Hypericin Fluorescence Research Articles

Effective transport of aggregated hypericin encapsulated in SBA-15 nanoporous silica particles for photodynamic therapy of cancer cells

Photodynamic therapy (PDT) represents an interesting modality for the elimination of damaged biomaterials and cells. This treatment takes advantage of the photosensitizing properties of molecules that are active only when irradiated with light. In the present work, a dual property of hypericin, a hydrophobic molecule with high performance in photodiagnostics and photodynamic therapy, was exploited. The non-fluorescent and photodynamically inactive form of hypericin aggregates was loaded into the nanopores of SBA-15 silica particles. The synthesized particles were characterized by infrared spectroscopy, thermogravimetry, differential thermal analysis, small-angle X-ray scattering and transmission electron microscopy. Hypericin aggregates were confirmed by absorption spectra typical of aggregated hypericin and by its short fluorescence lifetime. Release of hypericin from the particles was observed toward serum proteins, mimicking physiological conditions. Temperature- and time-dependent uptake of hypericin by cancer cells showed gradual release of hypericin from the particles and active cellular transport by endocytosis. A closer examination of SBA-15-hypericin uptake by fluorescence lifetime imaging showed that aggregated hypericin molecules, characterized by a short fluorescence lifetime (∼4 ns), were still present in the SBA-15 particles upon uptake by cells. However, monomerization of hypericin in cancer cells was observed by extending the hypericin fluorescence lifetime by ∼8 ns, preferentially in lipid compartments and the plasma membrane. This suggests a promising prognosis for delayed biological activity of the entire cargo, which was confirmed by effective PDT in vitro. In summary, this work presents an approach for safe, inactive delivery of hypericin that is activated at the target site in cells and tissues.

The Japanese quail chorioallantoic membrane as a model to study an amphiphilic gradient copoly(2-oxazoline)s- based drug delivery system for photodynamic diagnosis and therapy research

Amphiphilic gradient copoly(2-oxazoline)s are widely researched in the field of drug delivery. They could be used as a transport system for hydrophobic drugs such as hypericin (HYP). We prepared six gradient copolymers (EtOx)-grad-(ROPhOx) by living cationic ring-opening polymerization of a hydrophilic comonomer 2-ethyl-2-oxazoline (EtOx) and a hydrophobic comonomer 2-(4-alkyloxyphenyl)-2-oxazoline (ROPhOx), with different composition ratio (88:12 and 85:15) and three different alkyl chain lengths of alkyl (R) substituents. As an experimental model, Japanese quail chorioallantoic membrane (CAM) was used. The effect of nanoparticles loaded with HYP was evaluated by the changes of fluorescence intensity during photodynamic diagnosis (PDD) monitored under 405 nm LED light before administration, and 0,1,3 and 24 h after topical administration. The effectiveness of photodynamic therapy (PDT) (405 nm, 285 mW/cm2) applied 1h after the administration of HYP-loaded nanoparticles was evaluated using vascular damage score and histological sections. Molecular analysis was done by measuring angiogenesis-related gene expression by qPCR. The application of nanoparticles unloaded or loaded with HYP proved to be biocompatible, non-toxic, and undamaging to the CAM tissue, while they successfully altered the HYP fluorescence. We observed a possible anti-angiogenic potential of prepared nanoparticles, which could present an advantage for PDT used for tumour treatment.

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.

Subdiffraction localization of a nanostructured photosensitizer in bacterial cells.

Antibacterial treatments based on photosensitized production of reactive oxygen species is a promising approach to address local microbial infections. Given the small size of bacterial cells, identification of the sites of binding of the photosensitizing molecules is a difficult issue to address with conventional microscopy. We show that the excited state properties of the naturally occurring photosensitizer hypericin can be exploited to perform STED microscopy on bacteria incubated with the complex between hypericin and apomyoglobin, a self-assembled nanostructure that confers very good bioavailability to the photosensitizer. Hypericin fluorescence is mostly localized at the bacterial wall, and accumulates at the polar regions of the cell and at sites of cell wall growth. While these features are shared by Gram-negative and Gram-positive bacteria, only the latter are effectively photoinactivated by light exposure.

