Biodegradable Hypericin-Containing Nanoparticles for Necrosis Targeting and Fluorescence Imaging.

Necrosis targeting and imaging has significant implications for evaluating tumor growth, therapeutic response, and delivery of therapeutics to perinecrotic tumor zones. Hypericin is a hydrophobic molecule with high necrosis affinity and fluorescence imaging properties. To date, the safe and effective delivery of hypericin to areas of necrosis in vivo remains a challenge because of its incompatible biophysical properties. To address this issue, we have developed a biodegradable nanoparticle (Hyp-NP) for delivery of hypericin to tumors for necrosis targeting and fluorescence imaging. The nanoparticle was developed using methoxy poly(ethylene glycol)-b-poly(ε-caprolactone) and hypericin by a modified solvent evaporation technique. The size of Hyp-NP was 19.0 ± 1.8 nm from cryo-TEM and 37.3 ± 0.7 nm from dynamic light-scattering analysis with a polydispersity index of 0.15 ± 0.01. The encapsulation efficiency of hypericin was 95.05% w/w by UV-vis absorption. After storage for 30 days, 91.4% hypericin was retained in Hyp-NP with nearly no change in hydrodynamic size, representing nanoparticle stability. In an ovarian cancer cell line, Hyp-NP demonstrated cellular internalization with intracellular cytoplasmic localization and preserved fluorescence and necrosis affinity. In a mouse subcutaneous tumor model, tumor accumulation was noted at 8 h postinjection, with near-complete clearance at 96 h postinjection. Hyp-NP was shown to be tightly localized within necrotic tumor zones. Histological analysis of harvested organs demonstrated no gross abnormalities, and in vitro, no hemolysis was observed. This proof-of-concept study demonstrates the potential clinical applications of Hyp-NP for necrosis targeting.

Simultaneous Photodiagnosis and Photodynamic Treatment of Metastatic Melanoma.

Metastatic melanoma (MM) has a poor prognosis and is attributed to late diagnoses only when metastases has already occurred. Thus, early diagnosis is crucial to improve its overall treatment efficacy. The standard diagnostic tools for MM are incisional biopsies and/or fine needle aspiration biopsies, while standard treatments involve surgery, chemotherapy, or irradiation therapy. The combination of photodynamic diagnosis (PDD) and therapy (PDT) utilizes a photosensitizer (PS) that, when excited by light of a low wavelength, can be used for fluorescent non-destructive diagnosis. However, when the same PS is activated at a higher wavelength of light, it can be cytotoxic and induce tumor destruction. This paper focuses on PS drugs that have been used for PDD as well as PDT treatment of MM. Furthermore, it emphasizes the need for continued investigation into enhanced PS delivery via active biomarkers and passive nanoparticle systems. This should improve PS drug absorption in MM cells and increase effectiveness of combinative photodynamic methods for the enhanced diagnosis and treatment of MM can become a reality.

Hypocrellin B-loaded, folate-conjugated polymeric micelle for intraperitoneal targeting of ovarian cancer in vitro and in vivo.

Photodynamic therapy (PDT) is considered an innovative and attractive modality to treat ovarian cancer. In the present study, a biodegradable polymer poly (ethylene glycol) (PEG)-poly (lactic acid)(PLA)-folate (FA-PEG-PLA) was prepared in order to synthesize an active-targeting, water-soluble and pharmacomodulated photosensitizer nanocarrier. Drug-loading content, encapsulation efficiency, in vitro and in vivo release were characterized, in which hypocrellin B (HB)/FA-PEG-PLA micelles had a high encapsulation efficiency and much slower control release for drugs compared to free drugs (P < .05). To evaluate the targeting ability of the HB/FA-PEG-PLA micelles, a cellular uptake study in vitro was carried out, which showed significantly enhanced uptake of HB/FA-PEG-PLA micelles in SKOV3 (FR+) compared to A2780 cancer cells (FR-). The enhanced uptake of HB/FA-PEG-PLA micelles to cancer cells resulted in a more effective post-PDT killing of SKOV3 cells compared to plain micelles and free drugs. Binding and uptake of HB/FA-PEG-PLA micelles by SKOV3 cells were also observed in vivo after ip injection of folate-targeted micelles in tumor-bearing ascitic ovarian cancer animals. Drug levels in ascitic tumor tissues were increased 20-fold (P < .001), which underscored the effect of a regional therapy approach with folate targeting. Furthermore, the HB-loaded micelles were mainly distributed in kidney and liver (the main clearance organs) in biodistribution. These results showed that our newly developed PDT photosensitizer HB/FA-PEG-PLA micelles have a high drug-loading capacity, good biocompatibility, controlled drug release, and enhanced targeting and antitumor effect, which is a potential approach to future targeting ovarian cancer therapy.

