Preparation and In Vitro Evaluation of W/O Nanoemulsions for the Skin Permeation of 5-Aminolevulinic Acid.

We investigated the skin permeation of 5-aminolevulinic acid (ALA) through Yucatan micropig full-thickness skin by using ALA-loaded W/O nanoemulsions composed of Span/Tween/ethanol (EtOH)/isopropyl palmitate (IPP)/10 wt% aqueous ALA solution. The nanoemulsions were prepared using Span 20/Tween 20 (S20/T20), Span 80/Tween 80 (S80/T80), and Span 20/Tween 80 (S20/T80) mixed surfactant systems. Based on the results of the phase diagram study and hydrodynamic diameter measurement of the nanoemulsions, we decided that the optimal weight ratio of Span/Tween/EtOH/IPP/10 wt% aqueous ALA solution in the nanoemulsion was 0.8/0.2/14/19/1.4. The permeability coefficient of ALA in the S20/T80 system was approximately five times larger than those in the S20/T20 and S80/T80 systems. The high skin permeation of ALA afforded by the ALA-loaded W/O nanoemulsion in the S20/T80 system is attributable to a significant enhancement in the partitioning of ALA to the stratum corneum.

5-Aminolevulinic acid-based photodynamic diagnosis for detection of urothelial carcinoma cells in bladder washing sediment suspension: A pilot study.

BACKGROUND: Bladder cancer is a common malignant disease in developed countries. Early detection of malignancy is important using urine cytology. The 5-aminolevulinic acid (ALA)-based photodynamic diagnosis (ALA-PDD) has not been routinely applied in urine cytology analysis yet, although it has been well accepted for tumor lesion marking in cystoscopy. METHODS: A total of eight volunteers were enrolled in this study. The cells of sediment suspension from bladder washing fluid and random urine were stained by ALA-induced protoporphyrin IX (ALA-PpIX) and the fluorescent intensity of ALA-PpIX was analyzed by ImageJ. RESULTS: The cutoff value of fluorescent intensity was 90.260 per pixel. The proposed protocol provided an objective fluorescent intensity for evaluation. Sensitivity was 0.931 and specificity was 1.000. CONCLUSIONS: The staining procedure applied was ALA-PpIX for suspicious cells in the cellular suspension from bladder wash fluid and random urine. ImageJ was applied to the objective measurement for the fluorescent intensity of the stained cells. The cutoff value for the positive result was 90.260 per pixel. Therefore, the protocol proposed in this study provides a potential means to enhance accuracy for urine cytology analysis.

[Detection of ß-Lactamase Produced by Klebsiella pneumoniae Using High Performance Liquid Chromatography].

In order to develop a new method to detect ß-lactamase, we determined degradation of ß-lactam antibiotics, ampicillin sodium (ABPC) and cefotaxime sodium (CTX), by ß-lactamase-producing Klebsiella pneumoniae (K. pneumoniae) in terms of high performance liquid chromatography (HPLC). Using HPLC with an ODS column and an eluent composed of phosphate buffer and methanol, we could detect ABPC and CTX within 10 min. After cultured with K. pneumoniae, ABPC and CTX were degraded. The degradation rate corresponding to the rate of peak area incubated with and without bacteria increased with increasing McFarland No. of bacterial suspension and incubation time. Under the culture condition of McFarland No. 3.0 and 90-min incubation, the degradation rate of ABPC and CTX was 52.6 and 70.8% by K. pneumoniae, whereas it was less than 10% by susceptible bacteria. Consequently, these results confirmed that ß-lactamase produced by ABPC- and CTX-resistant bacteria could be detected within about 120 min through HPLC measurement.

Accumulation of porphyrins in Propionibacterium acnes by 5-aminolevulinic acid and its esters.

We have investigated the accumulation of porphyrins in Propionibacterium acnes (P.acnes) by 5-aminolevulinic acid hydrochloride (ALA) and its esters, ALA methyl ester hydrochloride (mALA), ALA octyl ester hydrochloride (oALA), and ALA benzyl ester hydrochloride (bALA). From the fluorescence spectra of porphyrins accumulated in P.acnes, the order of porphyrin accumulation is as follows: ALA≫mALA≈bALA>oALA (≈0). Moreover, the PDT efficacy is reduced in the order of ALA>mALA≈bALA>oALA (≈without additives). These results confirm that ALA is superior to ALA esters in accumulating porphyrins in P.acnes.

