POLYMER-PARTICLE LIGHT-CLEAVABLE CARRIER SYSTEMS FOR PHOTODYNAMIC THERAPY

Abstract

The present invention generally relates to the formation, chemistry and application of biologically active compositions. More particularly, the present invention relates to certain dyes, specifically porphyrin and chlorin derivatives, in combination with inventive polymers, i.e. light-cleavable polymers, that can be used as photosensitizer compositions for a wide range of light irradiation treatments such as photodynamic therapy of cancer, infections and other diseases. The dye derivatives may either be adsorbed on, or incorporated in, or attached to specific polymers, which as well form part of the invention.

Claims

1. A polymer, selected from the group consisting of a polycarbonate based on the formula 10 ##STR00050## wherein: n=0 or 1; x=1-1000; R.sub.1 is: H or an alkyl chain with 1 to 5 carbon atoms; R.sub.2 is selected from the group consisting of: ##STR00051## R.sub.3 is: a divalent radical bridging group formed from an alkyl, aryl, or alkylene group having 1 to 10 carbon atoms; a block copolymer based on the formula 12 ##STR00052## wherein: n=0 or 1; x=1-1000; y=1-1000; R.sub.1 is: H or an alkyl chain with 1 to 5 carbon atoms; R.sub.2 is selected from the groups consisting of: ##STR00053## R.sub.3 is: a divalent radical bridging group formed from an alkyl, aryl, or alkylene group having 1 to 10 carbon atoms; and a polyester based on the formula 13 ##STR00054## wherein: n=0 or 1; x=1-1000; R.sub.1 is: H or an alkyl chain with 1 to 5 carbon atoms; R.sub.2 is selected from the group consisting of ##STR00055## R.sub.3 is: formed from an alkyl, aryl, or alkylene group having 1 to 10 carbon atoms.

2. A pharmaceutical composition, comprising a light-cleavable polymer formed as nanometer particles, wherein the light-cleavable polymer comprises a polymer according to claim 1; a therapeutically effective amount of a photosensitizer; and optionally one or more auxiliary agents, such as a stabilizer.

3. The pharmaceutical composition according to claim 2, characterized in that the photosensitizer is a tetrapyrrole-based photosensitizer.

4. The pharmaceutical composition according to claim 3, characterized in that the photosensitizer is a chlorin or bacteriochlorin derivative according to formula A: ##STR00056## wherein: R.sup.1 is: H or OH; R.sup.2 to R.sup.5 are different or the same and comprise substituents either in the meta- or para-position of the phenyl ring with R.sup.2 to R.sup.5 independently of one another chosen from a group of substituents consisting of: —OH, —COOH, —NH.sub.2, —COOX, —NHX, OX, —NH—Y—COOH, or —CO—Y—NH.sub.2; wherein: X is a polyethyleneglycol-residue with (CH.sub.2CH.sub.2O).sub.nCH.sub.3 with n=1-30 or a carbohydrate moiety; Y is peptides or oligopeptides wherein n=1-30; ring D has a structure selected from: ##STR00057##

5. The pharmaceutical composition according to claim 2, characterized in that the stabilizing agent is selected from the group consisting of polyvinyl alcohol, polysorbate, poloxamer, and albumin.

6. The pharmaceutical composition according to claim 2, characterized in that the photosensitizer is present in an amount of 1 to 500 μg per mg light cleavable polymer.

7. The pharmaceutical composition according to claim 2, characterized in that the photosensitizer is a tetrapyrrolic compound based on the formulas 14, 15, 16, 17, 18 or 19: ##STR00058## ##STR00059## wherein: B is selected from: ##STR00060## X is: NH, O or S; R.sup.1 is a linear or branched alkyl chain with 3-4 carbon atoms and containing at least two hydroxyl moieties; R.sup.2 is: a substituent either in the meta- or para-position of the phenyl ring with R.sup.2 is —OH, —COOH, —COOY, —NHY, OY, —NH—Z—COOH, or —CO—Z—NH.sub.2; wherein: Y is a polyethyleneglycol-residue with (CH.sub.2CH.sub.2O).sub.nCH.sub.3 with n=1-30 or a carbohydrate moiety; and Z is selected from peptides or oligopeptides wherein n=1-30.