Single-Cell Real-Time Visualization and Quantification of Perylene Bioaccumulation in Microorganisms.

Bioaccumulation of perylene in Escherichia coli and Staphylococcus aureus was visualized and quantified in real time with high sensitivity at high temporal resolution. For the first time, single-molecule fluorescence microscopy (SMFM) with a microfluidic flow chamber and temperature control has enabled us to record the dynamic process of perylene bioaccumulation in single bacterial cells and examine the cell-to-cell heterogeneity. Although with identical genomes, individual E. coli cells exhibited a high degree of heterogeneity in perylene accumulation dynamics, as shown by the high coefficient of variation (C.V = 1.40). This remarkable heterogeneity was exhibited only in live E. coli cells. However, the bioaccumulation of perylene in live and dead S. aureus cells showed similar patterns with a low degree of heterogeneity (C.V = 0.36). We found that the efflux systems associated with Tol C played an essential role in perylene bioaccumulation in E. coli, which caused a significantly lower accumulation and a high cell-to-cell heterogeneity. In comparison with E. coli, the Gram-positive bacteria S. aureus lacked an efficient efflux system against perylene. Therefore, perylene bioaccumulation in S. aureus was simply a passive diffusion process across the cell membrane.

Analysis of Hypericin-Mediated Effects and Implications for Targeted Photodynamic Therapy.

The phototoxic effect of hypericin can be utilized for Photodynamic Therapy (PDT) of cancer. After intravenous application and systemic distribution of the drug in the patient's body, the tumor site is exposed to light. Subsequently, toxic reactive oxygen species (ROS) are generated, inducing tumor cell death. To prevent unwanted activation of the drug in other regions of the body, patients have to avoid light during and after the treatment cycles, consequently impairing quality of life. Here, we characterize toxicity and hypericin-mediated effects on cancer cells in vitro and confirm that its effect clearly depends on concentration and illumination time. To reduce side effects and to increase therapy success, selective accumulation of hypericin in the tumor region is a promising solution. Loading hypericin on superparamagnetic iron oxide nanoparticles (SPIONs) and guiding them to the desired place using an external magnetic field might accomplish this task (referred to as Magnetic Drug Targeting (MDT)). Thus, using a double targeting strategy, namely magnetic accumulation and laser induced photoactivation, might improve treatment effectivity as well as specificity and reduce toxic side effects in future clinical applications.

Hypericin fluorescence kinetics in the presence of low density lipoproteins: study on quail CAM assay for topical delivery.

There has been increasing interest in fluorescence-based imaging techniques in clinical practice, with the aim to detect and visualize the tumour configuration and the border with healthy tissue. Strong photodynamic activity of hypericin (Hyp) can be improved by various molecular transport systems (e.g. LDL). Our aim was to examine pharmacokinetics of Hyp in the presence of LDL particles on ex ovo chorioallantoic membrane (CAM) of Japanese quail with implanted TE1 tumour spheroids (human squamocellular carcinoma). Spheroids were implanted on CAM surface on embryonal day 7 and after 24 hours formulations of free Hyp and Hyp:LDL 100:1 and 200:1 were topically applied. All experimental formulations in the fluorescent image very well visualized the tumour spheroid position, with gradual increase of fluorescence intensity in 6-h observation period. LDL transportation system exhibited clear superiority in fluorescence pharmacokinetics than free Hyp formulation by increasing tumour-normal difference. Our experimental results confirm that Hyp and Hyp:LDL complex is potent fluorophore for photodynamic diagnosis of squamocellular carcinoma.