Fluorescence spectra of complexes of hypericin with s2 metal cations

The chlorides of s2 metal cations including Sn2+, Sb3+, Bi3+ and Te4+ form complexes with hypericin as indicated by a blue shift of the absorption and fluorescence of hypericin. It is suggested that this is caused by a stabilization of the aromatic HOMO of hypericin by the s2 metal centers. This, in turn, increases the HOMO/LUMO gap of hypericin. A concomitant decrease of the fluorescence intensity occurs, probably due to a heavy-atom effect.

Hypericin as a Marker for Determination of Myocardial Viability in a Rat Model of Myocardial Infarction

The aim of this study was to investigate the necrosis-avid agent hypericin as a potential indicator for determination of myocardial infarction (MI). Male Sprague-Dawley rats (n = 30) weighing 350 ± 20 g were subjected to acute reperfused MI. Animals were divided into four groups (n = 6), in which hypericin was intravenously injected at 0, 1, 2 and 5 mg kg(-1) respectively. One day after injection, rats were euthanized with their hearts excised for qualitative and quantitative studies by means of microscopic fluorescence examination to decide the dosage of hypericin. Another group was injected with hypericin at the decided dose and evaluated by fluorescence macroscopy in colocalization with triphenyltetrazoliumchloride (TTC) and histomorphology. Infarct-to-normal contrast ratio and relative infarct size were quantified. Hypericin-induced red fluorescence was significantly brighter in necrotic than in viable myocardium as proven by a six times higher mean fluorescence density. Mean MI area was 35.66 ± 22.88% by hypericin fluorescence and 32.73 ± 21.98% by TTC staining (R(2) = 0.9803). Global MI-volume was 34.56 ± 21.07% by hypericin and 35.11 ± 20.47% by TTC staining (R(2) = 0.9933). The results confirm that hypericin specifically labeled necrosis, and enhanced the imaging contrast between the infarcted and normal myocardium, suggesting its potential applications for the assessment of myocardial viability.

Microendoscopy for hypericin fluorescence tumor diagnosis in a subcutaneous glioma mouse model

New treatment strategies for malignant gliomas are indispensible, due to the poor prognosis for patients. Fluorescence diagnosis (FD) and photodynamic therapy (PDT) are currently under intensive investigation and seem to improve the prognosis. Especially for deep seated malignant brain lesions and in order to optimize therapy new diagnostic tools are needed. In a syngeneic subcutaneous glioma mouse model we investigated the time dependent hypericin (HYP) uptake in malignant tumor tissue by microendoscopically fluorescence measurements. The HYP fluorescence in tumor was also detected by fluorescence microscopy (FM) and was compared to endoscopic data. Both methods, microendoscopy and FM, demonstrated time dependent HYP uptake in subcutaneously implanted mouse glioma. Maximum of HYP uptake was achieved after 6h, measured with both methods. FM reached a 10-fold increase in fluorescence intensity compared to the autofluorescence. Measured by microendoscopy a 2.2-fold HYP fluorescence intensity compared to the autofluorescence was detected. Microendoscopy enables visualization of small vessels even in healthy brain tissue by intravascular HYP fluorescence. The new developed microendoscope enables not only fluorescence based discrimination of tumor and healthy tissue, but also semiquantitative measurements of fluorescence intensities in vivo. Individual repetitive fluorescence diagnosis will become possible by this method and opens up new possibilities for determining optimal settings of light applications for PDT.

Hypericin Fluorescence Imaging of Oral Cancer: From Endoscopy to Real-time 3-Dimensional Endomicroscopy

Hypericin Fluorescence Imaging of Oral Cancer: From Endoscopy to Real-time 3-Dimensional Endomicroscopy

Hypericin incorporation and localization in fixed HeLa cells for various conditions of fixation and incubation