8. A method of forming a pharmaceutical composition according to claim 2, comprising the steps of: a) dissolving the light-cleavable polymer alone or in combination with a further polymer in an organic solvent to form a polymer solution; b) optionally dissolving a stabilizer in an aqueous solution to form a stabilizing solution; c) optionally filtering said polymer solution and optionally said stabilizing solution through a filtration unit; d) mixing the optionally filtered polymer solution with an aqueous solution; e) evaporating the organic solvent; f) purifying the formed nanoparticles; and g) adding photosensitizer to the solution formed in step a) or to the purified nanoparticles formed in step f).

9. The method according to claim 8, characterized in that the stabilizing solution includes polyvinylalcohol.

10. The method according to claim 8, characterized in that the composition formed in step g) is freeze dried in the presence of cryoprotective agents.

11. The method according to claim 10, characterized in that the cryoprotective agents are selected from the group consisting of glucose, trehalose, sucrose, sorbitol, mannitol and combinations hereof.

12. The method according to claim 8, characterized in that the organic solvent used in step a) is a water-miscible compound, such as acetone or water immiscible solvents such as dichloromethane.

13. A monomer, usable for forming a polymer according to claim 1, wherein the monomer is selected from the group consisting of a cyclic carbonyl monomer based on the formula 20 ##STR00061## wherein: n=0 or 1 R.sub.1 is: H or an alkyl chain with 1 to 5 carbon atoms R.sub.2 is selected from the group consisting of: ##STR00062## and a diol monomer based on the formula 21 ##STR00063## wherein: n=0 or 1 R.sub.1 is: H or an alkyl chain with 1 to 5 carbon atoms R.sub.2 is selected from the group consisting of: ##STR00064##

14.-15. (canceled)

16. A method of treating a patient with photodynamic therapy comprising administering light and a photosensitizer to the patient, wherein the photosensitizer comprises the pharmaceutical formulation of claim 2.

17. The method of claim 16, wherein the photodynamic therapy treats a condition selected from the group consisting of tumors and other neoplastic diseases, dermatological disorders, opthalmological disorders urological disorders and arthritis and similar inflammatory diseases.

Description

[0117] In the drawings:

[0118] FIG. 1 shows (a).sup.1H NMR spectrum of PC01 in DMSO-d.sub.6 before irradiation and (b).sup.1H NMR spectrum of PC01 in DMSO-d.sub.6 after irradiation with UV light (320-480 nm, 0.603 W/cm.sup.2) for 15 min;

[0119] FIG. 2 shows APC traces of PC01 in THF after irradiation with UV light (320-480 nm, 0.603 W/cm.sup.2) for 0, 1, 5, 15 and 30 min;

[0120] FIG. 3 shows UV-VIS spectra of PC01 in DCM after irradiation with UV light (320-480 nm, 0.603 W/cm.sup.2) for 0, 10, 20, 30, 45, 60, 75, 90, 120 and 180 s;

[0121] FIG. 4 shows the light-induced desintegration of nanoparticles measured by variation of the PCS count rate (mean±S.D.; n=3) of different nanoparticle suspensions over 24 h after irradiation with light of a wavelength of 365 nm for 5 min;

[0122] FIG. 5 depicts the amount of the released photosensitizer mTHPC in dependence of particle light irradiation of mTHPC-PC-PLGA-NP100 (A), mTHPC-PC-(PLA-PEG)-PLGA-NP100 (B), and mTHPC-(PC-PEG)-PLGA-NP100 (C) over 24 h, and the release of the irradiated nanoparticles compared to each other and the non-light-cleavable control mTHPC-PLGA-NP100 (D);

[0123] FIG. 6 shows the light-induced variation of the count rate (mean±S.D.; n=3) of different nanoparticle suspensions over 24 h after irradiation with light of a wavelength of 365 nm for 5 min;