Transport and accumulation of PVP-Hypericin in cancer and normal cells characterized by image correlation spectroscopy techniques.

PVP-Hypericin (PVP: polyvinylpyrrolidone) is a potent anti-cancer photosensitizer for photodynamic diagnosis (PDD) and therapy (PDT). However, cellular targets and mechanisms involved in the cancer-selectivity of the photosensitizer are not yet fully understood. This paper gives new insights into the differential transport and localization of PVP-Hypericin in cancer and normal cells which are essential to unravel the mechanisms of action and cancer-selectivity. Temporal (TICS) and spatiotemporal (STICS) image correlation spectroscopy are used for the assessment of PVP-Hypericin diffusion and/or velocity in the case of concerted flow in human cervical epithelial HeLa and human lung carcinoma A549 cells, as well as in human primary dendritic cells (DC) and human peripheral blood mononuclear cells (PBMC). Spatiotemporal image cross-correlation spectroscopy (STICCS) based on organelle specific fluorescent labeling is employed to study the accumulation of the photosensitizer in nucleus, mitochondria, early-endosomes and lysosomes of the cells and to assess the dynamics of co-migrating molecules. Whereas STICS and TICS did not show a remarkable difference between the dynamics of PVP-Hypericin in HeLa, A549 and DC cells, a significantly different diffusion rate of the photosensitizer was measured in PBMC. STICCS detected a stationary accumulation of PVP-Hypericin within the nucleus, mitochondria, early endosomes and lysosomes of HeLa and A549 cells. However, significant flow due to the directed motion of the organelles was detected. In contrast, no accumulation in the nucleus and mitochondria of DC and PBMC could be monitored.

Liposomal hypocrellin B as a potential photosensitizer for age-related macular degeneration: pharmacokinetics, photodynamic efficacy, and skin phototoxicity in vivo.

Photodynamic therapy (PDT) has been successfully implemented as a treatment for wet age-related macular degeneration (AMD), but very few photosensitizers have been developed for clinical use. Herein, we describe a novel formulation of liposomal hypocrellin B (LHB) that was prepared by high-pressure homogenization. The encapsulation efficiency and PDT efficacy in vitro of this new preparation were found to remain nearly constant over 1 year. Moreover, LHB is rapidly cleared from the blood, with a half-life of 2.319 ± 0.462 h and a very low serum concentration at 24 h after injection. Testing in a rat model of choroidal neovascularization (CNV) showed that leakage of blood vessels in CNV lesions was significantly reduced when LHB PDT was given at a dose of 1 mg kg(-1) along with yellow laser irradiation; the damage to the collateral retina and the retinal pigment epithelium was minimal. Skin phototoxicity assays showed that only two of the 200 mice given a 4 mg per kg dose of LHB experienced an inflammatory reaction in the auricle irradiated at 24 h after dosing. These data collectively indicate that LHB may be a safe and effective photosensitizer for vascular-targeted PDT of AMD.

Permeation Characteristics of Hypericin across Caco-2 Monolayers in the Absence or Presence of Quercitrin - A Mass Balance Study.

Hypericin is a natural polycyclic quinone found in Hypericum perforatum. Although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably because hypericin is a highly lipophilic compound that is poorly soluble in a physiological medium. The major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the Caco-2 cell system under different experimental conditions. The permeation characteristics of hypericin (5 µM) in the absence or presence of the model flavonoid quercitrin (20 µM) were studied in the absorptive direction, without or with the addition of 10 % FBS to the transport buffer apically. Following the application of hypericin to the apical side of the monolayer, only negligible amounts of the compound were found in the basolateral compartment when the experiment was performed with a transport buffer. The amount of hypericin in the basolateral compartment increased in the presence of quercitrin (from 0 to 4 %). The majority of hypericin was found after cell extraction (44 % in the absence and 64 % in the presence of quercitrin). When 10 % FBS was added to the transport buffer in the apical compartment to improve the solubility of hypericin in the aqueous solution, around 68 % of hypericin was bound to the serum proteins. Under these experimental conditions, the amount of hypericin in the cells/cell membrane was only 13 % in the absence and 18 % in the presence of quercitrin. The low recovery and significant amounts of hypericin found after cell extraction and bound to the surface of the culture dish made a correct estimation of permeability constants impossible. Fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. The precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. However, our experiments demonstrated that regardless of the experimental conditions, the permeation characteristics of hypericin improved in the presence of the model flavonoid quercitrin.

