PHOTOSENSITIZER DISPERSION, AND USE THEREOF

Abstract

A photosensitizer-containing dispersion, and use thereof.

Claims

1. A dispersion, comprising: (a) at least one photosensitizer, (b) at least one liquid polar phase, and (c) at least one surfactant, and wherein the dispersion comprises a microemulsion, a gel or a mixture thereof, at a temperature in the range 2? C. to 50? C. and a pressure in the range 800 to 1200 mbar.

2. The dispersion as claimed in claim 1, wherein the photosensitizer is selected from the group which consists of phenalenones, curcumins, flavins, porphyrins, porphycenes, xanthene dyes, coumarins, phthalocyanines, phenothiazine compounds, anthracene dyes, pyrenes, fullerenes, perylenes and mixtures thereof.

3. The dispersion as claimed in claim 1, wherein the at least one liquid polar phase comprises at least one polar solvent.

4. The dispersion as claimed in claim 1, wherein the at least one surfactant is selected from the group which consists of non-ionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants and mixtures thereof.

5. The dispersion as claimed in claim 4, wherein the cationic surfactants are selected from the group which consists of quaternary alkylammonium salts, esterquats, acylated polyamines, benzylammonium salts and mixtures thereof.

6. The dispersion as claimed in claim 4, wherein the non-ionic surfactants are selected from the group which consists of polyalkyleneglycol ethers, alkylglucosides, alkylpolyglycosides, alkylglyco side esters and mixtures thereof.

7. The dispersion as claimed in claim 4, wherein the anionic surfactants are selected from the group which consists of alkylcarboxylates, alkylsulphonates, alkylsulphates, alkylphoshates, alkylpolyglycolethersulphates, sulphonates of alkylcarboxylic acid esters, N-alkyl-sarcosinates and mixtures thereof.

8. The dispersion as claimed in claim 1, wherein the dispersion further comprises at least one liquid non-polar phase, wherein the at least one liquid non-polar phase comprises at least one non-polar solvent.

9. The dispersion as claimed in claim 1, wherein the dispersion further contains at least one alkanol containing 2 to 12 carbon atoms.

10. The dispersion as claimed in claim 1, wherein the dispersion at least comprises a microemulsion at a pressure in the range 800 to 1200 mbar and a temperature in the range 2? C. to 50? C.

11. The dispersion as claimed in claim 10, wherein the microemulsion is an O/W-microemulsion, a water-in-oil (W/O) microemulsion or a bicontinuous microemulsion.

12. The dispersion as claimed in claim 1, wherein the dispersion further contains at least one pH-regulating substance.

13. The dispersion as claimed in claim 1, wherein the dispersion further comprises at least one gelling agent which is selected from the group which consists of carboxyvinyl polymers, polyacrylamides, polyvinyl alcohols, acylated polyethylene amines, alginates, cellulose ethers and mixtures thereof.

14. The dispersion as claimed in claim 1, wherein the dispersion at least comprises a gel at a pressure in the range 800 to 1200 mbar and a temperature in the range 2? C. to 50? C.

15. A method for the photodynamic inactivation of microorganisms selected from the group which consists of viruses, archaeae, bacteria, bacterial spores, fungi, fungal spores, protozoa, algae and blood-borne parasites, wherein the method comprises contacting said microorganisms with the dispersion as claimed in claim 1.

16. The method as claimed in claim 15, wherein the method is used for the surface cleaning and/or surface coating of an article.

17. The method as claimed in claim 15, wherein the method is used for the surface cleaning and/or surface coating of at least one which is selected from the group which consists of medical products, food packaging, foodstuffs, beverage packaging, beverage containers, textiles, building materials, electronic devices, household appliances, furniture, windows, floors, walls pal and hygiene articles,

18. The method as claimed in claim 15, wherein the method is used for the decontamination of liquids.

19. A method for the photodynamic inactivation of microorganisms selected from the group consisting of viruses, archaeae, bacteria, bacterial spores, fungi, fungal spores, protozoa, algae and bloodborne parasites, wherein the method comprises the following steps: (A) bringing the microorganisms into contact with a photosensitizer-containing dispersion as claimed in claim 1, and (B) irradiating the microorganisms and the at least one photosensitizer with electromagnetic radiation of a suitable wavelength and energy density for inactivating the microorganisms.

20. A method of photodynamic therapy for the inactivation of microorganisms which are selected from the group which consists of viruses, archaeae, bacteria, bacterial spores, fungi, fungal spores, protozoa, algae and blood-borne parasites, wherein the method comprises contacting said microorganisms with the dispersion as claimed in claim 1.

21. The dispersion as claimed in claim 1, adapted for use during photodynamic therapy for the inactivation of microorganisms which are selected from the group which consists of viruses, archaeae, bacteria, bacterial spores, fungi, fungal spores, protozoa, algae and blood-borne parasites, in the treatment and/or prophylaxis of a disease of dental tissue and/or of the periodontium.

Description

[0365] FIG. 1 shows the mean value of the contact angle for the photosensitizer-free microemulsions E1 to E4 as well as aqueous ethanol solutions with the concentrations given.

[0366] FIG. 2 shows the mean values of the measured contact angle measured in Example 1 of the dilution of a microemulsion E3 (DMS; TWEEN? 20/1,2-pentanediol (1:3); water) with water.

[0367] FIG. 3 shows the measured time-resolved singlet oxygen spectra for the photosensitizer TMPyP in water (w), microemulsion E1 (E1) or microemulsion E2 (E2).

[0368] FIG. 4 shows the time-resolved singlet oxygen spectra measured in Example 1 for the photosensitizer SA-PN-01a in water (w), microemulsion E1 (E1) or microemulsion E2 (E2).

[0369] FIG. 5 shows the time-resolved singlet oxygen spectra measured in Example 1 for the photosensitizer FL-AS-H-1a in water (w), microemulsion E1 (E1) or microemulsion E2 (E2).

