Photodynamic Therapy of Melanoma B16 with Chlorin E6 Conjugated with a PSMA-Ligand.

The efficacy of a new photosensitizer of chlorin E6 conjugated with a prostate-specific membrane antigen (PSMA) in photodynamic therapy of murine melanoma B16 was studied in in vivo experiments. The dynamics of photosensitizer accumulation in the tumor and surrounding tissues was evaluated and antitumor efficacy of photodynamic therapy was assessed by parameters of regression and morphological characteristics of experimental transplanted melanoma B16. The inhibitory effect of photodynamic therapy on melanoma was evaluated by complete regression of the tumor, absolute tumor growth coefficient in animals with continuation of tumor growth, and the increase in life span in comparison with the control; the criterion of cure was the absence of signs of tumor recurrence in mice within 90 days after therapy. The therapeutic potential of photodynamic therapy was determined by devitalization of tumor cells (histological examination of the zones of laser exposure on day 21 after treatment). The photosensitizer with PSMA-ligand exhibited high antitumor activity in photodynamic therapy for melanoma B16. Photodynamic therapy carried out at the optimum time after photosensitizer injection with experimentally determined parameters of laser exposure allows achieving the maximum inhibitory effect on melanoma. Pathomorphological study in the zones of exposure detected no survived tumor cells.

Efficient Delivery of Chlorin e6 by Polyglycerol-Coated Iron Oxide Nanoparticles with Conjugated Doxorubicin for Enhanced Photodynamic Therapy of Melanoma.

Chlorin e6 (Ce6) is a promising photosensitizer for tumor photodynamic therapy (PDT). However, the efficacy of Ce6 PDT is limited by Ce6's poor water solubility, rapid blood clearance, and inadequate accumulation in the tumor tissue. This problem is tackled in this work, wherein functionalized superparamagnetic iron oxide nanoparticles (IO-NPs) were used as carriers to deliver Ce6 to melanoma. The IO-NPs were coated with polyglycerol (PG) to afford good aqueous solubility. The chemotherapeutic agent doxorubicin (DOX) was attached to the PG coating via the hydrazone bond to afford affinity to the cell membrane and thereby promote the cell uptake. The hydrophobic nature of DOX also induced the aggregation of IO-NPs to form nanoclusters. Ce6 was then loaded onto the IO nanoclusters through physical adsorption and coordination with surface iron atoms, yielding the final composites IO-PG-DOX-Ce6. In vitro experiments showed that IO-PG-DOX-Ce6 markedly increased Ce6 uptake in mouse melanoma cells, leading to much-enhanced photocytotoxicity characterized by intensified reactive oxygen species production, loss of viability, DNA damage, and stimulation of tumor cell immunogenicity. In vivo experiments corroborated the in vitro findings and demonstrated prolonged blood clearance of IO-PG-DOX-Ce6. Importantly, IO-PG-DOX-Ce6 markedly increased the Ce6 distribution and retention in mouse subcutaneous melanoma grafts and significantly improved the efficacy of Ce6-mediated PDT. No apparent vital organ damage was observed at the same time. In conclusion, the IO-PG-DOX NPs provide a simple and safe delivery platform for efficient tumor enrichment of Ce6, thereby enhancing antimelanoma PDT.

The Metabolism and Potential Bioactivity of Chlorophyll and Metallo-chlorophyll Derivatives in the Gastrointestinal Tract.

Chlorophyll is the vivid chromophore which imparts the green color to plant leaves, and is consumed by humans through green vegetables. The basic porphyrin structure of chlorophyll binds magnesium in plants, but can bind different divalent metals (e.g., copper, zinc, iron) facilitated by food processing techniques and/or chemical synthesis. This review covers the known elements of chlorophyll and metallo-chlorophyll absorption, distribution, metabolism, excretion in vitro and in vivo. The review discusses what is understood about the ability of these novel metallo-chlorophyll derivatives to deliver essential metals. This review also detail chlorophyll and metallo-chlorophyll toxin binding properties which largely occur during digestion, focusing on toxins including dioxins, heterocyclic aromatic amines, polyaromatic hydrocarbons, and aflatoxin. Finally, the article highlights the gaps in the understanding of the metabolism and metal and toxin-binding bioactivity of this family of molecules.

A photoactivatable antibody-Chlorin e6 conjugate enabling singlet oxygen production for tumor-targeting photodynamic therapy.

Photodynamic therapy is a new technology for disease diagnosis and treatment in modern medical clinics. The main advantages of photodynamic therapy are low toxicity and side effects, a wide range of applications, no drug resistance, and no obvious trauma in the treatment process. However, to achieve effective photodynamic therapy, new photosensitizer carriers need to be constructed, which can selectively deliver photosensitizers into tumor tissues. In this work, a photoactivatable antibody-Chlorin e6 conjugate with a dual-function to target tumor tissue and realize cancer photodynamic therapy is constructed. Bothin vitroandin vivoexperiments indicate that the antibody-Chlorin e6 conjugate has the ability to target tumors rapidly and efficiently, and has the ability to generate reactive oxygen species and kill tumor cells. Overall, this photoactivable antibody-Chlorin e6 conjugate may provide a promising strategy to address the current challenges of cancer photodynamic therapy.