Synthesis of oxovanadium complexes and their apoptosis-inducing activity in leukemia cells.

Vanadium complexes with different ligands were synthesized and evaluated for antiproliferative activity on U937 cells. The alkyl chain length of the ligands affected the antiproliferative activity, and two complexes-3b and 4-exhibited strong activities with IC(50) values of 6.02 and 3.90 µM respectively. Annexin V staining and DNA ladder formation indicated that these complexes induced apoptosis in U937 cells.

Oxovanadium complexes with quinoline and pyridinone ligands: syntheses of the complexes and effect of alkyl chains on their apoptosis-inducing activity in leukemia cells.

Vanadium complexes with quinoline ligands (1b-g) and pyridinone ligands (2b-d) were synthesized, and the effect of the length and shape of alkyl chains on the antiproliferative activity toward U937 cells was studied. For the synthesis of the vanadium complexes, quinoline and pyridinone ligands were prepared and then treated with VOSO(4) or VO(acac)(2). The vanadyl(IV) complexes were characterized by IR, ESR, and UV-vis spectroscopy and elemental analyses. The antiproliferative activity of 1a-g toward U937 cells showed little dependence on the length and shape of the alkyl chain. In contrast, a good correlation was found between the IC(50) values and partition coefficients (logP) values of 2a-c. Among them, 2c showed the highest inhibitory activity, and its IC(50) value was smaller than that of cisplatin. The apoptosis-inducing ability of 2b and 2c was supported by annexin V-propidium iodide staining experiments and agarose gel electrophoresis analysis. Inhibitors of caspase-3, -8, and -9 did not affect the antiproliferative activity of 2c, indicating that the apoptosis induced by 2c was via a caspase-independent pathway.

Enhancement in skin permeation of 5-aminolevulinic acid using l-menthol and its derivatives.

Enhancing effect of l-menthol and its derivatives, l-menthyl formate, l-menthyl acetate, and l-menthyl propionate, on skin permeation of 5-aminolevulinic acid (ALA) through Yucatan micropig full-thickness skin was investigated using a Franz-type diffusion cell. ALA solutions were prepared using ethanol-water mixed solvents with l-menthol or the derivative. Skin permeation coefficients (Kp) of ALA with more than 3.0 wt% of l-menthol was significantly larger than that without l-menthol. In addition, Kp of ALA with the derivative increased as follows: l-menthol approximately l-menthyl propionate < l-menthyl formate < l-menthyl acetate. These results suggest that l-menthol and the derivative are effective to enhance ALA skin permeation.

Oxidation decomposition of unsaturated fatty acids by singlet oxygen in phospholipid bilayer membranes.

Oxidation decomposition of unsaturated fatty acids with singlet oxygen generated from a photosensitizing agent was investigated in liposome bilayer membranes under a light irradiation condition. The liposome of which the bilayer membrane was composed of L-alpha-dipalmitoylphosphatidylcholine (DPPC), protoporphyrin IX (PpIX), and an unsaturated fatty acid (oleic acid, linoleic acid, alpha-linolenic acid, or arachidonic acid) were prepared with Bangham's method. In irradiating the liposome dispersion with light ranged from 550 to 750 nm, the unsaturated fatty acid was decomposed through an oxidation reaction with singlet oxygen. The decomposition rate constant was obeyed as the following order: arachidonic acid > oleic acid > alpha-linolenic acid > linoleic acid. This result indicates that oleic acid is readily degraded despite its lower unsaturated degree. In addition, micropolarity and microfluidity of the hydrocarbon region in the liposome bilayer membrane including the unsaturated fatty acid and PpIX decreased with an increase in light irradiation time. These findings suggest that interaction among the hydrocarbon chains of DPPC in the liposome bilayer membrane is promoted by migration of the oxidized unsaturated fatty acid from the hydrocarbon region, leading to form close-packed and well-ordered orientation of the hydrocarbon chains.

Immobilization and enzymatic activity of glucose oxidase on polystyrene surface modified with ozone aeration and UV irradiation in distilled water and/or aqueous ammonia solution.