Hypericin is a photosensitizer expressing high affinity for cancerous cells in vivo. Diagnosis of cancer based on hypericin fluorescence imaging has been successfully assessed in several clinical trials. Our final objective will be to evaluate the potential of hypericin fluorescence imaging to improve the efficacy of cervical cancer diagnosis performed on fixed cell smears obtained from liquid-based cytology. For this purpose, the mechanism of hypericin incorporation and localization in fixed HeLa cells using different incubation media and fixation conditions was investigated. Since the duration of fixation may play an important role, the influence of fixation time on hypericin incorporation in fixed HeLa cells was studied. The uptake and distribution of hypericin in fixed HeLa cells were found to be strongly dependent on the hypericin incubation medium: for a polar organic solvent such as the alcohol-based fixative, the localization was essentially perinuclear and nuclear; for cell culture medium supplemented with serum, the localization was cytoplasmic and non-specific; the highest incorporation was observed for the serum-free culture medium but mainly as non-fluorescent aggregates. The hypericin aggregation in the incubation medium, the passive diffusion and the partitioning between the cells and hypericin carriers seemed to be the major factors accounting for these results. The localization was found to be weakly dependent on fixation time, whereas fluctuations of hypericin fluorescence at short fixation time and stabilization after two days of fixation were observed. These results suggest that the fixed cells reached a steady state after two days of fixation.

Photodynamic therapy using topically applied hypericin: Comparative effect with methyl-aminolevulinic acid on UV induced skin tumours

Photodynamic therapy (PDT) is a treatment option particularly well-suited for superficial (pre)malignant skin lesions due to the skin’s accessibility to light. In the present study, the efficacy of topical hypericin-PDT was evaluated using a mouse model for actinic keratosis. For comparison, similar experiments were conducted with methyl-aminolevulinic acid (Me-ALA). Small skin tumours (1–2 mm) were induced in hairless mice by chronic UV irradiation. After topical application of hypericin (0.1% in gelcream for 24 h) or Me-ALA (Metvix® for 4 h), the lesional/non-lesional skin surface fluorescence ratio was determined and fluorescence microscopy was used to study the skin penetration of the photosensitizers. The antitumour activity of topical PDT (20 mW cm −2, 40 J cm −2) was evaluated by measurement of the lesional diameters. Moreover, biopsies were taken at various time points after PDT for histological evaluation of the therapy. Our results demonstrate that after topical application of hypericin and Me-ALA, tumour selectivity is limited in mouse skin. The microscopic distribution of hypericin fluorescence showed an accumulation in the stratum corneum and low fluorescence levels in the rest of the lesions, whereas the distribution of PpIX in the skin was more homogenous. Topical hypericin-PDT was found to be less efficient (44% total lesional clearance) as compared to Me-ALA-PDT (80% total lesional clearance). Full lesional necrosis was observed in responsive lesions, and the atypical cells of actinic keratosis were replaced by normal keratinocytes 3 weeks later, both after hypericin-PDT and Me-ALA-PDT.

Altered expression of cell adhesion molecules leads to differential uptake of hypericin in urothelial cancer

ObjectiveTo elucidate the mechanism behind selective uptake of hypericin in bladder cancer after intravesical instillation for photodynamic diagnosis of urothelial cell carcinoma of bladder. Patients and methodsClinical studies were done on a series of 60 bladder cancer biopsies obtained from 28 patients who received intravesical instillations with 8 μM hypericin. Serial 5 μm cryosections were cut from 43 biopsies, and expression of the E-cadherin and associated catenins were determined using immunohistochemical and immunofluorescence staining. Hypericin was assessed using fluorescence confocal laser scanning microscopy. In addition, mRNA expression of these cell-adhesion molecules was analyzed in 17 biopsies using reverse transcription-PCR. ResultsIncreased variability in the expression of E-cadherin and associated molecules was found in high-grade, advanced stage bladder carcinoma. An inverse association was found between immunoreactivity for E-cadherin, β- and γ-catenin, and both stage and grade of cancer (P < 0.05). A positive association was observed between the hypericin fluorescence and tumor grade. There was a significant down-regulation of E-cadherin and β-catenin mRNA in grade 2 and 3 tumors. Although a small sample size was studied, it provided sufficient proof to support the hypothesis that altered expression of cell adhesion molecules would lead to preferential hypericin uptake in urothelial cell carcinoma. ConclusionsOur study has unraveled one of the many factors contributing to the selective uptake of hypericin in bladder cancer. We have thus identified the effects of alteration of E-cadherin–catenin complex and transformed intercellular junction in the modified paracellular uptake of hypericin that provides the rationale for using this photosensitizer in photodynamic diagnosis of bladder carcinoma.

