Microemulsions Improve Topical Protoporphyrin IX (PpIX) Delivery for Photodynamic Therapy of Skin Cancer

Abstract We report here microemulsions (MEs) for topical delivery of protoporphyrin IX (PpIX) for Photodynamic Therapy (PDT) of skin cancers. Selected MEs consisting of Oil/Water (O/W) bicontinuous (BC) and Water/Oil (W/O) preparations were characterized as to pH, nanometric size, zeta potential, drug content, and viscosity. Sustained in vitro PpIX release was achieved from MEs 2A (O/W), 10B (BC) and 16B (W/O) through an artificial membrane for up to 24 h, characterizing MEs as drug delivery systems. None of these MEs showed permeation through the skin, demonstrating the required topical effect. After 4 h, in vitro retention of PpIX in the stratum corneum (SC) was higher from both ME 10B and control (PpIX at 60 µg/mL in PEG 300). However, in the Epidermis + Dermis ([Ep + D]), retention from ME 10B and ME 16B was ~40 times higher compared to control. Confocal Laser Scanning Microscopy (CLSM) showed higher fluorescence intensity in the SC for both control and ME 10B, whereas ME 10B fluorescence was higher in [Ep+D]. The results indicate that ME 10B is suitable for PpIX encapsulation, showing good characteristics and a localized effect for a potential delivery system for PDT-associated treatments of skin cancers.

Effects of serum albumin on the photophysical characteristics of synthetic and endogenous protoporphyrin IX

The study of the interaction of synthetic protoporphyrin IX (PpIXs) and protoporphyrin IX extracted from Harderian glands of ssp Rattus novergicus albinus rats (PpIXe) with bovine serum albumin (BSA) was conducted in water at pH 7.3 and pH 4.5 by optical absorption and fluorescence spectroscopies. PpIXs is present as H- and J-aggregates in equilibrium with themselves and with monomers. The PpIXs charge is 2− at pH 7.3 and 1− at pH 4.5. This increases its aggregation at pH 4.5 and shifts the equilibrium in favor of J-aggregates. In spite of electrostatic attraction at pH 4.5, where BSA is positive, the binding constant (Kb) of PpIXs to BSA is 20% less than that at pH 7.3, where BSA is negative. This occurs because higher aggregation of PpIXs at pH 4.5 reduces the observed Kb value. At both pHs, water-soluble PpIXe exists in the monomeric form with the charge of 1− and its Kb exceeds that of PpIXs. At pH 4.5, its Kb is 12 times higher than that at pH 7.3 due to electrostatic attraction between the positively charged BSA and the negatively charged PpIXe. The higher probability of PpIXe binding to BSA makes PpIXe more promising as a fluorescence probe for fluorescence diagnostics and as a photosensitizer for photodynamic therapy. The existence of PpIXe in the monomeric form can explain its faster cell internalization. Aggregation reduces quantum yields and lifetimes of the PpIXs excited states, which explains higher phototoxicity of PpIXe toward malignant cells compared with PpIXs.

In vitro Photodynamic Antimicrobial Activity of Protoporphyrin IX in the Presence of Hydrogen Peroxide against Staphylococcus aureus and Pseudomonas aeruginosa.

Aims: This study reports on In vitro investigation of photodynamic antimicrobial activity of protoporphyrin IX (PPIX) in the presence and absence of Hydrogen peroxide (H2O2) against S. aureus and P. aeruginosa. Place and Duration of Study: Department of Medical Physics, Anna University, Chennai between December 2013 and February 2014. Methodology: A light-emitting diode (LED) was used as a light source to irradiate PPIX. The antibacterial effect was analyzed by standard plate counting method. Steady-state fluorescence spectroscopy technique was used to monitor the damage at protein level. Results: We found that the antibacterial effect is dependent on PPIX concentration as well as H2O2 concentration and light dose. PPIX-H2O2 combination showed higher bacterial reduction of 6.5 log10 and 2.7 log10 for S. aureus and P. aeruginosa respectively, when the light dose increased to 70 J/cm2. Fluorescence spectroscopic characterization showed a considerable change in the intensity of emission of tryptophan present in the microorganisms between pre- and post- APDT. Conclusion: PPIX-H2O2 is a promising combination for APDT against Gram positive and Gram negative bacteria. The LED seems to be a very good option for PDT because of its low cost and miniature in size.