[0370] FIG. 6a shows the results measured in Example 1 of the phototoxicity tests for the photosensitizer SA-PN-01a in water in concentrations given.

[0371] FIG. 6b shows the results measured in Example 1 of the phototoxicity tests for the photosensitizer SA-PN-01a in microemulsion E2 (E2) in the concentrations given.

[0372] FIG. 7 shows the mean values for the contact angle measured in Example 3 of the photosensitizer-free gels G2 and G3, to which the relevant quantity of the given surfactant had been added.

[0373] FIG. 8 shows the time-resolved singlet oxygen spectrum measured in Example 3 for the photosensitizer TMPyP in gel G3.

[0374] FIG. 9 shows the wavelength-resolved singlet oxygen spectrum measured in Example 3 for the photosensitizer TMPyP in gel G3.

EXAMPLES

[0375] All of the chemicals were purchased from conventional suppliers (TCI, ABCR, Acros, Merck and Fluka) and used without further purification. The solvents were distilled before use and if required, were dried in the normal manner. Dry DMF was purchased from Fluka (Taufkirchen, DE). Thin film chromatography was carried out on thin film aluminium foils coated with silica gel 60 F254, from Merck (Darmstadt, DE). Preparative thin film chromatography was carried out on commercially available glass plates coated with silica gel 60 (20 cm?20 cm, Carl Roth GmbH & Co. KG, Karlsruhe, DE). The compounds were detected with UV light (?=254 nm, 333 nm) and some detected with the naked eye or stained with ninhydrin. The chromatography was carried out with silica gel (0.060-0.200) from Acros (Waltham, US). NMR spectra were recorded on a Bruker Avance 300 spectrometer (300 MHz [.sup.1H-NMR], 75 MHz [.sup.13C-NMR]) (Bruker Corporation, Billerica, US). All of the chemical displacements are given in ? [ppm] relative to an external standard (tetramethylsilane, TMS). The coupling constants are respectively given in Hz; characterization of the signals: s=singlet, d=doublet, t=triplet, m=multiplet, dd=doublet of doublets, br=broad. Integration determined the relative number of atoms. The definitive identification of the signals in the carbon spectra was carried out using the DEPT method (pulse angle: 135?). Error limits: 0.01 ppm for .sup.1H-NMR, 0.1 ppm for .sup.13C-NMR and 0.1 Hz for coupling constants. The solvent used is noted for each spectrum. The IR spectra were recorded on a Biorad Excalibur FTS 3000 spectrometer (Bio-Rad Laboratories GmbH, Munich, DE). ES-MS was measured using a ThermoQuest Finnigan TSQ 7000 spectrometer, all of the HR-MS were determined on a ThermoQuest Finnigan MAT 95 (respectively Thermo Fisher Scientific Inc, Waltham, US) spectrometer; argon was used as the ionization gas for FAB ionization (fast atom bombardment). The melting points were determined with the aid of the Buchi SMP-20 melting point instrument (Buchi Labortechnik GmbH, Essen, DE) using a glass capillary. All of the UV/VIS spectra were recorded using a Varian Cary 50 Bio UV/VIS spectrometer; the fluorescence spectra were recorded with a Varian Cary Eclipse spectrometer. The solvents for absorption and emission measurements were purchased in special spectroscopic purity grade from Acros or Baker, or Uvasol from Merck. Millipore water (18 M?, Milli Q.sub.Plus) was used for all of the measurements.

[0376] The following photosensitizers were used in the examples below:

1.) 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-porphyrin-tetra-(p-toluenesulphonate) (TMPyP, M=1363.65 g/mol)

[0377] ##STR00039##

[0378] TMPyP was purchased from TCI Germany GmbH (Eschborn, DE).

2.) 2-(4-pyridinyl)methyl)-1H-phenalen-1-on-chloride

[0379] (SA-PN-01a, M=307.78 g/mol),

[0380] Chloride of the compound with formula (24)

##STR00040##

[0381] SA-PN-01a was produced in accordance with the synthesis described in EP 2 678 035 A2, Example 7. The .sup.1H-NMR spectrum in DMSO-d6 was identical to the spectrum known from the literature.

3a) 10-[2-({[(tert-butyl)oxy]carbonyl}amino)eth-1-yl]-7,8-dimethyl-[3H,10H]-benzo[g]pteridine-2,4-dione (Flavin 32a)

[0382] ##STR00041##

[0383] The synthesis was carried out as published by Butenandt, J. et al. (2002) using commercially available precursors. The .sup.1H-NMR spectrum in DMSO-d6 was identical to the spectrum known from the literature.

3b) 10-(2-aminoeth-1-yl)-7,8-dimethyl-[3H,10H]-benzo[g]pteridine-2,4-dione hydrochloride (FL-AS-H-1a; M=321.77 g/mol)

[0384] Chloride of the Compound with Formula (32)

##STR00042##

[0385] Flavin 32a (2.0 mmol) was dissolved in dichloromethane (100 mL); HCl in diethyl ether (10 mL) was added dropwise and the reaction mixture was stirred overnight in the dark with the exclusion of moisture. The precipitate was aspirated off, washed with diethyl ether and dried. The .sup.1H-NMR spectrum in DMSO-d6 was identical to the spectrum known from the literature.

4a) 3,10-bis[2-(tert-butyloxycarbonylamino)eth-1-yl]-7,8-dimethylbenzo[g]-pteridine-2,4-dione (Flavin 64a)

[0386] ##STR00043##

[0387] The synthesis of flavin 64a was carried out as described in the publication by Svoboda J. et al. (2008) using flavin 32a. The .sup.1H-NMR spectrum in DMSO-d6 was identical to the spectrum known from the literature.