Adsorption condition and enzymatic activity of glucose oxidase (GOD) on polystyrene (PS) film surfaces modified with ozone aeration and UV irradiation (O3/UV) treatment were investigated. The total amount of GOD immobilized on the PS film modified with the O3/UV treatment in distilled water (PS-W film) was approximately twice as large as that on the film treated in an aqueous ammonia solution (PS-A film), whereas the specific activity of GOD on the PS-A film was four times higher than that on the PS-W film. In contrast, no enzymatic activity of GOD on the non-treated PS film was observed because of irreversible denaturation of the adsorbed GOD. We therefore conclude that the PS films modified by the O3/UV treatment in the aqueous media are effective in immobilizing GOD.

Size and surface charge effect of 5-aminolevulinic acid-containing liposomes on photodynamic therapy for cultivated cancer cells.

5-Aminolevulinic acid (ALA)-containing liposomes having various average diameters and/or positive surface charges were prepared, and their photodynamic therapy (PDT) efficacy for murine thymic lymphoma cells, EL-4 cells, cultivated in vitro was investigated. The PDT efficacy for EL-4 cells and the accumulation of ALA-induced protoporphyrin IX (PpIX) in the cells increased with a decrease in the average diameter of liposomes. In particular, the ALA-containing liposomes smaller than 63.5 nm in diameter promoted the PDT efficacy in comparison with that of ALA alone. We also found no significant changes in PDT efficacy and PpIX accumulation with increasing positive surface charges of liposomes.

Sintering behavior and apatite formation of diopside prepared by coprecipitation process.

Sintering behavior and bioactivity of diopside, CaMgSi(2)O(6), prepared by a coprecipitation process were examined for its biomedical applicability. As-prepared powder was synthesized by adding aqueous ammonia to an ethanol solution containing Ca(NO(3))(2).4H(2)O, Mg(NO(3))(2).6H(2)O, and Si(OC(2)H(5))(4) and characterized by means of TG-DTA, XRD, and TG-MS. The dried powder was X-ray amorphous and crystallized into diopside at 845.5 degrees C. The glass network formation by SiO(4) tetrahedra was almost completed below 800 degrees C. The bioactivity of the diopside prepared by sintering the compressed powder at 1100 degrees C for 2h was evaluated by immersion of the sintered body in a simulated body fluid (SBF) at 36.5 degrees C. Leaf-like apatite particles were found to be formed on the surface of the sintered body and grew with passage of soaking time. This apatite-forming behavior in the SBF is related to the dissolution of Ca(II) ions from the sintered body in the early stage of immersion. Thus, diopside prepared by the coprecipitation process using the metal alkoxide and the metal salts was found to have an apatite-forming ability.

Delta-aminolevulinic acid-based photodynamic therapy for acne on the body.

Intractable acne on the body was clinically treated with photodynamic therapy (PDT) combining oral administration of delta-aminolevulinic acid (ALA) with polychromatic visible light from a metal halide lamp. The total number of body acne patients was 51. A 10 mg/kg B.W. of ALA was administered to the patients, and the acne lesions were exposed to the light emitted from the metal halide lamp. The wavelengths of the light ranged from 540 to 800 nm. The diameter of the irradiated area and the irradiating distance from the lamp were 100 mm and 130 mm, respectively. Under the irradiation conditions, the total fluence rate and the energy-density uniformity of the light were 69.2 mW/cm2 and 11.5%. The total light energy dose in one session was 60-80 J/cm2 for the body. All patients underwent two sessions of PDT and received no other treatments after PDT or during the follow-up period. The interval between the two PDT sessions was 2-4 weeks. The physician's clinical assessment scores as worsened, unchanged, improved, and markedly improved were 0 (0%), 4 (7.8%), 16 (31.4%), and 31 (60.8%) patients, respectively. Adverse effects of very slight discomfort, burning, and stinging were reported during the irradiation. The blood tests of patients before and after oral administration of ALA demonstrated that no liver dysfunction occurred in this study with ALA dosages within 10 mg/kg B.W. Consequently, orally administered ALA-PDT with a metal halide lamp was considered to be effective for treatment of acne on the body.