Radiopharmaceutical study on Iodine-131-labelled hypericin in a canine model of hepatic RFA-induced coagulative necrosis.

PURPOSE: Hypericin (HYP) has been found avid to necrosis in small animal studies. We sought to evaluate the tissue distribution of (131)I-HYP in a large animal model and to explore the theranostic utilities of (131)I-HYP after radiofrequency ablation (RFA). MATERIALS AND METHODS: This animal experiment was approved by the institutional ethics committee. Twenty-five male dogs were enrolled and subjected to transabdominal hepatic RFA. (131)I-HYP was prepared by an electrophilic substitution method and intravenously administered at 0.5 mCi/kg. Systemic and regional distributions of (131)I-HYP were monitored dynamically by single-photon emission computed tomography/computed tomography (SPECT-CT), gamma counting, autoradiography, and fluorescent and light microscopy at different time points up to 14 days. Experimental data were quantified and statistically analysed. RESULTS: Most of the tissues and organs retained (131)I-HYP only transiently. (131)I-HYP was mainly metabolised in the liver and excreted into the bile. (131)I-HYP gradually accumulated in the RFA-induced necrosis with a peak concentration occurring within 2 days and lasting over 2 weeks as visualised by in vivo SPECT-CT and ex vivo autoradiography and fluorescent microscopy, and quantified by radioactivity and fluorescence measurements. Accumulation of (131)I-HYP was low in both the necrosis centre and normal liver tissue. CONCLUSION: (131)I-HYP showed persistent high affinity to hepatic thermo-coagulative necrosis, but only a transient uptake by normal liver in dogs. Necrosis caused by RFA could be indicated by (131)I-HYP on nuclear imaging, which suggests a supplementary measure for tumour detection and therapy.

Hypericum perforatum: pharmacokinetic, mechanism of action, tolerability, and clinical drug-drug interactions.

Hypericum perforatum (HP) belongs to the Hypericaceae family and is one of the oldest used and most extensively investigated medicinal herbs. The medicinal form comprises the leaves and flowering tops of which the primary ingredients of interest are naphthodianthrones, xanthones, flavonoids, phloroglucinols (e.g. hyperforin), and hypericin. Although several constituents elicit pharmacological effects that are consistent with HP's antidepressant activity, no single mechanism of action underlying these effects has thus far been found. Various clinical trials have shown that HP has a comparable antidepressant efficacy as some currently used antidepressant drugs in the treatment of mild/moderate depression. Interestingly, low-hyperforin-content preparations are effective in the treatment of depression. Moreover, HP is also used to treat certain forms of anxiety. However, HP can induce various cytochrome P450s isozymes and/or P-glycoprotein, of which many drugs are substrates and which are the main origin of HP-drug interactions. Here, we analyse the existing evidence describing the clinical consequence of HP-drug interactions. Although some of the reported interactions are based on findings from in vitro studies, the clinical importance of which remain to be demonstrated, others are based on case reports where causality can, in some cases, be determined to reveal clinically significant interactions that suggest caution, consideration, and disclosure of potential interactions prior to informed use of HP.

Comparative study of iodine-123-labeled hypericin and (99m)Tc-labeled hexakis [2-methoxy isobutyl isonitrile] in a rabbit model of myocardial infarction.