4b) 3,10-bis(2-aminoeth-1-yl)-7,8-dimethylbenzo[g]pteridine-2,4-dion-dihydrochloride (FL-AS-H-2; M=401.29 g/mol)

[0388] Dichloride of Compound (64)

##STR00044##

[0389] Flavin 64a (2.0 mmol) was dissolved in dichloromethane (100 mL); HCl in diethyl ether (10 mL) was added dropwise and the reaction mixture was stirred overnight in the dark with the exclusion of moisture. The precipitate was aspirated off, washed with diethyl ether and dried. The .sup.1H-NMR spectrum in DMSO-d6 was identical to the spectrum known from the literature.

5) Synthesis of Compounds with Formula (26), (27), (28a) and (28)

[0390] ##STR00045##

##STR00046##

5a) N-methyl-N-(1-oxo-1H-phenalen-2-yl)methanaminium Chloride Chloride of the Compound with Formula (27)

[0391] An ice-cold solution of methylamine in methanol (40 mL, 10%) was added dropwise over 1 h to 2-chloromethyl-1H-phenalen-1-one (1) (113 mg, 0.5 mmol) in methanol (10 mL). After stirring for 30 h at room temperature, the excess amine and the solvent were driven off in a stream of nitrogen. The residue was dissolved in 4:1 dichloromethane (DCM)/ethanol and precipitated by adding diethyl ether. The product was centrifuged (60 min, 4400 rpm, 0? C.) and the supernatant was discarded. This step was repeated once more. The residue was suspended in diethyl ether. After the yellow solid had settled out, the supernatant was decanted off and discarded. This step was repeated twice more. The product (101 mg, 0.39 mmol) was a yellowish-brown powder.

[0392] .sup.1H-NMR (300 MHz, CDCl.sub.3): ?[ppm]=8.66 (d, J=7.4 Hz, 1H), 8.28-8.20 (m, 2H), 8.08 (d, J=8.3 Hz, 1H), 7.94 (d, J=7.0 Hz, 1H), 7.80 (t, J=7.7 Hz, 1H), 7.67-7.59 (m, 1H), 4.20 (s, 2H), 2.79 (s, 3H). MS (ESI-MS, CH.sub.2Cl.sub.2/MeOH+10 mmol NH.sub.4OAc): e/z (%)=224.1 (MH.sup.+, 100%); molecular weight (MW)=224.28+35.45 g/mol; empirical formula (MF)=C.sub.15H.sub.14NOCl.

5b) N,N,N-trimethyl-1-(1-oxo-1H-phenalen-2-yl)methanaminium chloride (SA-PN-02a)

[0393] Chloride of the compound with formula (26)

[0394] 2-(chloromethyl)-1H-phenalen-1-on (1) (230 mg, 1 mmol) in ethanol (60 mL) was placed in a Schlenk flask. Trimethylamine in ethanol (5 mL, 5.6 M, 23 mmol) was added via the septum using a syringe. The solution was stirred overnight in the dark. Stirring was then continued at 50? C. for 30 h. The solvent volume was reduced to 3 mL. Diethyl ether (50 mL) was added in order to completely precipitate the product. The product was centrifuged (60 min, 4400 rpm, 0? C.) and the supernatant was discarded. The residue was suspended in diethyl ether. After the yellow solid had settled out, the supernatant was decanted off and discarded. This step was repeated twice more. The solid was dried under reduced pressure and a yellow powder was obtained (210 mg, 0.73 mmol).

[0395] .sup.1H-NMR (600 MHz, D.sub.2O): ?[ppm]=8.02 (d, J=8 Hz, 1H), 7.97 (d, J=6.3 Hz, 1H), 7.92 (d, J=8.2 Hz, 1H), 7.77 (s, 1H), 7.62 (d, J=7 Hz, 1H), 7.50 (t, J=7.8 Hz, 1H) 7.45 (t, J=7.8 Hz, 1H), 4.12 (s, 2H), 2.98 (s, 9H). MS (ESI-MS, CH.sub.2Cl.sub.2/MeOH+10 mmol NH.sub.4OAc): e/z (%)=252.1 (100, M+); MW=287.79 g/mol; MF=C.sub.17H18NOCl;

5c) 1-((1-oxo-1H-phenalen-2-yl)methyl)-1-methyl-2,3-di(tert-butoxycarbonyl)guanidine

[0396] Compound with Formula (28a)

[0397] N,N-di-Boc-N-triflylguanidine (0.41 g, 1.05 mmol) in dichloromethane (10 mL) was placed in a dry 25 mL round bottom flask. Triethylamine (0.3 g, 0.39 mL, 3 mmol) was slowly added at 2-5? C. with the exclusion of moisture. Compound 3 (130 mg, 0.5 mmol) was added all at once. After stirring for 5 h at room temperature, it was diluted with dichloromethane (30 mL) and the solution was transferred into a separating funnel. The organic phase was washed with aqueous potassium hydrogen sulphate (10 mL, 5%), saturated sodium bicarbonate solution (10 mL) and saturated sodium chloride solution (20 mL), dried over MgSO.sub.4, filtered and rotary evaporated. The crude product was purified by column chromatography using 1:2 acetone/petroleum ether and the product was obtained as a yellow solid (0.21 g). To purify it further, the material was dissolved in acetone (1 mL) and precipitated with petroleum ether (14 mL). The precipitate was aspirated off and washed with petroleum ether.

