative release rate of 60.3% within 24 h. It exhibited much higher phototoxicity against melanoma B16-F10 and liver cancer HepG2 cells than talaporfin and its precursor compound 3, with IC50 value being 0.73 μmol/L for B16-F10 cells and 0.90 μmol/L for HepG2 cells, respectively. Upon light irradiation, it also could significantly induce cell apoptosis and intracellular ROS level and block cell cycle in S phase. Its structure was confirmed by UV, 1H-NMR, ESI-MS and elemental analysis data. Conclusion The conjugate compound 1 of compound 3 and 5-Fu has the advantages of strong PDT anticancer activity, high therapeutic index (i.e. dark toxicity/phototoxicity ratio) and responsively release 5-Fu at pH 5.0 etc. which shows “unimolecular” dual antitumor effects of PDT and chemotherapy and is worthy of further research and development.

Brain endothelial cell-derived extracellular vesicles with a mitochondria-targeting photosensitizer effectively treat glioblastoma by hijacking the blood‒brain barrier

Glioblastoma (GBM) is the most aggressive malignant brain tumor and has a high mortality rate. Photodynamic therapy (PDT) has emerged as a promising approach for the treatment of malignant brain tumors. However, the use of PDT for the treatment of GBM has been limited by its low blood‒brain barrier (BBB) permeability and lack of cancer-targeting ability. Herein, brain endothelial cell-derived extracellular vesicles (bEVs) were used as a biocompatible nanoplatform to transport photosensitizers into brain tumors across the BBB. To enhance PDT efficacy, the photosensitizer chlorin e6 (Ce6) was linked to mitochondria-targeting triphenylphosphonium (TPP) and entrapped into bEVs. TPP-conjugated Ce6 (TPP-Ce6) selectively accumulated in the mitochondria, which rendered brain tumor cells more susceptible to reactive oxygen species-induced apoptosis under light irradiation. Moreover, the encapsulation of TPP-Ce6 into bEVs markedly improved the aqueous stability and cellular internalization of TPP-Ce6, leading to significantly enhanced PDT efficacy in U87MG GBM cells. An in vivo biodistribution study using orthotopic GBM-xenografted mice showed that bEVs containing TPP-Ce6 [bEV(TPP-Ce6)] substantially accumulated in brain tumors after BBB penetration via transferrin receptor-mediated transcytosis. As such, bEV(TPP-Ce6)-mediated PDT considerably inhibited the growth of GBM without causing adverse systemic toxicity, suggesting that mitochondria are an effective target for photodynamic GBM therapy.

Photodynamic effects of gold nanostars loading chlorin e6 on lung cancer A549 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To prepare GNS (gold nanostars) loading photosensitizer chlorin e6 (Ce6) and to investigate its photodynamic effects on lung cancerA549 cells. Methods: GNS was firstly modified by SH-PEG-NH2 and then mixed with Ce6 and shaken overnight to prepare GNS-PEG@Ce6, which had photodynamic therapy effects. The characterization, morphology and encapsulation rate were detected. The difference between the phagocytosis of Ce6 and GNS-PEG@Ce6 by A549 cells were observed with a Leical TCS SP8 confocal laser scanning microscope. MTT assay was used to examine the inhibitory effect of GNS-PEG@Ce6 on the proliferation of A549 cells while FCM was used to detect the effect of probe GNS-PEG@Ce6 on the apoptosis ofA549 cells. Results: The particle size of the GNS-PEG@Ce6 was about 100 nm. The prepared GNS-PEG@Ce6 nanoparticles exhibited good dispersion and stability and the encapsulation rate of Ce6 was about 50%. GNS-PEG@Ce6 entered the cells by endocytosis and mainly distributed in the cytoplasm; compared with Ce6, GNS-PEG@Ce6 could enter the cells more effectively. The proliferation-suppression effect of GNS-PEG@Ce6 on A549 cells was significantly stronger than that of Ce6 (P<0.05). The results of flow cytometry showed that the probe exhibited strong apoptotic effect on A549 cells. Conclusion: GNS, as the drug carrier, could effectively increase the Ce6 uptake efficacy in A549 cells, thus further enhancing the killing effects of Ce6 on lung cancerA549 cells.