Identification of myocardial infarction (MI) by imaging is critical for clinical management of ischemic heart disease. Iodine-123-labeled hypericin (¹²³I-Hyp) is a new potent infarct avid agent. We sought to compare target selectivity and organ distribution between ¹²³I-Hyp and the myocardial perfusion agent, technetium-99m-labeled hexakis [2-methoxy isobutyl isonitrile] ((99m)Tc-Sestamibi) in rabbits with acute MI. Hypericin was radiolabeled with I using iodogen as oxidant, and (99m)Tc-Sestamibi was prepared from a commercial kit and radioactive sodium pertechnetate. Rabbits (n = 6) with 24-hour-old MI received ¹²³I-Hyp intravenously and received (99m)Tc-Sestamibi 9 hours later. They were studied by dual-isotope simultaneous acquisition micro single photon emission computed tomography/computed tomography (DISA-µSPECT/CT), tissue gamma counting (TGC), autoradiography, and histology. After purification, ¹²³I-Hyp was obtained with radiochemical purity around 99%. DISA-µSPECT/CT images showed ¹²³I-Hyp retention in infarcted but not in normal myocardium. By TGC, accumulation values reached 1.175 ± 0.096 percentage of injected dose per gram (%ID/g) and 0.028 ± 0.007%ID/g in infarcted myocardium and normal myocardium with high tracer concentration in liver, intestines, and gallbladder. (99m)Tc-Sestamibi was prepared with radiochemical purity over 95%. DISA-µSPECT/CT showed no accumulation in MI and high initial radioactivity levels in normal myocardium that were rapidly cleared as confirmed by TGC (0.011 ± 0.003%ID/g). Liver and intestines were clearly visualized. By TGC, gallbladder and kidneys show moderate (99m)Tc-Sestamibi uptake. The selectivity of ¹²³I-Hyp for infarcted myocardium and (99m)Tc-Sestamibi for normal myocardium was confirmed. ¹²³I-Hyp distribution in rabbits is characterized by hepatobiliary excretion. (99m)Tc-Sestamibi undergoes hepatorenal elimination.

Laser light activation of a second-generation photosensitiser and its use as a potential photomodulatory agent in skin rejuvenation.

Photodynamic rejuvenation therapy (PDRT) is a growing field in cosmetic dermatology. In this study, different sources of light (a yellow laser, a red laser and ultraviolet A (UVA) lamps) were used to activate a second-generation photosensitiser, hypericin. Uptake of hypericin was monitored over 24 h and efficacy of PDRT was assessed using cell viability and reactive oxygen species (ROS) quantification assays. In addition, we show for the first time, a quantifiable assay for ROS production in human dermal fibroblasts incubated with hypericin and exposed to yellow laser light or UVA lamps. Furthermore, we optimised a protocol with regard to hypericin concentration and irradiation parameters using the XTT cell viability kit. This study showed that this photosensitiser, hypericin, was taken up by the cells in a concentration-dependent manner over 24 h with cell saturation occurring after approximately 16 h. The uptake seemed to be localised to the cell cytoplasm with no hypericin appearing in the nucleus. The levels of ROS increased in the cell when irradiated with the yellow laser (561 nm) however, it did not increase further with the addition of hypericin. Hypericin and UVA showed a significant increase in the amount of ROS produced. The results also show that cell viability is not affected by low power light (2 mW) from the yellow laser irrespective of the dose used. However, an increase to 10 mW power with 5 J/cm(2) light dose, resulted in a significant drop (p < 0.05) in cell viability at both 0.5 (77.53 ± 9.67 %) and 1 µM (48.51 ± 13.27 %) hypericin concentrations. In contrast, a 20 % increase in cell viability was seen with 1 J/cm(2) and 20 mW and 0.25 µM hypericin. Overall, this study highlights an optimised protocol for hypericin-induced photorejuvenative therapy using laser light and proposes that parameters of 0.25 µM hypericin as a photosensitiser activated via a dosage of 1 J/cm(2) yellow laser light produces an effective in vitro outcome to be considered as an important contribution towards optimising PDRT.

A phase 1/2 study of orally administered synthetic hypericin for treatment of recurrent malignant gliomas.