[0398] .sup.1H-NMR (300 MHz, CDCl.sub.3): ?[ppm]=8.63 (d, J=7.3 Hz, 1H), 8.21 (d, J=7.9 Hz, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.85-7.70 (m, 3H), 7.67-7.54 (m, 1H), 4.59 (s, 2H), 3.01 (s, 3H), 1.50 (s, 9H), 1.48 (s, 9H). MS (ESI-MS, CH.sub.2Cl.sub.2/MeOH+10 mmol NH.sub.4OAc): e/z (%)=466.1 (MH.sup.+, 100%); MW=465.53 g/mol; MF=C.sub.26H.sub.31N.sub.3O.sub.5

5d) 1-((1-oxo-1H-phenalen-2-yl)methyl)-1-methylguanidinium chloride

[0399] (SA-PN-24d)

[0400] Chloride of the Compound with Formula (28)

[0401] The compound was produced and purified, protected from light. Compound 5 (200 mg, 0.45 mmol) was placed in dichloromethane (20 mL, dried over CaCl.sub.2). A saturated solution of HCl in diethyl ether (2 mL) was added dropwise. After stirring for 4 h at room temperature with the exclusion of moisture, the solution was distributed into two Blue Caps and each filled with diethyl ether to 15 mL. The product was centrifuged (60 min, 4400 rpm, 0? C.) and the supernatant was discarded. The residue was suspended in diethyl ether. After the yellow solid had settled out, the supernatant was decanted off and discarded. This step was repeated twice more. Next, the product was dried under reduced pressure in order to obtain 130 mg of a yellow powder.

[0402] .sup.1H-NMR (300 MHz, DMSO-d6): ?[ppm]=8.60-8.47 (m, 4H), 8.33-8.24 (m, 2H), 8.16-8.09 (m, 2H), 7.98-7.89 (m, 2H), 7.84-7.73 (m, 4H), 7.57-7.48 (m, 7H), 4.55-4.42 (m, 4H), 3.05 (s, 6H). MS (ESI-MS, CH.sub.2Cl.sub.2/MeOH+10 mmol NH.sub.4OAc): e/z (%)=266.1 (MH.sup.+, 100%); MW=266.3+35.45=301.75 g/mol; MF=C.sub.16H.sub.16N.sub.3OCl

Example 1

[0403] A) Production of Various Water-Containing Microemulsions

[0404] The % by weight of the components of the microemulsions E1 to E4 given below are with respect to the total weight of the relevant microemulsion without photosensitizer.

[0405] Microemulsion E1: microemulsion consisting of DMS, SDS and 1-pentanol with a constant weight ratio of SDS to 1-pentanol of 1:2, as well as water.

[0406] 20.0% by weight dimethylsuccinate (DMS)

[0407] 8.33% by weight sodium dodecylsuiphate (SDS)

[0408] 16.67% by weight 1-pentanol

[0409] 55.0% by weight water

[0410] Microemulsion E2: microemulsion consisting of DMS, SDS and 1,2-pentanediol with a constant weight ratio of 1:2 SDS to 1,2-pentanediol, as well as water.

[0411] 20.0% by weight dimethylsuccinate (DMS)

[0412] 8.33% by weight sodium dodecylsuiphate (SDS)

[0413] 16.67% by weight 1,2-pentanediol

[0414] 55.0% by weight water

[0415] Microemulsion E3: microemulsion consisting of DMS, TWEEN? 20 and 1,2-pentanediol with a constant weight ratio of TWEEN? 20 to 1,2-pentanediol of 1:3, as well as water.

[0416] 10.0% by weight dimethylsuccinate (DMS)

[0417] 3.75% by weight TWEEN? 20

[0418] 11.25% by weight 1,2-pentanediol

[0419] 75.0% by weight water

[0420] Microemulsion E4: microemulsion consisting of DMS, TWEEN? 20 and 1,2-propanediol with a constant weight ratio of TWEEN? 20 to 1,2-propanediol of 1:3, as well as water

[0421] 10.0% by weight dimethylsuccinate (DMS)

[0422] 3.75% by weight TWEEN? 20

[0423] 11.25% by weight 1,2-propanediol

[0424] 75.0% by weight water

[0425] The relevant microemulsions E1 to E4 were initially produced without photosensitizer, wherein all of the components were measured without water and then mixed together one after the other. After a homogeneous mixture had been obtained, the appropriate quantity of water was added, with constant stirring.

[0426] As an example, 100 g of microemulsion E4 was produced by weighing out 3.75 g of TWEEN? 20, 11.25 g of 1,2-propanediol and 10 g of DMS. The resulting solution was stirred until a homogeneous mixture had been obtained. Next, 75 g of water was added, with stirring.

[0427] For the further experiments, the photosensitizers were dissolved in the appropriate concentration in the respective microemulsion and stirred until the photosensitizer had been completely dissolved.

[0428] B) Contact Angle Test

[0429] Wetting of the surfaces by the microemulsions used was determined with the aid of the contact angle test.

[0430] For the contact angle test, the emulsions given above were used without photosensitizer, as well as photosensitizer-containing emulsions which contained the photosensitizers TMPyP, SA-PN-01a, SA-PN-02a, SA-PN-24d, FL-AS-H-1a or FL-AS-H-2.

[0431] In order to compare the novel dispersions with conventional, alcohol-containing disinfecting solutions, furthermore, aqueous ethanol solutions with various ethanol concentrations in the range 10% by weight ethanol to 90% by weight ethanol were used as comparative solutions.

[0432] Furthermore, dilutions of the aforementioned emulsions without photosensitizer, as well as photosensitizer-containing emulsions were used, in which the relevant microemulsion was diluted in 5 steps to a water content of 99% by weight.

[0433] The contact angle was determined with the aid of the DataPhysics OCA 35 contact angle measuring instrument from DataPhysics Instruments GmbH (Filderstadt, DE), following the manufacturer's instructions.

[0434] For the measurement, 2.5 ?L of each test solution was applied at room temperature with full climate control (temperature: 25? C., pressure: 1013 mbar, relative humidity: 50%) to a glass slide as the test surface, using an automatic Hamilton syringe in the form of a droplet and photographed at one second intervals.