[0124] FIG. 7 shows the amount of the released PS mTHPC in dependence of particle light irradiation of mTHPC-(PC-PEG).sub.100%-NP100 and mTHPC-(PC-PEG).sub.50%-PLGA.sub.50%-NP100, and mTHPC-(PC-PEG)-PLGA-NP100 over 24 h;

[0125] FIG. 8 shows the biological safety of unloaded light-cleavable nanoparticles before (A) and after (B) 5 min of irradiation with light of a wavelength of 365 nm measured by WST-1 cell viability assay;

[0126] FIG. 9 depicts the intracellular uptake of mTHPC-loaded light-cleavable nanoparticles compared to free photosensitizer mTHPC and non-light cleavable mTHPP-PLGA-NP100 by HT-29-MTX cells over 24 h quantified via HPLC-FLD analysis;

[0127] FIG. 10 shows the cellular association of mTHPC-loaded light-cleavable nanoparticles compared to mTHPC and non-light cleavable mTHPP-PLGA-NP100 by HT-29-MTX cells over 24 h quantified via red fluorescence of the photosensitizer using IncuCyte® Live-Cell Imaging;

[0128] FIG. 11 depicts dark toxicity (A) and phototoxicity (B) of mTHPC-loaded light-cleavable nanoparticles. mTHPC and non-light-cleavable mTHPP-PLGA-NP100 served as control; and

[0129] FIG. 12 shows the phototoxicity of mTHPC-(PC-PEG).sub.50%-PLGA.sub.50%-NP100 after irradiation with light of a wavelength of 365 nm and 652 nm, separately and in combination.

[0130] The following examples are presented to provide those of ordinary skill in the art with a full and illustrative disclosure and description of how to make the biologically active compositions of the invention and show their photodynamic activity and are not intended to limit the scope of what the inventor regards as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature etc.), but some experimental errors and deviations should be accounted for. Also, best measures have been taken to name the compounds with their systematic IUPAC name, nevertheless the basic reference are the given structural formulas based on the experimental spectroscopic data.

EXAMPLES

Materials for Monomer and Polymer Synthesis and Characterization

[0131] All reagents were used as purchased from commercial suppliers. Benzyl alcohol (BnOH) (99%, Acros Organics) was distilled over calcium hydride and stored under Ar atmosphere. Dichloromethane (DCM) (98%, Stockmeier Chemie) was dried over CaCl.sub.2 then distilled over calcium hydride. Tetrahydrofuran (THF) (98%, Stockmeier Chemie) was dried over KOH then distilled over calcium hydride. 1,3-dioxan-2-one (TMC) (99%, Shanghai Worldyang Chemical Co.) was purified by column chromatography with ethyl acetate/n-hexane (3:1).

[0132] .sup.1H and .sup.13C nuclear magnetic resonance (NMR) spectra were recorded using Bruker AV 500 spectrometer at 500 MHz and 125 MHz, respectively. Chloroform-d (CDCl.sub.3-d, 99.8 D %) or dimethylsulfoxide-d.sub.6 (DMSO-d.sub.6, 99.8 D %) were used as solvent for NMR measurements. ESI-ToF-mass spectra were measured on a SYNAPT G2 HDMS™ from Waters. The mass spectrometric parameters were the following: capillary voltage: 3 kV; sampling cone voltage: 45 V; extraction cone voltage: 1.7 V; cone gas flow: 30 L/h; source temperature: 120° C.; desolvation gas flow: 650 L/h; desolvation temperature: 350° C.; helium cell gas flow: 180 mL/min. The sample was dissolved in acetonitrile (2 g/L) and then mixed with NaI 0.1 g/L in methanol and methanol in the ratio of 5:5:990. Data were obtained with Mass Lynx 4.1. Light degradation experiments were performed using an OmniCure S1500 Curing System from Lumen Dynamics with a power of 0.607 W/cm.sup.2 (320 to 480 nm). UV/VIS spectra were recorded on Specord 50 PLUS UV-VIS spectrophotometer from Analytik Jena using Aspect UV 1.1 software. The molar masses and dispersities (Ð.sub.M) were analyzed employing an advanced polymer chromatography (APC) system equipped with two consecutive columns (Acquity APC XT columns filled with polyethoxysilane with a defined porosity of 125 Å and 45 Å, respectively) and an Acquity APC RI-detector. The system was operated at a flow rate of 0.7 mL/min with THF/DMF (v/v=80/20) as solvent. Poly(methyl methacrylate) (PMMA) standards were used for calibration.