BACKGROUND: Hypericin is a potent inhibitor of glioma growth in vitro. To examine whether synthetic oral hypericin can be tolerated by patients with recurrent malignant gliomas (anaplastic astrocytoma and glioblastoma) and to investigate its efficacy against these tumors, the authors undertook an open-label, sequential dose escalation/de-escalation tolerance study. METHODS: Patients with documented recurrent or progressive malignant gliomas who had received standard radiation therapy with or without chemotherapy were included. Patients were excluded for previous treatment with agents known to contain hypericin or treatment within 30 days with medications known to cause photosensitivity. Enrolled patients were given gradually increasing dosages of oral synthetic hypericin (0.05-0.50 mg/kg) for up to 3 months if no toxicity was observed, and patient response to treatment was noted. The patients were examined each month and underwent magnetic resonance imaging to evaluate tumor status at 3 months. RESULTS: Synthetic hypericin administered orally appeared to provide stabilization or a slight (<50%) decrease in tumor volume (coded as stable disease) at 3 months for 7 of 42 patients (17%) and produced a tumor reduction >50% (partial response) in 2 patients (5%). Seventeen patients (40%) survived for 3 months on daily synthetic hypericin at dose levels of 0.33 ± 0.070 mg/kg daily. The mean maximum tolerated dose was 0.40 ± 0.098 mg/kg daily. Twelve patients continued on hypericin therapy beyond 3 months. The median survival was 26 weeks (Kaplan-Meier method). CONCLUSIONS: The results of this study indicated that synthetic, oral hypericin is well tolerated in this patient group. The response results were comparable to those reported from other studies of salvage therapies for recurrent malignant brain tumors.

Photocytotoxicity of hypocrellin B (HB) was enhanced by liposomalization in vitro.

Hypocrellin B (HB) was encapsulated into a phosphatidylcholine liposome. Encapsulation of HB into liposomes not only improved the delivery of this photosensitizer but also increased its photodynamic efficacy compared to free HB molecules. Liposomal HB showed a higher cellular uptake than free HB as measured by confocal microscopy and was internalized into cultured HeLa cells by caveolar endocytosis, which was lipid-raft-dependent. Cell viability measurements demonstrated that liposomal HB was more phototoxic to HeLa cells than free HB as a result of the higher concentration of intracellular HB delivered by the liposomal formulation. The encapsulation of HB influenced the cell death pathway by an increased rate of necrotic cells after irradiation versus free HB, and a Type II (singlet oxygen) mechanism was responsible for the photocytotoxicity.

Disposition of charged nanoparticles after their topical application to the skin.

The objective of this preliminary investigation was to examine the disposition of charged nanoparticles on and within the skin following their topical application and to explore whether the formulations have potential utility for the local delivery of an associated 'active' substance. Three nanoparticles (approx. 100 nm in diameter) were investigated: cationic amino-functionalized polystyrene, an anionic carboxyl-functionalized polystyrene and anionic poly-(L-lactide), into each of which the fluorophore N-(2,6-diisopropylphenyl)perylene-3,4-dicarboximine (PMI) was incorporated. Formulations were applied to excised porcine skin in vitro for 6 h. After cleaning the skin surface following treatment, the skin was either examined by laser scanning confocal microscopy or subjected to repeated tape-stripping and subsequent analysis of the removed stratum corneum (SC) for the presence of PMI. The cationic nanoparticles showed clear affinity for the negatively charged skin surface (in contrast to the anionic carriers) and delivered a significantly greater amount of the model 'active' agent (PMI) into the SC.

Photodynamic efficacy of hypericin targeted by two delivery techniques to hepatocellular carcinoma cells.

The photocytotoxic effect of hypericin (Hyp) targeted by two different delivery techniques, namely, liposomes and anti-hepatocyte specific antigen (anti-HSA) was investigated. Optical absorption and steady-state fluorescence were used to analyze the conjugation of Hyp with anti-HSA model and to evaluate the encapsulation capacity and drug release in a liposome model. Particle size and thermal analysis of the prepared liposomes were performed using laser-light scattering and differential scanning calorimetry (DSC), respectively. Viability study of HepG2 cells exposed to Hyp in the two delivery systems, in the dark and following visible light irradiation, was performed in comparison to free Hyp. The intracellular uptake and localization of Hyp in HepG2 cells were analyzed by means of spectrofluorometry and fluorescence microscopy. Spectroscopic measurements demonstrated that Hyp binds to anti-HSA in its monomeric form. The photocytotoxic effect of Hyp depended clearly on the form of Hyp administered, either in free form, loaded into liposomes or conjugated with anti-HSA. While liposomes loaded with Hyp (Lip-Hyp) did not induce significant phototoxicity, both free Hyp or anti-HSA-Hyp inflicted substantial cell mortality, after photoirradiation. The intracellular uptake of Lip-Hyp by HepG2 cells was estimated to be 20% less compared to free Hyp or anti-HSA-Hyp. In spite of the equal uptake of both free Hyp and anti-HSA-Hyp, HepG2 cells demonstrated a relatively higher mortality with anti-HSA-Hyp compared to free Hyp.