[0435] Next, for each image, both the left and also the right contact angle between the droplet and the test surface was determined using SCA 20 software from DataPhysics Instruments GmbH, along with the mean of the measured contact angle. Each measurement was carried out 4 times.

[0436] FIG. 1 shows the mean of the measured contact angle for aqueous ethanol solutions with various ethanol concentrations in the range from 10% by weight of ethanol to 80% by weight of ethanol.

[0437] By way of example, FIG. 1 also shows the means of the measured contact angle for the photosensitizer-free microemulsions E1 to E4.

[0438] The means of the measured contact angle for microemulsions E1 to E4, which each contained 100 ?m of one of the photosensitizers used, deviated only insignificantly from the measured contact angles for the photosensitizer-free microemulsions E1 to E4.

[0439] The various microemulsions with SDS and TWEEN? 20 exhibited a significantly reduced contact angle compared with pure water. More than 40% by weight of ethanol had to be used in order to obtain a comparable wetting of the glass surface employed.

[0440] The effect of the dilution of a microemulsion with water is shown by way of example in FIG. 2 on the photosensitizer-free microemulsion used (DMS; TWEEN? 20/1,2-pentanediol (1:3); water).

[0441] As can be seen in FIG. 2, microemulsion E3 can be diluted with an approximately 8-fold quantity of water without the contact angle of the dilution obtained increasing significantly in the test described above. Even a 16-fold dilution still exhibited sufficient wetting of the glass plate used in the test.

[0442] Similar results were obtained for microemulsions E1, E2 and E4 as well as for microemulsions E1 to E4, which respectively contained 5 ?M of one of the photosensitizers TMPyP, SA-PN-01a, SA-PN-02a, SA-PN-24d, FL-AS-H-1a or FL-AS-H-2 employed.

[0443] C) UV/VIS Measurements

[0444] The absorption of the photosensitizers TMPyP, SA-PN-01a and FL-AS-H-1a used in the respective microemulsions E1 to E4 were determined by recording an absorption spectrum for a wavelength range of 250 nm to 600 nm.

[0445] In this regard, the photosensitizers SA-PN-01a and FL-AS-H-1a were dissolved in a concentration of 20 ?M in water and in the respective microemulsions E1 to E4.

[0446] Because of the higher absorption of TMPyP in solution, the photosensitizer TMPyP was respectively used in a concentration von 5 ?M.

[0447] Absorption spectra were measured using a Varian Cary BIO UV/VIS/IR spectrometer (Agilent Technologies Inc., Santa Clara, Calif., USA), wherein a 10 mm Hellma quartz cell (SUPRASIL, Type 101-QS, Hellma GmbH & Co. KG, M?hlheim, DE) was used.

[0448] The respective absorption spectra of TMPyP, SA-PN-01a and FL-AS-H-1a in the microemulsions E1 to E4 were almost identical, within the margin of error, to the corresponding absorption spectra of TMPyP, SA-PN-01a and FL-AS-H-1a in water.

[0449] There was no difference between the intensity of the signal, nor were there any modifications to the spectrum.

[0450] D) Determination of Singlet Oxygen Formed Following Irradiation

[0451] The formation of singlet oxygen following irradiation of a photosensitizer-containing microemulsion was determined using time-resolved singlet oxygen luminescence measurements.

[0452] For the relevant measurements, 5 ?M of the respective photosensitizers used were dissolved in water or in the emulsions E1 to E4.

[0453] The time-resolved singlet oxygen luminescence measurements were carried out in accordance with the methods described in S. Y. Egorov et al., 1999.

[0454] A tuneable laser system was used to produce the singlet oxygen (model: NT242-SH/SFG, serial number: PGD048) from EKSPLA (Vilnius, Lettland). A portion of the monochromatic laser beam produced was directed onto a photodiode which acted as a trigger signal for the time-correlated single photon measurement.

[0455] The other part of the laser beam was directed onto a 1 cm thick quartz cell (SUPRASIL, Type 101-QS, Hellma GmbH & Co. KG, M?hlheim, DE), in which the solution to be tested had been disposed.

[0456] The formation of singlet oxygen was detected by direct detection of the time- and spectrally-resolved singlet oxygen luminescence.

[0457] Singlet oxygen luminescence was carried out by means of a nitrogen-cooled photomultiplier (model R5509-42, Hamamatsu Photonics, Hamamatsu, Japan) and a multiscaler (7886S, FAST Corn Tec GmbH, Oberhaching, Germany).

[0458] The singlet oxygen luminescence was detected at a wavelength in the range 1200 nm to 1400 nm using interference filters which were disposed in front of the photomultiplier.

[0459] The time-resolved singlet oxygen spectra are shown in FIGS. 3 to 5 by way of example for the respective photosensitizers TMPyP, SA-PN-01a and FL-AS-H-1a.

[0460] FIG. 3 shows the measured time-resolved singlet oxygen spectra for the photosensitizer TMPyP in a concentration of respectively 5 ?M, in water (w), microemulsion E1 (E1) or microemulsion E2 (E2). FIG. 4 shows the measured time-resolved singlet oxygen spectra for the photosensitizer SA-PN-01a in a concentration of respectively 5 ?M, in water (w), microemulsion E1 (E1) or microemulsion E2 (E2). FIG. 5 shows the measured time-resolved singlet oxygen spectra for the photosensitizer FL-AS-H-1a in a concentration of respectively 5 ?M, in water (w), microemulsion E1 (E1) or microemulsion E2 (E2).

[0461] A summary of the singlet oxygen detection is shown in Table 1.

[0462] Each of the photosensitizers used, TMPyP, SA-PN-01a and FL-AS-H-1a, produced singlet oxygen, following irradiation with electromagnetic radiation. The quantum yield was determined in accordance with the method described in Baier J. et al. (Singlet Oxygen Generation by UVA Light Exposure of Endogenous Photosensitizers, Biophys. J. 91(4), 2006, pages 1452 to 1459; doi. 10.1529/biophysj.106.082388).