Investigation of the absorption of hypericin into the skin of hairless mice.

The skin absorption of hypericin was evaluated in hairless mice to develop an optimised hypericin topical formulation that could be used in the clinical study of psoriasis. Hypericin (0.01-1.0%) in Beeler basis, polyethylene glycol ointment, carbopol gel, cetomacrogol cream, petrolatum or emulsifying ointment, with and without skin-absorption enhancers (isopropylidene glycerol and diethylene glycol monoethyl ether), was tested in-vivo on hairless mice skin. Using a skin-stripping technique and the intrinsic fluorescence of hypericin under standardised UV365 irradiation, it was demonstrated that the absorption of hypericin very much depended on the vehicle used. The concentrations of hypericin in the skin were then estimated by HPLC analysis. For this purpose, two vehicles were employed, with which hypericin penetrated the skin of hairless mice well (emulsifying ointment with isopropylidene glycerol) or very poorly (polyethylene glycol ointment). In the case of emulsifying ointment with isopropylidene glycerol (0.05% hypericin), a substantial concentration of hypericin (8.6+/-3.2 microg g(-1)) (mean +/- s.d., n = 5) was found in the skin. With polyethylene glycol ointment, however, only a limited hypericin skin concentration (0.38+/-0-34 microg g(-1), n = 5) was achieved. These results show that emulsifying ointment with polyethylene glycol holds promise as an effective topical vehicle for the treatment of skin diseases, such as psoriasis, with hypericin.

Clinical application of fluorescence endoscopic imaging using hypericin for the diagnosis of human oral cavity lesions

Background:Diagnosis of oral cancer is conventionally carried out using white light endoscopy and histopathology of biopsy samples. However, oral tumours are mostly superficial and the lesion and its margins can be difficult to visualise under white light. We present clinical data on fluorescence diagnostic imaging of oral lesions using hypericin, a plant-based photosensitiser.Methods:Fluorescence images of lesions and normal tissue were captured using an endoscope after hypericin administration. The images were analysed to extract their colour parameters, which, along with the red-to-blue intensity ratios, were analysed and used to discriminate between tissue types. The results were correlated with those from histopathology.Results:The red-to-blue intensity ratio increased from normal to hyperplastic to cancerous tissue and was a good parameter to discriminate between these tissue types, with sensitivity and specificity levels of 90% and above.Conclusion:Our results show that hypericin fluorescence imaging has the potential to be used for the clinical diagnosis of oral cancer. Further study to enhance the clinical potential of this technique includes the development of a real-time image processing and analysis system interfaced to the endoscope to enable same-day cancer diagnosis and demarcation of lesion margins in a clinical setting.

Quenching-induced deactivation of photosensitizer by nanoencapsulation to improve phototherapy of cancer

Photodynamic therapy has emerged as a promising alternative to current cancer treatment. However, conventional photosensitizers have several limitations due to their unsuitable pharmaceutical formulations and lack of selectivity. Our strategy was to exploit the advantages of nanoparticles and the quenching-induced deactivation of the model photosensitizer hypericin to produce “activatable” drug delivery systems. Efficient fluorescence and activity quenching were achieved by increasing the drug-loading rate of nanoparticles. In vitro assays confirmed the reversibility of hypericin deactivation, as the hypericin fluorescence and photodynamic activity were recovered upon cell internalization.