An Albumin-Based Therapeutic Nanosystem for Photosensitizer/Protein Co-Delivery to Realize Synergistic Cancer Therapy.

Oxygen-dependent photodynamic therapy (PDT) is hindered by the limited availability of endogenous oxygen in solid tumors and low tumor accumulation of photosensitizers. Herein, we developed a biocompatible cancer-targeted therapeutic nanosystem based on cRGD conjugated bovine serum albumin (CBSA) co-loaded with a photosensitizer (chlorin e6, Ce6) and a therapeutic protein (cytochrome c, Cytc) for synergistic photodynamic and protein therapy. The nanosystem (Ce6/Cytc@CBSA) can target αVß3 integrin overexpressed cancer cells to improve tumor accumulation due to incorporation of cRGD. In the intracellular environment, Ce6 is released to produce toxic singlet oxygen upon near-infrared irradiation. At the same time, the therapeutic protein, Cytc, can induce programmed cell death by activating the downstream caspase pathway. Most importantly, Cytc with the catalase-like activity accelerates O2 generation by decomposing excess H2O2 in cancer cells, thereby relieving the PDT-induced hypoxia to enhance therapeutic efficacy. Both in vitro and in vivo studies reveal the significantly improved antitumor effects of the combined photodynamic/protein therapy, indicating that Ce6/Cytc@CBSA shows great potential in synergetic cancer treatments.

Development and Validation of HPLC Method for Determination of Sodium Copper Chlorophyllin - A Food Colorant and Its Application in Pharmacokinetic Study.

A simple, accurate, and precise bioanalytical method was developed and validated for the determination of pharmacokinetic parameters of sodium copper chlorophyllin, a USFDA approved food additive and colorant in rat plasma. The column used was Luna® C18 250×4.6 mm, 100 Å, having particle size 4.5 µm, and the mobile phase used was methanol (MeOH), and 10 mM ammonium acetate buffer in the ratio of 90 : 10, the flow rate was 1 ml/min, and the injection volume of 20 µL. The retention time of sodium copper chlorophyllin was obtained at 9 min. The method was found to be linear at the range of 0.50-8.00 µg mL-1 .

HPLC-hrTOF-MS study of copper chlorophylls: Composition of food colorants and biochemistry after ingestion.

Despite the daily consumption of copper chlorophylls (E-141i), the green food colorants in foods high in fats, there is a general need for knowledge regarding their exact composition. Consequently, we have analyzed by HPLC-ESI(+)/APCI(+)-hrTOF-MS2 the accurate composition of different commercial copper chlorophyll colorants for the first time. Data showed a favored yield of copper pheophytins from a series, while pheophytins from b series are preferentially no complexed with copper. The copper pheophytins present in the food colorants consisted mainly of three structural rearrangements. New fragmentation patterns and structural assignments have been described for several copper pheophytins. During the ingestion of copper chlorophylls, no chlorophyll derivative was present in serum nor urine except a new copper-pyroporphyrin a accumulated in a few livers. In any case, this green additive could represent the ideal food colorant, as most of the copper pheophytins are excreted in the feces showing almost no absorption of copper-chlorophylls compounds.

In vitro bioavailability of iron from the heme analogue sodium iron chlorophyllin.

The use of heme analogues from vegetable origin could provide an alternative iron source of potentially high bioavailability. Sodium iron chlorophyllin is a water-soluble semisynthetic chlorophyll derivative where the magnesium in the porphyrin ring has been substituted by iron. We have used an in vitro model that combines gastric and intestinal digestion followed by intestinal iron uptake in Caco-2 cells to determine the bioavailability of iron from sodium iron chlorophyllin. Our results demonstrate that sodium iron chlorophyllin is stable under simulated gastrointestinal conditions and is able to deliver bioavailable iron to Caco-2 cells. Similar to the heme, the bioavailability of iron from sodium iron chlorophyllin is dependent on the food matrix, and it was inhibited by calcium. Potentially, sodium iron chlorophyllin could be used as an iron fortificant from vegetable origin with high bioavailability.

The mutagenic potentiator effect of chlorophyllin by the HGPRT assay.

Chlorophyllin, a sodium-copper salt derivative of chlorophyll-a and -b, was evaluated for antimutagenic activity against ethyl methane sulfonate by the hypoxanthin-guanine-phosphoribosyl transferase (HGPRT) assay. The results obtained suggest that this chlorophyllin can potentiate the mutagenicity of an alkylating agent which induces DNA damage.