[0463] The singlet oxygen formed in the respective microemulsion exhibited a significantly longer half-life compared with water. The microemulsion almost doubled the half-life of the singlet oxygen compared with the half-life for the singlet oxygen formed in water, which was approximately 3.5 ?s.

[0464] The relative yield of singlet oxygen for each photosensitizer with respect to the quantity of singlet oxygen formed in water was calculated from the ratio of the integrals.

[0465] The quantum yield of singlet oxygen in the microemulsions is at least twice as high as in water.

[0466] The formation of singlet oxygen in the microemulsions used was 5-times higher with FL-AS-H-1a and in fact 7 times higher with SA-PN-01a than in water.

TABLE-US-00003 TABLE 1 Results for singlet oxygen measurements for the photosensitizers TMPyP, SA-PN-01a and TMPyP (each 5 ?M) in water, microemulsion E1 (DMS; SDS/1-pentanol (1:2); water) and microemulsion E2 (DMS; SDS/1,2-pentanediol (1:2); water). Decay Relative yield Photo- Formation period with respect sensitizer Solvent time (?s) (?s) Integral to H.sub.2O TMPyP H.sub.2O 1.9 3.8 985 TMPyP E1 1.7 8.9 2874 2.92 TMPyP E2 2.3 7.2 1955 1.98 SA-PN-01a H.sub.2O 2.5 3.2 571 SA-PN-01a E1 1.0 9.2 4413 7.73 SA-PN-01a E2 1.6 7.4 3933 6.89 FL-AS-H-1a H.sub.2O 3.6 3.6 366 FL-AS-H-1a E1 3.0 8.6 1752 4.79 FL-AS-H-1a E2 4.0 6.7 1695 4.63

[0467] In summary, it can be seen that the use of a microemulsion has a positive influence on the photophysics of the photosensitizer used.

[0468] Significantly larger quantities of singlet oxygen were formed in one of the microemulsions used and the light absorption of the respective photosensitizers used in the microemulsion remained essentially unchanged.

[0469] E) Phototoxicity Measurements

[0470] In order to investigate the phototoxicity of the microemulsions in accordance with the invention, a MTT test was used. Assaying cell vitality using a MTT test is based on the reduction of the yellow, water-soluble dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT, Sigma-Aldrich Chemie GmbH, Munich, DE) into a blue-violet 2,3,5-triphenyltetrazolium chloride (formazan) which is insoluble in water. MTT is a dye which can pass through membranes, which is metabolized by mitochondrial dehydrogenases in living cells, which in the end leads to the formation of formazan crystals.

[0471] Formazan crystals can no longer pass through the membranes and accumulate in proliferating undamaged cells. After cell lysis and dissolving the crystals, the dye is then quantified by colorimetric measurement at 550 nm in a multi-well spectrophotometer (ELISA reader). The quantity of formazan formed is determined as the optical density (OD). The measured quantity of formazan is directly proportional to the number of proliferating cells, so that this test is suitable for the measurement of the phototoxicity of the microemulsions used. The measured OD can be assigned a cell count by means of a previously determined calibration curve.

[0472] The concentration of the respective photosensitizers TMPyP, SA-PN-01a, SA-PN-02a, SA-PN-24d, FL-AS-H-1a or FL-AS-H-2 in the microemulsions E1 to E4 was 0 ?M, 10 ?M, 25 ?M, 50 ?M, 100 ?M, 250 ?M and 500 ?M.

[0473] Furthermore, the respective microemulsions E1 to E4 without photosensitizer were used as a control.

[0474] The phototoxicity measurements were carried out on Escherichia coli (E. coli; ATCC Number: 25922) and Staphylococcus aureus (S. aureus; ATCC Number: 25923), as described by Mosmann (1983). (Mosmann T.: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays: J. Immunol. methods. 1983 (65); pages 55-63).

[0475] 25 ?L of a suspension of the bacteria used were grown overnight in M?ller-Hinton liquid medium (Merck KGaA, Darmstadt, Germany) with an optical density of 0.6 at 600 nm were incubated with 25 ?L of the test solution at room temperature for 10 seconds in darkness in a 96-well microtitre plate (Cellstar, Greiner Bio-One, Frickenhausen, Germany).

[0476] Next, the microtitre plate was irradiated for 40 s. For irradiation, the light source Blue V from Waldmann (Villingen-Schwenningen, Germany) was used, which emits light at 380 to 480 nm (emission maximum at approximately 420 nm). The applied power was 20 mW/cm.sup.2.

[0477] For each experiment, three controls were carried out at the same time in order to exclude side effects of the irradiation/photosensitizer (PS) on survival of the bacteria: (i) no PS, only light (=light control), (ii) no light, only PS (=dark control), and (iii) neither light nor PS (=reference control).

[0478] After irradiation had been completed, 75 ?L of a 25% by weight SDS solution was added to each well of the microtitre plate and the bacterial cells were lysed overnight at 37? C. in an incubator.

[0479] Finally, the optical density (OD) was determined with the aid of a microtitre plate photometer (model EAR 400 AT, SLT Laborinstruments Austria, Salzburg, AT).

[0480] After lysis of the cells and dissolution of the crystals, the dye could then be quantified in a multi-well spectrophotometer (ELISA-Reader) by colorimetric measurement at 550 nm.

[0481] The determination of the colony forming units was carried out in accordance with the method published by Miles and Misra (Miles, AA; Misra, SS, Irwin, JO (1938 November). The estimation of the bactericidal power of the blood The Journal of hygiene 38 (6): 732-49). In this regard, serial dilutions from 10.sup.?2 to 10.sup.?9 of the corresponding bacterial suspension were produced. In each case, 3?20 ?L of the corresponding bacterial dilutions were then dropped onto M?ller-Hinton plates and incubated at 37? C. for 24 h. Next, the number of surviving colony forming units (CFU) was determined. All of the experiments were carried out three times.