Time‐resolved Luminescence and Singlet Oxygen Formation After Illumination of the Hypericin–Low‐density Lipoprotein Complex

Time-resolved fluorescence and phosphorescence study of hypericin (Hyp) in complex with low-density lipoproteins (LDL) as well as the evolution of singlet oxygen formation and annihilation after illumination of Hyp/LDL complexes at room temperature are presented in this work. The observed shortening of the fluorescence lifetime of Hyp at high Hyp/LDL molar ratios (>25:1) proves the self-quenching of the excited singlet state of monomeric Hyp at these concentration ratios. The very short lifetime ( approximately 0.5 ns) of Hyp fluorescence at very high Hyp/LDL ratios (>150:1) suggests that at high local Hyp concentration inside LDL molecules fast and ultrafast nonradiative decay processes from excited singlet state of Hyp become more important. Contrary to the lifetime of the singlet excited state, the lifetime (its shorter component) of Hyp phosphorescence is not dependent on Hyp/LDL ratio in the studied concentration range. The amount of singlet oxygen produced as well as the integral intensity of Hyp phosphorescence after illumination of Hyp/LDL complexes resemble the dependence of the concentration of molecules of Hyp in monomeric state on Hyp/LDL until a concentration ratio of 60:1. This fact confirms that only monomeric Hyp is able to produce the excited triplet state of Hyp, which in aerobic conditions leads to singlet oxygen production. The value of singlet oxygen lifetime ( approximately 8 micros) after its formation from the excited triplet state of Hyp in LDL proves that molecules of singlet oxygen remain for a certain period of time inside LDL particles and are not immediately released to the aqueous surrounding. That Hyp exists in the complex with LDL in the monodeprotonated state is also demonstrated.

Hypericin as a marker for determination of tissue viability after radiofrequency ablation in a murine liver tumor model

In this proof-of-principle study, the necrosis avid agent hypericin was investigated as a potential indicator for early therapeutic response following radiofrequency ablation (RFA) of murine liver tumors. Eight mice bearing intrahepatic RIF-1 tumors were intravenously injected with hypericin 1 h before or 24 h after RFA treatment. Mice were euthanized 24 h after hypericin injection and excised livers were investigated by means of fluoromacroscopic and fluoromicroscopic examinations in combination with conventional histomorphology. Significant differences in hypericin fluorescence were found in necrosis, viable tumor and normal liver tissue in a decreasing order: in necrosis, mean fluorescence densities were about 5 times higher than in viable tumor and approximately 12 times higher than in normal liver (p<0.05). Mean fluorescence densities were not significantly different when hypericin was injected 24 h after or 1 h before RFA treatment (p>0.05). As a conclusion, hypericin features the property to specifically enhance the imaging contrast between necrotic and viable tissues and to non-specifically distinguish viable tumor from normal liver. The results suggest that hypericin offers significant potential in the early assessment of response following necrosis-inducing antineoplastic treatments such as RFA.

Endoscopic image analysis of photosensitizer fluorescence as a promising noninvasive approach for pathological grading of bladder cancer in situ

Our aim is to apply image analysis on photosensitizer fluorescence and compare the relationship between histopathology and endoscopic fluorescence imaging. The correlation between hypericin fluorescence and histopathology of diseased tissue was explored in a clinical study involving 58 fluorescence cystoscopic images from 23 patients. Based on quantification of fluorescence colorimetric parameters extracted from the image analysis, diagnostic functions were developed to pathologically classify the bladder cancer. Our preliminary results show that the differences in fluorescence intensity ratios among the three different grades of bladder cancer are statistically significant. The results also show a decrease in macroscopic fluorescence intensity that correlated with higher cancer grades. By combining both the red-to-green and red-to-blue fluorescence intensity ratios into a 2-D scatter plot and defining diagnostic linear discrimination functions on the data points, this technique is able to yield an average sensitivity and specificity of around 68.6% and 86.1%, respectively, for pathological cancer grading of the three different grades of bladder cancer in our study. We conclude that our proposed approach in applying colorimetric intensity ratio analysis on hypericin fluorescence shows potential to optically grade bladder cancer in situ.