Optimization of synthesis process of photosensitizer chlorin f / 药学实践杂志

Objective To improve the synthesis process of chlorine f (1).Methods A "one-pot"method was applied to prepare Photosensitizer component (1),using pheophorbide a (3) as raw material by oxidating and cracking of the E-ring of (3) with bubbling oxygen in alcoholic solution of potassium hydroxide at 0 ℃ followed by refluxing in nitrogen atmosphere.In order to obtain the optimal synthetic procedure,the orthogonal experimental design of L9 (34 ) was adopted to investigate three different levels of four main factors i.e.ring opening reaction time,alcoholic variety,alkali concentration and refluxing reaction time.Results The target compound (1) was optimizedly synthesized through treatment of raw material (3) with bubbling oxy-gen in 25% ethanol solution of potassium hydroxide at 0℃ for 30 min,followed by refluxing in nitrogen atmosphere for 20 min in yield of 40.8%.Conclusion The procedure developed has some advantages of simple and safty operation,and high synthetic yield.

Identificação e propriedades físico-químicas da clorofilina cúprica de sódio e da clorina cúprica e6 / Identification and physicochemical properties of sodium copper chloruphyllin and copper chlorin e6

A clorofilina cúprica de sódio (CuChl) é um corante semissintético derivado da clorofila. Quimicamente é constituído de diversas clorinas, em especial a clorina cúprica e4 (CuCe4), a clorina cúprica e6 (CuCe6), e possíveis clorinas e porfirinas não cúpricas em proporções variáveis. Além do seu uso como corante alimentar, são atribuídas atividades biológicas à CuChl, tais como, antimutagênica, anticarcinogênica e antioxidante. Em decorrência destes potenciais efeitos benéficos, sua comercialização sob a forma de suplementos é crescente. Todavia, curiosamente, informações sobre a absorção e biodisponibilidade da CuChl são escassas. Além disso, até o momento nenhum estudo avaliou o impacto da composição da CuChl em sua bioatividade e eficácia. Assim, o presente estudo teve como objetivo identificar e caracterizar quimicamente duas amostras de CuChl (Sigma® e Chr. Hansen®) e o padrão de CuCe6 (Frontier Scientific®). Para tanto, empregou-se técnicas cromatográficas e espectrofotométricas, determinou-se a lipofilicidade em modelos miméticos de membrana, cinética de degradação e avaliou-se a interação CuCe6/BSA. A análise elementar da CuChl resultou em teores de cobre total inferiores aos recomendados pela United States Pharmacopeia (U.S.P.). Os elementos (CHN) e a razão Cu/N não foram coerentes com os valores teóricos da molécula de CuChl. Apenas uma amostra de CuChl apresentou razão Soret/Q dentro dos valores preconizados pela U.S.P. A titulação base-ácido da CuCe6 revelou dois valores de pkas (10,62 e 6,41) que foram similares para as amostras de CuChl. A determinação de log P da CuCe6 mostrou que a hidrofobicidade é máxima em pH 3 (log P = 1,49±0,09) e sua hidrofilicidade ocorre em pHs > 7. Esse comportamento foi confirmado nos ensaios de incorporação em lipossomas em função do pH. A degradação térmica da CuChl (25 a 95 °C) avaliada por HPLC foi drástica a partir de 75 °C. A energia necessária para que ocorra a degradação da CuChl e CuCe6 é Ea = 16,1 e 9,3kcal/mol, respectivamente. A meia-vida a 35 °C é de 6 horas para a CuChl e 2 horas e meia para a CuCe6. A separação mais eficiente dos componentes da CuChl por HPLC foi conseguida utilizando coluna C30 e a identificação dos principais constituintes CuCe6, CuCe4 e a clorina cúprica p6 (CuCp6), ocorreu por HPLC/MSMS. No estudo da ligação entre CuCe6 e proteína BSA foram obtidos os valores de KD = 0,38 ± 0,07 µM, KA = 3,3 ± 0,28 x 106 M-1 e número de sítios de ligação ~1 (N = 0,75 ± 0,09), indicativo de alta afinidade entre a clorina e a proteína. Assim, o comportamento químico dos principais componentes da CuChl e sua interação com os componentes do soro tornaram inviáveis a identificação e quantificação destas moléculas em ensaios in vivo. Os resultados aqui apresentados servem de subsídio para o desenvolvimento de outras pesquisas que visem o estudo específico da associação e dissociação da CuChl em material biológico