Example 1a

Synthesis of Cyclic Carbonyl Monomer with Light-Cleavable Protecting Group

[0133] ##STR00024##

Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanol (1)

[0134] 1 was synthesized according to the literature, see Z. Jia, D. E. Lonsdale, J. Kulis, and M. J. Monteiro, Construction of a 3-Miktoarm Star from Cyclic Polymers, ACS Macro Lett., 2012, 1, 780-783.

[0135] In a round bottom flask 80 g 2-(hydroxymethyl)-2-methylpropane-1,3-diol (0.67 mol), 320 mL dry acetone, and 100 mg p-toluenesulfonic acid monohydrate (TsOH.H.sub.2O) (0.53 mmol) were stirred overnight at room temperature. The reaction was quenched by addition of 150 μL triethylamine (TEA) (1.1 mmol). The organic solvent was removed under reduced pressure and the residue was taken up in 200 mL DCM. The precipitate was filtered off, the DCM was removed under reduced pressure, and the residue was distilled at 0.2 mbar to achieve 86 g of a slightly viscous liquid. Yield: 81%

##STR00025##

[0136] .sup.1H-NMR (500 MHz, CDCl.sub.3): δ (ppm)=0.81 (s, 3H, .sup.5CH.sub.3), 1.37 (s, 3H, .sup.1CH.sub.3), 1.42 (s, 3H, .sup.1CH.sub.3), 2.47 (b, 1H, .sup.7OH), 3.58 (m, 2H, .sup.3CH.sub.2), 3.64 (m, 2H, .sup.3CH.sub.2), 3.66 (s, 2H, .sup.6CH.sub.2). .sup.13C-NMR (125 MHz, CDCl.sub.3): δ (ppm)=17.70 (1C, .sup.5CH.sub.3), 20.33 (1C, .sup.1CH.sub.3), 27.33 (1C, .sup.1CH.sub.3), 34.87 (1C, .sup.4C.sub.q), 66.01 (1C, .sup.6CH.sub.2), 66.46 (2C, .sup.3CH.sub.2), 98.10 (1C, .sup.2C.sub.q).

Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methyl-4-methylbenzenesulfonate (2)

[0137] 2 was synthesized according to the literature. see V. W. Gash, Dienoalkyl ethers. General synthesis of the symmetrical ethers, J. Org. Chem., 1972, 37, 2197-2201.

[0138] A solution of 108 g 4-toluenesulfonyl chloride (TsCl) (0.57 mol) in 140 mL pyridine was dropped over 30 min to a solution of 86.26 g of 1 (0.54 mol) in 270 mL pyridine. The resulting solution was stirred for 40 min at 100° C., cooled and poured in an excess of ice water. The precipitate was collected, washed with water and dried in vacuo to yield 140 g of an off-white solid. The crude product was used without further purification in the next step. Yield: 83%

##STR00026##

[0139] .sup.1H-NMR (500 MHz, CDCl.sub.3): δ (ppm)=0.81 (s, 3H, .sup.5CH.sub.3), 1.22 (s, 3H, .sup.1CH.sub.3), 1.36 (s, 3H, .sup.1CH.sub.3), 2.43 (s, 3H, .sup.13CH.sub.3), 3.54 (s, 4H, .sup.3CH.sub.2), 4.07 (s, 2H, .sup.6CH.sub.2), 7.34 (d, .sup.3J.sub.HH=8.3 Hz, 2H, .sup.9,11CH), 7.79 (d, .sup.3J.sub.HH=8.3 Hz, 2H, .sup.8,10CH).