Fluorometric quantification of protoporphyrin IX in biological skin samples from in vitro penetration/permeation studies

A fluorometric analytical method was developed for quantification of protoporphyrin IX (PpIX) in skin samples and receptor phase solution after in vitro cutaneous penetration/permeation studies. Analytical conditions used were: excitation and emission wavelengths: 400 nm and 632 nm; bandwidth: 0.5 nm; excitation and emission slits: 10/10. PpIX was recovered from two different layers of skin, the stratum corneum (SC) and the epidermis plus dermis ([E+D]), by vortex homogenization, probe and bath sonication, using DMSO as an extraction solvent. The detection and quantification limits were 0.002 and 0.005 μg/mL, respectively. The assay was linear from 0.005 - 0.5 μg/mL. The within-day and between-day assay precision and accuracy in DMSO and receptor phase solution were each studied at the two concentration levels 0.04 and 0.2 μg/mL, and 0.01 and 0.08 μg/mL, respectively. The coefficients of variation and deviation from the theoretical values were lower than 5%. The skin recovery of PpIX from SC and [E+D] layers using two different concentrations (0.5 and 1.0 μg/mL) were all above 90.0%. The method described has potential application to in vitro penetration/permeation studies of PpIX using porcine skin as a biological membrane model.

Um método analítico por espectrofluorimetria foi desenvolvido para quantificar a protoporfirina IX (Pp IX) em amostras de pele e fase receptora após a realização de testes in vitro de penetração/permeação cutâneas. As condições analíticas utilizadas foram: comprimentos de onda de excitação e emissão: 400 nm e 632 nm; largura de banda: 0,5 nm; fendas de excitação e emissão: 10/10. A PpIX foi extraída de amostras de estrato córneo (EC) e da epiderme sem estrato córneo + derme ([E+D]) através da agitação em vórtex e sonicação por haste e banho, utilizando-se o DMSO como solvente extrator. O limite de detecção e quantificação foram, respectivamente, de 0,002 e 0,005 μg/mL. O método mostrou-se linear da faixa de 0,005 - 0,5 μg/mL. A precisão e exatidão intra e inter-ensaio em DMSO e na fase receptora foram validadas utilizando-se duas concentrações distintas, respectivamente, de 0,004 e 0,2 μg/mL, e 0,01 e 0,08 μg/mL. Os valores de coeficiente de variação e o desvio do valor teórico foram inferiores a 5%. A recuperação da PpIX das camadas da pele (EC e [E+D]) utilizando-se duas concentrações distintas (0,5 e 1,0 μg/mL) foram todas acima de 90,0%. O método descrito pode ser utilizado para determinação da PpIX após estudos de penetração/permeação cutânea in vitro utilizando pele de porco como modelo de membrana.

A Comparative Study on the Enhancement Efficacy of Specific and Non-specific Iron Chelators for Protoporphyrin IX Production and Photosensitization in HaCat Cells / 华中科技大学学报（医学）（英德文版）

The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). The purpose of this study is to compare the effect of two kinds of iron chelators,desferrioxamine (DFO) and ethylenediaminetetraacetic acid (EDTA) on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark,the concentration of cellular protoporphyrin IX (PpIX) was detected by high performance liquid chromatography (HPLC),and the fluorescence of PpIX was observed at 630 nm emission under confocal laser scanning microscope.For PDT,HaCat cells were irradiated using 632.8 nm laser,and the fractions of apoptotic and necrotic cells were flow cytometricaUy assayed. Related differences in morphology and ultrastructure of HaCat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone,the addition of DFO or EDTA increased the concentration of cellular PpIX and the fluorescent density of PpIX,and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpIX level,greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpIX production and PDT. Compared to the non-specific iron chelator of EDTA,the specific chelator,DFO,showed more potential for the enhancement.

Protoporphyrin IX Synthesis Induced by 5-aminolevulinic Acid and Its Esters in Normal Mouse Skin / 대한피부과학회지

BACKGROUND: Photodynamic therapy with using 5-aminolevulinic acid (ALA) is a good noninvasive treatment modality that can treat several cutaneous diseases. Yet hydrophilic ALA is not able to easily penetrate the cellular membrane or the lipid layer of the skin. Thus, various ALA alkyl esters been developed. OBJECTIVE: We studied whether novel ALA unsaturated alkyl esters synthesize more protoporhyrin IX (PpIX) than does ALA in mouse skin tissues. METHODS: We applied 5, 10, 15% ALA and ALA esters ointment to the skin of shaved mice, respectively. We harvested the skin tissue, made a preparation and did quantitative analysis according to the fluorescent spectrum for determining the amount of PpIX synthesized in the skin after the elapse of 1 hour and 3 hours, respectively. RESULTS: No matter what the sort or the concentration of the ALA and ALA ester was, the amount of synthesized PpIX increased more after the elapse of 3 hours than that after 1 hour. ALA (hetenyl-, petenyl-, butenyl-, methyl-) esters tended to produce a much greater amount of PpIX than ALA. According to the results of the comparative study with using 5%, 10% and 15% of ALA and ALA esters ointment, the amount of synthesized PpIX was not proportional to the concentration of the ALA and the ALA esters, even though the ALA esters produced more PpIX than the ALA. CONCLUSION: We found that the ALA unsaturated alkyl esters are a good photosensitizer and they are similar to ALA-me, which is an ALA saturated alkyl ester that induces more PpIX, an actual photosensitizer, than ALA can.