[0482] E. coli and S. aureus were destroyed by the singlet oxygen formed by the irradiation in a concentration range of 10 ?M to 100 ?M of the photosensitizer TMPyP both in water and in the microemulsions E1 to E4 used.

[0483] A shielding effect occurred at a concentration of more than 100 ?M of the photosensitizer TMPyP in water. TMPyP can absorb 25 to 30 times more light. Thus, the formation of singlet oxygen at high concentrations is more than 100 ?M less and corresponding concentrated aqueous solutions could reduce the quantity of E. Coli and S. Aureus only by 2 log units.

[0484] In contrast, when using TMPyP in one of the microemulsions E1 to E4, significantly less shielding occurred. Thus, the quantity of singlet oxygen formed at high concentrations of TMPyP (more than 100 ?M to 500 ?M) is higher compared with aqueous solutions.

[0485] Corresponding concentrated microemulsions with TMPyP in a concentration of more than 100 ?M to 500 ?M could reduce the quantity of E. coli and S. aureus by only 5 log.sub.10 units.

[0486] The photosensitizers SA-PN-01a, SA-PN-02a and SA-PN-24d were more effective against E. coli and S. aureus when used in a microemulsion than when used in water.

[0487] S. aureus was completely destroyed in water (reduction in quantity following irradiation of more than 6 log.sub.10 units) when SA-PN-01a, SA-PN-02a and SA-PN-24d were used in a concentration in the range 50 to 500 ?M.

[0488] When using SA-PN-01a in one of the microemulsions E1 to E4, even from a concentration of 25 ?M of SA-PN-01a, a reduction in the quantity of E. coli and S. aureus following irradiation of more than 6 log.sub.10 units was obtained.

[0489] Furthermore, a concentration of 10 ?M SA-PN-01a in one of the microemulsions E1 to E4 was sufficient to obtain a reduction in the quantity of E. coli and S. aureus of 3 log 10 units following irradiation.

[0490] FIGS. 6a and 6b show the action of SA-PN-01a in water or SA-PN-01a in microemulsion E2 (E2) on Staphylococcus aureus, by way of example.

[0491] FIG. 6a shows the action of an aqueous solution of the photosensitizer SA-PN-01a in the given concentrations on Staphylococcus aureus following irradiation (hatched bars) with the light source Blue V (irradiation period: 40 s). The applied power was respectively 20 mW/cm.

[0492] As a control, two non-irradiated samples (black bars) were also included, in which Staphylococcus aureus was treated respectively with pure water without SA-PN-01a (concentration: 0 ?M) or SA-PN-01a in water in a concentration of 500 ?M.

[0493] FIG. 6b shows, by way of example, the action of the photosensitizer SA-PN-01a in microemulsion E2 in the concentrations given on Staphylococcus aureus following irradiation (hatched bars) with the light source Blue V (irradiation period: 40 s). The applied power was respectively 20 mW/cm.

[0494] As a control, two non-irradiated samples (black bars) were also included, in which Staphylococcus aureus was treated respectively with microemulsion E2 without SA-PN-01a (concentration: 0 ?M) or SA-PN-01a in microemulsion E2 in a concentration of 500 ?M.

[0495] The measured colony forming units of surviving bacteria are shown in each case using the test in accordance with the method published by Miles and Misra, shown in colony forming units per millilitre (CFU/mL).

[0496] For the photosensitizer FL-AS-H-1a, at a concentration of 10 ?M FL-AS-H-1a in one of the microemulsions E1 to E4, a reduction in the quantity of E. coli and S. aureus of approximately 2 log.sub.10 units was measured.

Example 2

[0497] A) Production of Various Oil-Containing Microemulsions

[0498] In addition, the oil containing microemulsions E5 and E6 were produced.

[0499] The % by weight of the components of the microemulsions E5 to E6 given below are respectively with respect to the total weight of the corresponding microemulsion without photosensitizer.

[0500] Microemulsion E5:

[0501] 66% by weight dodecane

[0502] 29% by weight Lutensol AO7

[0503] 5% by weight water

[0504] Microemulsion E6:

[0505] 66% by weight paraffin oil

[0506] 4% by weight water

[0507] 10% by weight Lutensol AO 7

[0508] 20% by weight Kosteran SQ/O VH

[0509] The surfactant Lutensol AO7 is commercially available from BASF SE (Ludwigshafen, DE).

[0510] Lutensol AO7 is an ethoxylated mixture of fatty acids containing 13 to 15 carbon atoms with an average of 7 ethyl oxide units (PEG 7).

[0511] The surfactant Kosteran SQ/O VH is commercially available from Dr. W. Kolb AG (Hedingen, CH). Kosteran SQ/O VH is a sorbitan-oleic acid ester with an average of 1.5 oleic acid molecules per molecule (sorbitan sesquioleate).

[0512] B) UV/VIS Measurements

[0513] The absorption of the FL-AS-H-1a photosensitizer used in the microemulsions E5 and E6 was determined by recording an absorption spectrum for a wavelength range of 250 nm to 600 nm, as described in Example 1. To this end, the FL-AS-H-2 photosensitizer was dissolved in a concentration of 10 ?M in the microemulsions E5 and E6, as well as in water.

[0514] The absorption spectrum of FL-AS-H-2 in microemulsion E6 did not exhibit any displacement of the spectrum compared with the spectrum measured in water. Only the intensity of the absorption signal was higher than in water or in microemulsion E5.

[0515] Furthermore, an absorption spectrum of FL-AS-H-2 in microemulsions E5 and E6 as well as in water was measured following irradiation with varying doses of light.