[0140] .sup.13C-NMR (125 MHz, CDCl.sub.3): δ (ppm)=17.34 (1C, .sup.5CH.sub.3), 19.50 (1C, .sup.1CH.sub.3), 21.72 (1C, .sup.13CH.sub.3), 27.71 (1C, .sup.1CH.sub.3), 34.06 (1C, .sup.4C.sub.q), 65.65 (2C, .sup.3CH.sub.2), 72.61 (1C, .sup.6CH.sub.2), 98.18 (1C, .sup.2C.sub.q), 128.18 (2C, .sup.8,10CH), 129.94 (2C, .sup.9,11CH), 132.85 (1C, .sup.7C.sub.q), 144.87 (1C, .sup.12C.sub.q).

Synthesis of 5-(azidomethyl)-2,2,5-trimethyl-1,3-dioxane (3)

[0141] 3 was synthesized according to the literature, see S. T. Liu and C. Y. Liu, Synthesis of amino-containing phosphines. The use of iminophosphorane as a protecting group for primary amines, J. Org. Chem., 1992, 57, 6079-6080.

[0142] 37.72 g of 2 (120 mmol), 23.41 g sodium azide (360 mmol), 20 mL water and 200 mL dimethylformamide (DMF) were stirred at 100° C. for 68 h. The mixture was poured in water and extracted 4× with 130 mL diethyl ether. The organic phase was dried over anhydrous MgSO.sub.4 and removed under reduced pressure. The residue was purified by column chromatography with 100 g silica gel and ethyl acetate/n-hexane (1:4) to give 20.9 g of a colorless liquid. Yield: 94%

##STR00027##

[0143] .sup.1H-NMR (500 MHz, CDCl.sub.3): δ (ppm)=0.81 (s, 3H, .sup.5CH.sub.3), 1.38 (s, 3H, .sup.1CH.sub.3), 1.41 (s, 3H, .sup.1CH.sub.3), 3.50 (s, 2H, .sup.6CH.sub.2), 3.57 (m, 4H, .sup.3CH.sub.2).

[0144] .sup.13C-NMR (125 MHz, CDCl.sub.3): δ (ppm)=18.13 (1C, .sup.5CH.sub.3), 19.81 (1C, .sup.1CH.sub.3), 27.73 (1C, .sup.1CH.sub.3), 34.55 (1C, .sup.4C.sub.q), 56.14 (1C, .sup.6CH.sub.2), 66.78 (2C, .sup.3CH.sub.2), 98.22 (1C, .sup.2C.sub.q).

Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanamine (4)

[0145] 4 was synthesized similar to the literature, see A. D. Ure, I. A. Lázaro, M. Cottera and A. R. McDonald, Synthesis and characterisation of a mesocyclic tripodal triamine ligand Org. Biomol. Chem., 2016, 14, 483-494.

[0146] 9.26 g of 3 (50 mmol) and 12.6 g ammonium formate (200 mmol) were dissolved in 130 mL dry methanol. The solution was purged with Ar for 20 min. The freshly degassed mixture was held under Ar atmosphere while 0.9 g Pd/C (10%) was added. The reaction mixture was stirred at room temperature in the opened reaction vessel. After a few minutes the solution became slightly warm and a large gas formation was noticed. From now the reaction mixture was stirred at room temperature for further 4.5 h. Pd/C was removed by a pad of Celite and the solvent was removed under reduced pressure. The residue was taken up in 25 mL water and the resulting solution was extracted 3× with 50 mL diethyl ether. The aqueous phase was basified with 25 g KOH and extracted 5× with 25 mL MeCN. The organic phase was dried over anhydrous MgSO.sub.4 and removed under reduced pressure to yield 5.91 g of a colorless liquid. Yield: 74%

##STR00028##

[0147] .sup.1H-NMR (500 MHz, CDCl.sub.3): δ (ppm)=0.78 (s, 3H, .sup.5CH.sub.3), 1.21 (b, 2H, .sup.7NH.sub.2), 1.36 (s, 3H, .sup.1CH.sub.3), 1.40 (s, 3H, .sup.1CH.sub.3), 2.76 (s, 2H, .sup.6CH.sub.2), 3.58 (m, 4H, .sup.3CH.sub.2).