Hypericin as a Marker for Determination of Tissue Viability After Intratumoral Ethanol Injection in a Murine Liver Tumor Model

In this preclinical proof-of-principle study, the necrosis avid agent hypericin was investigated as a potential early indicator for therapeutic response after ethanol-mediated chemical ablation in murine liver tumors. Seven mice bearing intrahepatic radiation-induced fibrosarcoma-1 tumors were intravenously injected with hypericin 1 hour before (n = 3) or 24 hours after (n = 4) intratumoral ethanol injection. Mice were euthanized 24 hours after hypericin injection and, taking advantage of the fluorescent property of the compound, the excised livers were investigated qualitatively and quantitatively by means of fluoromacroscopic and fluoromicroscopic examinations, colocalized with conventional histomorphology. Significant differences in hypericin fluorescence were found in necrosis, viable tumor and normal liver tissue in decreasing order (P < .05) (ie, in necrosis, mean fluorescence densities were about 4.5 times higher than in viable tumor and approximately 14 times higher than in normal liver). When hypericin was injected 1 hour before, maximal blood concentrations were achieved at the time of ethanol treatment, so that on ablation an outstanding extravasation took place in the entire necrotic area in comparison with accumulation of hypericin only at the peripheral zone of necrosis when it was injected 24 hours after ablation. Hypericin specifically enhanced the imaging contrast between necrotic and viable tissues and nonspecifically distinguished viable tumor from normal liver. Injection of hypericin shortly before ablation is more favorable than after ablation, because it circumvents difficulties with no-entry zones for hypericin and requires shorter intervals between ethanol ablation and imaging.

Influence of application and formulation factors on the penetration of hypericin in normal mouse skin and UV induced skin tumors

Hypericin, a naturally occurring photosensitizer, is currently being investigated for topical use in photodynamic therapy (PDT). In a previous study, it was found that hypericin can be delivered in the epidermis of hairless mouse skin after a 4-h application in Beeler base. With the intention to further optimize the penetration conditions, the present study examines the effect of the concentration of hypericin in the cream, the application time, the presence of penetration enhancers and occlusion on the penetration of hypericin in the skin of hairless mice. Experiments with different hypericin concentrations and application times indicated that application of 0.1% hypericin for 12–24 h maximizes the accumulation of hypericin-related fluorescence in the skin, as estimated by fluorescence microscopy with image analysis. Depending on the formulation, the use of an occlusive dressing did not alter, or even reduced the accumulation of hypericin in the viable layers. Also, the addition of propylene glycol (30%) and oleic acid (5%) did not change hypericin fluorescence levels in the epidermis. Conversely, incorporation of ethanol (40%, integrated in a gel, and added to Beeler base) increased dramatically the fluorescence levels in all skin layers. Consequently, the optimized conditions were used to investigate the penetration of hypericin into UV induced skin tumors. It was found that application under occlusion of hypericin, formulated in the gelcream containing ethanol, during 24 h enabled the penetration of hypericin in the entire skin lesions with a relatively homogenous distribution. In conclusion, our results suggest the possible use of 0.1% hypericin in a gelcream containing ethanol for PDT of skin lesions.

Effects of N-Methyl Pyrrolidone on the Uptake of Hypericin in Human Bladder Carcinoma and Co-staining with DAPI Investigated by Confocal Microscopy

Photodynamic diagnosis (PDD) using hypericin (HY), a natural photosensitizer, detects bladder cancer significantly better than white light endoscopy. However, the lipophilicity of HY complicates its administration for clinical applications. Currently, pharmaceutical preparations for HY without plasma protein are being developed. Formulations containing a biocompatible solvent, N-methyl pyrrolidone (NMP) have been shown to enhance the photodynamic therapeutic effects of HY. It was recently reported that, NMP formulations of HY were able to produce significantly higher contrast for fluorescence detection of tumors than albumin-containing HY formulations. This present work hypothesizes that NMP acts both as a solvent and penetration enhancer to improve the delivery of HY into cells by increasing the permeability of cell membranes. This paper reports the use of 3-D confocal microscopy to monitor real-time uptake of HY in human carcinoma. 3-D confocal microscopy was used to investigate the possibility of nuclear localization of HY in MGH cells. The fluorescence of HY was confirmed to be emitted from HY containing cells using spectrometry. The localization of a DNA fluorescent probe 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to confirm the possibility of colocalization of DAPI and HY. The colocalization analysis in the present study suggests that it was very unlikely that HY colocalized in the nucleus that was stained by DAPI. Fluorescein leakage tests showed that 1% NMP changes the permeability of cell membranes, and enhanced the delivery of HY into cells resulting in lower cell survival ratios. Thus, NMP was able to enhance the photodynamic therapeutic effects of HY on cancer cells.