Sodium copper chlorophyllin (CuChl) is a semisynthetic derivative of chlorophyll dye. It is composed chemically by several chlorins, especially copper chlorin e4 (CuCe4), copper chlorin e6 (CuCe6), and possible others no copper porphyrins and chlorins in different proportions. In addition to its use as a food coloring, CuChl may have interesting biological effects as antimutagenic, anticarcinogenic and antioxidant. Because of these potential benefits, its use as a dietary supplement is increasing. However, information on the absorption and bioavailability of CuChl is scarce. Furthermore, no studies have evaluated the impact of CuChl composition in its bioactivity and efficacy. Thus, the present study aimed to identify and chemically characterize two samples of CuChl (Sigma® and Hansen®) and the standard of CuCe6 (Frontier Scientific®). Chromatographic and spectrometric techniques as well as mimetics models membrane were used. The CuCe6/BSA interaction was also evaluated. The elemental analysis of CuChl showed that the total copper content of it was smaller that the one recommended by United States Pharmacopeia (USP). The elements (CHN) and the ratio Cu / N were not consistent with the theoretical values of the molecule CuChl. Only one CuChl sample showed Soret / Q ratio within the range recommended by USP. The acid-base titration of CuCe6 revealed two pKas values (10.62 and 6.41), which were similar for CuChl samples. The log P determination of CuCe6 showed that its hydrophobicity is maximal at pH 3 (log P = 1.49 ± 0.09) and its hydrophilicity occurs at pH> 7. These results were confirmed using the incorporation into liposomes assay in function of pH. Using HPLC, it was observed that thermal degradation of CuChl (25 to 95 °C) hardly occurred from 75 °C. The energy necessary for CuChl and CuCe6 degradation is Ea = 16.1 and 9.3 kcal/mol, respectively. The half-life at 35°C for CuChl and CuCe6 is 6 hours and 2 ½ hours, respectively. A more efficient separation of the CuChl components by HPLC was achieved using a C30 column while its major constituents CuCe6, CuCe4 and copper chlorin p6 (CuCp6) were identified by HPLC / MS-MS. In binding analysis of CuCe6 and BSA, it was observed KD = 0.38 ± 0.07 mM, KA = 3.3 ± 0.28 x 106 M-1, and number of binding sites ~ 1 (N = 0.75 ± .09), indicating high affinity between BSA and chlorine. Thus, due to the chemical characteristics of the main components of CuChl and their interaction with serum components the identification and quantification of these molecules in vivo is unviable. Future studies should investigate the association and dissociation of CuChl in biological samples

Estudio fitoquímico de hojas de Uncaria guianensis y evaluación de actividad antibacteriana / Phytochemical study of Uncaria guianensis leaves and antibacterial activity evaluation

Del extracto de éter de petróleo de hojas de Uncaria guianensis (Rubiaceae), se aisló un compuesto tipo clorina denominado éster etílico de feoforbida a y una mezcla de esteroles conocidos como ß-sitosterol y estigmasterol. Sus estructuras fueron elucidadas por análisis detallado de RMN, incluyendo técnicas bidimensionales, y por comparación con datos reportados en la literatura. Posteriormente, se evaluó la actividad antibacteriana al éster etílico de feoforbida a contra dos cepas Gram(+): S. aureus ATCC 6538 y E. faecalis ATCC 29212 y contra tres cepas Gram (-): E. coli ATCC 25922, S. typhimurium ATCC 14028s y S. typhimurium MS7953. Se encontró actividad significativa contra S. aureus, E. faecalis, E. coli y S. tiphymurium MS7953.

A chlorin compound, pheophorbide a ethyl ester and a mixture of sterols known as ß-sitosterol and stigmasterol, were isolated from the petroleum ether extract of Uncaria guianensis (Rubiaceae) leaves. Their structures were elucidated by detailed analysis of NMR spectra, including bidimensional techniques and by comparison with literature data. The antibacterial activity for the pheophorbide a ethyl ester was evaluated against two Gram (+) strains: S. aureus ATCC 6538 y E. faecalis ATCC 29212 and three Gram (-) strains: E. coli ATCC 25922, S. typhimurium ATCC 14028s y S. typhimurium MS7953S. aureus ATCC 6538 and E. fecalis ATCC 29212, finding significant activity against S. aureus 6538, E. faecalis 29212, S. tiphymurium MS7953 and E. coli 25922.