[0516] For the irradiation, the light source Blue V from Waldmann, which emits light at 380 to 480 nm (emission maximum at approximately 420 nm) was used. The applied light dose was from 5.5 J to 990 J.

[0517] It was shown that the FL-AS-H-2 photosensitizer was degraded both in water as well as in the microemulsions E5 and E6. The degradation in water occurred significantly faster than in the respective microemulsion E5 or E6.

Example 3

[0518] A) Production of Photosensitizer-Containing Gels

[0519] The following percentages by weight for the components of gels G1 to G3 are respectively with respect to the total weight of the original aqueous solution used.

[0520] Gel G1: (Comparative exampleno surfactant)

TABLE-US-00004 Quantity Component [mL] Carbopol SF-1 (4% by weight aqueous solution) 6.25 Sodium hydroxide (2% by weight aqueous solution) 2 Sodium chloride (10% by weight aqueous solution) 4

[0521] Carbopol Aqua SF-1 polymer, an acrylate copolymer, obtained from Lubrizol Corporation (Wickliffe, Ohio, USA), was used as the gelling agent.

[0522] Gel G2:

TABLE-US-00005 Quantity Component [mL] Carbopol SF-1 (4% by weight aqueous solution) 6.25 Sodium hydroxide (2% by weight aqueous solution) 2 Sodium chloride (20% by weight aqueous solution) 2 Brij 35 (6% by weight aqueous solution) 2

[0523] Brij 35, a polyoxyethylene (23) lauryl ether, obtained from Merck KGaA (Darmstadt, DE), was used as the surfactant.

[0524] Gel G3:

TABLE-US-00006 Quantity Component [mL] Carbopol SF-1 (4% by weight aqueous solution) 6.25 Sodium hydroxide (2% by weight aqueous solution) 2 Sodium chloride (20% by weight aqueous solution) 2 PLANTACARE 818 UP (6% by weight aqueous solution) 2

[0525] PLANTACARE 818 UP, a C8 to C16 fatty alcohol glucoside of D-glucopyranose, obtained from BASF SE (Ludwigshafen, DE), was used as the surfactant.

[0526] According to the manufacturer, the distribution of the lengths of the fatty alcohol portion is as follows:

TABLE-US-00007 C6 max. 0.5% C8 24-30% C10 15-22% C12 37-42% C14 12-18% C16 max. 4%

[0527] Firstly, the aforementioned quantity of a 2% by weight aqueous NaOH solution was added in portions to a corresponding quantity of a 4% by weight aqueous solution of Carbopol Aqua SF-1 in a graduated flask, with stirring. After a clear gel had been formed, the aforementioned quantity of a sodium chloride solution was added in order to adjust the viscosity.

[0528] Next, the respective aforementioned quantity of a 6% by weight aqueous solution of one of the aforementioned surfactants was added dropwise, with stirring.

[0529] The photosensitizer TMPyP used was added to the relevant gel in a final concentration of 100 ?M.

[0530] The gels G1, G2 and G3, respectively with and without photosensitizer TMPyP, were transparent and exhibited pseudo-elastic behaviour.

[0531] Furthermore, the consistency of the gels G2 and G3 did not change after storage for 24 hours at 50? C. as well as at 0? C.

[0532] B) Contact Angle Test

[0533] The wetting of surfaces by the gels which were produced was determined with the aid of the contact angle test.

[0534] For the contact angle test, the gels mentioned above were used, without photosensitizer as well as photosensitizer-containing gels.

[0535] The contact angle test was carried out as described in Example 1, wherein a polyethylene test plate was used as the test surface.

[0536] By way of example, FIG. 7 shows the measured contact angle for the photosensitizer-free gels G2 and G3, in which the relevant quantity of the given surfactant was added. The measured contact angles for the respective photosensitizer-containing gels G2 and G3 were identical.

[0537] The measurements show that, for a proportion of 0.5% by weight with respect to the total weight of the gel, a minimum contact angle and thus a maximum wetting was obtained.

[0538] In order to detach any aggregates of bacteria present, the proportion of the surfactants was then raised to 1.0% by weight with respect to the total weight of the gel.

[0539] C) UV/VIS Measurements

[0540] The absorption of the TMPyP photosensitizer used in the respective gels G1 to G3 as well as in water was determined by recording an absorption spectrum for a wavelength range of 250 nm to 600 nm, as described in Example 1.

[0541] In this regard, the photosensitizer TMPyP was dissolved in a concentration of 10 ?M in the gels G1 to G3 as well as in water.

[0542] The absorption spectrum of TMPyP in gels G1 to G3 did not exhibit any displacement of the spectrum compared with the spectrum measured in water.

[0543] D) Determination of Singlet Oxygen Formed Following Irradiation

[0544] The formation of singlet oxygen following irradiation of a photosensitizer-containing microemulsion was determined using time-resolved singlet oxygen luminescence measurements, as described in Example 1.

[0545] In gels G1, G2 and G3, in the presence of TMPyP (final concentration 10 ?M), the formation of singlet oxygen could be detected following irradiation.

[0546] By way of example, FIG. 8 shows the time-resolved singlet oxygen spectrum for the photosensitizer TMPyP in gel G3. The measured rise time for the signal (t.sub.R) was 2.7 ?s.

[0547] The measured decay time for the signal (t.sub.D) was 7.4 ?s.

[0548] By way of example, FIG. 9 shows the wavelength-resolved singlet oxygen spectrum for the photosensitizer TMPyP in gel G3.

[0549] The distinct peak in the wavelength-resolved spectrum at 1270 nm definitively shows that singlet oxygen is formed by TMPyP in the gel following irradiation.

[0550] The measured decay time for the singlet oxygen signal in the gel (7.4 ?s), compared with the measured decay time for the singlet oxygen signal in water (?3.5 ?s) was significantly longer, so that the singlet oxygen formed in one of the tested gels G1 to G3 was active for longer.

LITERATURE

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