Expression of Protoporphyrin IX by Methyl 5-aminolevulinate in Various Dermatologic Diseases and Cells / 대한피부과학회지

BACKGROUND: 5-aminolevulinic acid (ALA), is the precursor of heme biosynthesis and used in photodynamic therapy (PDT). When applied exogenously, it is converted to protoporphyrin IX (PpIX) that is an immediate photosensitizer. PpIX is accumulated selectively in various cells including tumor cells. Because most of photosensitizers emit fluorescence of a specific wavelength, it is very important to identify the fluorescence in the tissues and cells targeted for therapy. OBJECTIVE: This study was undertaken to identify the presence of PpIX fluorescence image and localization in various dermatologic diseases with methyl 5-aminolevulinate (MAL), that satisfactorily penetrates cutaneous lipid layers and cell membranes. METHODS: Various skin diseases such as basal cell carcinoma (6), squamous cell carcinoma (4), keratoacanthoma (6), malignant melanoma (3), extramammary Paget's disease (4), verruca (5), psoriasis vulgaris (5), rosacea (2), and acne (5) were included. We applied MAL, ALA ester, ointment to cutaneous lesions and perilesional area for at least 3 hours. After that, we identified the fluorescence image with Wood's lamp (wavelength 320~400 nm) and photographed fluorescence image. Also, we performed skin biopsies of fluorescence site and investigated the PpIX fluorescent location with a fluorescence microscope. In addition, we treated three cultured cell lines (HaCaT cells, human dermal fibroblasts, A431 cells) with MAL and investigated PpIX fluorescence. RESULTS: The PpIX fluorescence images were observed significantly in basal cell carcinoma, squamous cell carcinoma, extramammary Paget's disease, keratoacanthoma, psoriasis, rosacea, and acne. In tissues, PpIX fluorescence was expressed mainly in the pilosebaceous unit, abnormal keratinocytes, tumor cells including basal cell carcinoma, and extramammary Paget's disease. In addition, PpIX was expressed in the cytoplasm of HaCaT cells, human dermal fibroblasts, and A431 cells in vitro. CONCLUSION: In applieation of photodynamic therapy, this study is expected to be helpful in enhancement of therapeutic effectiveness and increase of indications of diseases.

Heme Oxygenase-1 is Involved in the Down-regulation of Nuclear Transcription Factor kappa B Activation in the Colonic Epithelium During Inflammation / 대한해부학회지

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with anti-inflammatory activity, but the mechanisms underlying this activity are incompletely understood. Nuclear transcription factor kappa B (NF-kappa B) activation is an important factor in the pathogenesis of inflammatory bowel disease (IBD). We investigated the suppressive effects of HO-1 on the activation of NF-kappa B by pro-inflammatory cytokines in cultured colonic epithelial cells and by trinitrobenzene sulfonic acid (TNBS) in the colon of mice. The expression level of HO-1 in the colonic epithelium of a patient with inflammatory bowel disease and pseudo-membranous colitis was lower than that in a healthy control subject. In cultured human colonic epithelial HT-29 cells, pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha ) and IL-1 beta down-regulate HO-1 expression. The HO-1 inducer, cobalt protoporphyrin IX (CoPPIX), dramatically down-regulated NF-kappa B activation in HT-29 cells by TNF-alpha. In addition, bilirubin-a product of heme catabolism by HO-1-and the carbon monoxide donor tricarbonyldichlororuthenium (II) dimer also suppressed NF-kappa B activation by TNF-alpha. However, iron, another heme metabolite, did not suppress NF-kappa B activation by TNF-alpha. Furthermore, CoPPIX diminished the macroscopic and histopathological symptoms of TNBS-induced colitis and down-regulated NF-kappa B activation in mice. In conclusion, this study suggests that HO-1 plays an important role in the down-regulation of NF-kappa B activation, which is a key factor in the pathogenesis of IBD and is thus an excellent therapeutic target for the treatment of IBD.