The sonodynamic effect of chlorin-e6 on the proliferation of human breast cancer cells MDA-MB-231 / 肿瘤

Objective:To evaluate the sonodynamic effect of chlorin-e6, a sonosensitizing agent, on the proliferation of MDA-MB-231 cells. Methods:MDA-MB-231 and normal peripheral mononuclear cells (PMNCs) were treated with chlorin-e6 alone or combined with ultrasound. Cell proliferation was determined by MTT assay and cell morphology was observed under inversed fluorescence microscope. Results:Treatment with ultrasound (1.0 MHz, 1.0-2.0 W/cm~2,60 s) or chlorin-e6 (0.10-1.60 mg/mL) alone significantly inhibited the cell proliferation of both MDA-MB-231 and PMNC cells in a intensity-dependent and a dose-dependent manner, respectively. The 50% intensity of ultrasound for inhibiting the growth of MDA-MB-231 and PMNC cells was 1.23 W/cm~2 and 1.25 W/cm~2, respectively (P>0.05) and the IC_(50) of chlorin-e6 was 0.38 mg/mL and 0.77 mg/mL, respectively (P0.05). Compared with single ultrasound treatment (1.0 MHz, 0.5 W/cm~2,60 s) and single chlorin-e6 treatment (0.20 mg/mL), combination of ultrasound and chlorin-e6 significantly increased the death rate of MDA-MB-231 cells (P<0.05). Conclusion:Ultrasound combined with chlorin-e6 exerted specific inhibitory effect on the proliferation of MDA-MB-231 cells. Chlorin-e6 may be a promising sonosensitizing agent for the treatment of breast cancer.

Therapeutic effect of photodynamic therapy with chlorin e6 and 5-aminolevulinic acid on malignant melanoma in mice / 第三军医大学学报

Objective To investigate the effect of photodynamic therapy with chlorin e6 (Ce6) and 5-aminolevulinic acid (5-ALA) on mouse model of malignant melanoma. Methods The mouse model of malignant melanoma was established by injecting 0.1 ml A-375 cells (about 2?10 6 cells) under the right hind leg of BALB/c-6 mice,and that the yellowish white node appears at injection site proves the successful model. Twenty-four of 27 successful mouse models were irradiated at the tumor site with semiconductor laser (wavelength 652 nm) with a total dose of 100 J/cm 2 . Before laser exposure,the mice were treated with 10% 5-ALA by topical compress for 2 h or 7.5 mg/kg Ce6 by intraperitoneal injection for 1 hour or 5-ALA topical application combined with intraperitoneal injection of Ce6 (n=6 in each group). Another six mice as control only underwent PDT. One week after PDT,the mice were killed,the tumor mass was peeled off and weighed,whether the metastasis occurred or not was detected,and the tumor,liver,spleen,lung,kidney were sent to histopathological examination. Results The tumor weight in 5-ALA group,Ce6 group,and the combined group had significant difference as compared with control group (P0.05). The dehydration and scab formation and necrosis could be seen in tumor sites at 1 week after PDT. The cell collapse and necrosis,subdermal thrombosis and cell outline clouding could be observed by histopathological examination. Metastasis of melanoma were found in 5-ALA group,Ce6 group,and the combined group. Conclusion PDT with Ce6 and 5-ALA could kill the malignant melanoma effectively in animal experiment but could not affect the metastasis of melanoma.

Identification of chlorin metal complexes by the convolution spectrum method / 第二军医大学学报

Objective: To qualitatively identify 5 chlorin metal complexes which had similar structures. Methods: The compounds were identified by the convolution curve transformation technology and computer information process technology. Results: The results were demonstrated by match comparison and three dimensional differential diagram of convolution spectra. Five chlorin metal complexes were identified satisfactorily and 10 nonidentity identification results were acquired. Conclusion: The convolution spectrum method is simple and feasible for the identification of the compounds which have similar structures. [