Naturally available hypericin undergoes electron transfer for type I photodynamic and photothermal synergistic therapy.

Naturally available compounds with bioactivity are potential candidates for cancer treatment. In this paper, we isolated hypericin (HC) from Hypericum sinense L. and investigated its antitumor activity both in vitro and in vivo. The nanoparticles (NPs) of HC were prepared by a nanoprecipitation process with 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2000). With light irradiation, HC NPs not only undergo efficient electron transfer to generate the superoxide radical (O2-˙) and the hydroxyl radical (OH˙) as well as energy transfer producing singlet oxygen (1O2) for photodynamic therapy (PDT), but also non-radiative decay to produce heat for photothermal therapy (PTT) with a photothermal conversion efficiency of 29.3%. This synergistic therapy, therefore, largely boosts the phototherapy efficacy of HC NPs on human cervical cancer cells (HeLa), guaranteeing a low half maximal inhibitory concentration (IC50) of only 5.6 µg mL-1. Furthermore, in vivo studies suggest that HC NPs are capable of inhibiting tumor proliferation after laser irradiation, and the main organs remain healthy, including the heart, kidneys, liver, lungs and spleen. Our results indicate that HC NPs derived from nature with excellent phototherapy efficacies are biocompatible candidates for type I PDT/PTT synergistic cancer therapy.

Study of interaction of hypericin and its pharmaceutical preparation by fluorescence techniques.

We report the detection of interactions between a photosensitizer, hypericin (HY), and its solvent system prepared with a formulation additive, polyvinylpyrrolidone (PVP), a commonly used pharmaceutical excipient. Fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) were used to study aggregation and binding of HY in the presence of PVP. Digitized fluorescence endoscopic imaging (DFEI) was used to study the effect of the pharmaceutical formulation in the in vivo tumor implanted chick chorioallantoic membrane (CAM) model. The results presented reveal the coordination of HY-PVP binding, HY disaggregation in the presence of PVP, and strengthened HY tumor uptake selectivity. PVP is thus suggested as a potential adjuvant to previously investigated N-methyl pyrrolidone (NMP) in the HY delivery system as well as a replacement for the conventionally used albumin in the HY bladder instillation fluids preparation for clinical use.

Permeation characteristics of hypericin across Caco-2 monolayers in the presence of single flavonoids, defined flavonoid mixtures or Hypericum extract matrix.

OBJECTIVES: The major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the Caco-2 cell system when combined with various flavonoids, mixtures of flavonoids or Hypericum perforatum extract matrix (STW3-VI). METHODS: The permeation characteristics of hypericin in the absence or presence of quercetin, quercitrin, isoquercitrin, hyperoside and rutin were tested. Hypericin (5 µm) was mixed with single flavonoids (20 µm) or with different flavonoid combinations (each flavonoid 4 or 10 µm, total flavonoid concentration: 20 µm). Further, the uptake of hypericin (5 µm) in the presence of H. perforatum extract matrix (7.25, 29 and 58 µg/ml) was studied. KEY FINDINGS: Following application of hypericin to the apical side of the monolayer, only negligible amounts of the compound were found in the basolateral compartment. From all tested flavonoids, only quercitrin increased the basolateral amount of hypericin. Dual flavonoid combinations were not superior compared to the single combinations. The amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of extract matrix (from 0 to 7.5%). CONCLUSION: Comparing the effects of various flavonoid mixtures vs the extract matrix, it can be concluded that, besides flavonoids, the extract seems to contain further compounds (e.g. phenolic acids or proanthocyanidins) which substantially improve the permeation characteristics of hypericin.