Chemoprevention with chlorophyllin in individuals exposed to dietary aflatoxin.

Because of the multiplicative interaction between dietary aflatoxins and hepatitis B virus infection in the etiology of liver cancer, efforts to reduce the consequences of either chemical or viral component are likely to have substantial public health benefit. Chlorophyllin (CHL), a water-soluble form of chlorophyll, was recently evaluated as a chemopreventive agent in a population at high risk for exposure to aflatoxin and subsequent development of hepatocellular carcinoma. CHL, which is used extensively as a food colorant and has numerous medicinal applications, is an effective anticarcinogen in experimental models including aflatoxin-induced hepatocarcinogenesis. CHL is thought to form molecular complexes with carcinogens, thereby blocking their bioavailability. In the clinical trial, administration of CHL three times a day led to a 50% reduction in the median level of urinary excretion of aflatoxin-N(7)-guanine compared to placebo. This excreted DNA adduct biomarker is derived from the ultimate carcinogenic metabolite of aflatoxin B(1), aflatoxin-8,9-epoxide, and is associated with increased risk of developing liver cancer in prospective epidemiologic studies. Compliance in the intervention was outstanding and no toxicities were observed. Thus, CHL has been found to be a safe and effective agent suitable for use in individuals unavoidably exposed to aflatoxins.

Identification of pelvic lymph nodes with chlorophyllin after injection into the uterine cervix: an experimental and clinical study.

We investigated the value of staining retroperitoneal lymph nodes with chlorophyllin in normal dogs and in women with malignant uterine tumors undergoing lymphadenectomy. In dogs, after 0.3% chlorophyllin (sodium copper chlorophyllin) was injected into the canine uterus, the concentration of dye in the bloodstream was measured with a spectrophotometer and sections of stained retroperitoneal lymph nodes were examined using light and electron microscopy. The highest blood levels were detected at 4 hrs and nearly all of the chlorophyllin was gone from the bloodstream by 18 hrs but was retained in nodal macrophages for at least 4 days. No morphological changes were found in the excised lymph nodes. Twenty-four patients with cervical carcinoma and 20 patients with endometrial carcinoma undergoing radical hysterectomy and lymphadenectomy were divided into a lymphatic coloration group (23 patients) and a non-coloration (control) group (21 patients). In the lymphatic coloration group (0.3% chlorophyllin) was injected into the cervix 5 days before elective lymphadenectomy. There were no complications attributed to injection of the chlorophyllin. The number of dissected lymph nodes in the coloration group were greater than the control group (p<0.01) and the time of operation was shorter (p<0.01). These results suggest that chlorophyllin is safe and facilitates identification of retroperitoneal lymph nodes, allows more complete nodal excision and shortens the time of operation in patients undergoing radical hysterectomy with lymphadenectomy.

Mechanisms of chlorophyllin anticarcinogenesis: dose-responsive inhibition of aflatoxin uptake and biodistribution following oral co-administration in rainbow trout.

Chlorophyllin (CHL) is a potent blocking agent against aflatoxin B(1) DNA adduction and tumorigenesis in the trout model, but mechanisms responsible for this chemoprotection in vivo are not well established. This study employed aflatoxin B(2) (AFB(2)), a structural analogue of AFB(1) that cannot be metabolized directly to the 8,9-exo-epoxide electrophile, to investigate CHL effects on carcinogen uptake and distribution kinetics following oral exposure in trout. CHL was shown to form an AFB(2) complex in vitro with a dissociation constant (K(d) = 1.92 +/- 0.13 microM) comparable to that with AFB(1). Following gavage, [(3)H]AFB(2) equivalents distributed rapidly from the stomach to other organs including blood, liver, and eventually to bile as a major repository. Bile was found to contain almost entirely parent AFB(2) 1 h after gavage, with a single metabolite dominating 3-24 h and an additional metabolite prominent by 48 h after gavage. Addition of sufficient CHL (>/=13.9 mM) to assure >99% complexation of AFB(2) (0.906 microM) in the gavage mix resulted in 80-90% reduction in AFB(2) equivalents in liver and bile 3 h after gavage. In three separate kinetic studies of up to 120 h postgavage, addition of >/=13.9 mM CHL to the gavage mix reproducibly and markedly delayed the rate of AFB(2) loss from stomach, retarded its appearance in blood, liver, and bile, and reduced peak AFB(2) concentrations in those tissues by up to 60%. Introduction of a food bolus immediately after gavage prolonged AFB(2) residence in stomach and intestine but did not abrogate the inhibitory effects of CHL on AFB(2) uptake and distribution. These results demonstrate that oral co-treatment with CHL under conditions where complex formation is initially assured, substantially reduces AFB(2) systemic uptake and target organ bioavailability in the trout.