[0148] .sup.13C-NMR (125 MHz, CDCl.sub.3): δ (ppm)=18.03 (1C, .sup.5CH.sub.3), 21.12 (1C, .sup.1CH.sub.3), 26.61 (1C, .sup.1CH.sub.3), 34.34 (1C, .sup.4C.sub.q), 46.41 (1C, .sup.6CH.sub.2), 67.34 (2C, .sup.3CH.sub.2), 97.96 (1C, .sup.2C.sub.q).

Synthesis of 4,5-dimethoxy-2-nitrobenzyl ((2,2,5-trimethyl-1,3-dioxan-5-yl)methyl)carbamate (5)

[0149] A mixture of 2.954 g of 4 (18.55 mmol), 5.965 g of 8 (15.77 mmol), 4.9 mL TEA (35.16 mmol), and 120 mL dry MeCN was stirred at room temperature overnight. After adding 100 mL DCM the organic phase was washed 2× with 100 mL 0.1 M Na.sub.2CO.sub.3 solution and 2× with 100 mL 0.3 M NaCl solution. The organic phase was dried over anhydrous MgSO.sub.4 and removed under reduced pressure to yield 7.68 g of a viscous yellow liquid. The crude product was used without further purification in the next step. Yield: 84%

##STR00029##

[0150] .sup.1H-NMR (500 MHz, CDCl.sub.3): δ (ppm)=0.81 (s, 3H, .sup.5CH.sub.3), 1.40 (s, 3H, .sup.1CH.sub.3), 1.43 (s, 3H, .sup.1CH.sub.3), 3.40 (d, .sup.3J.sub.HH=6.1 Hz, 2H, .sup.6CH.sub.2), 3.60 (m, 4H, .sup.3CH.sub.2), 3.95 (s, 3H, O.sup.16CH.sub.3), 3.97 (s, 3H, O.sup.17CH.sub.3), 5.22 (b, 1H, .sup.7NH), 5.52 (s, 2H, .sup.9CH.sub.2), 7.01 (s, 1H, .sup.13CH), 7.71 (s, 1H, 12CH).

Synthesis of 4,5-dimethoxy-2-nitrobenzyl (3-hydroxy-2-(hydroxymethyl)-2-methyl-propyl)carbamate (6)

[0151] To a mixture of 5.465 g of 5 (13.70 mmol), 25 mL THF and 25 mL 1 N HCl was added. The suspension was stirred at room temperature overnight. After addition of 100 mL DCM the mixture was washed 3× with 100 mL 0.1 M Na.sub.2CO.sub.3 solution and 2× with 100 mL 0.3 M NaCl solution. The organic phase was dried over anhydrous MgSO.sub.4 and evaporated under reduced pressure.

[0152] Purification method 1: diethyl ether was added to the crude product and stirred/triturated until a fine precipitate appears. A large excess of diethyl ether was reduced before the precipitate was collected by filtration and dried in vacuo to achieve 4.326 g of a yellow solid. Yield: 88% Purification method 2: The crude product was purified by column chromatography with silica gel and ethyl acetate to give 4.410 g of a yellow solid. Yield: 90%

##STR00030##

[0153] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.73 (s, 3H, .sup.4CH.sub.3), 2.98 (d, .sup.3J.sub.HH=6.3 Hz, 2H, .sup.5CH.sub.2), 3.21 (d, .sup.3J.sub.HH=5.5 Hz, 4H, .sup.2CH.sub.2), 3.87 (s, 3H, O.sup.15CH.sub.3), 3.91 (s, 3H, O.sup.16CH.sub.3), 4.32 (t, .sup.3J.sub.HH=5.5 Hz, 2H, .sup.1OH), 5.34 (s, 2H, .sup.8CH.sub.2), 7.19 (s, 1H, .sup.12CH), 7.22 (t, .sup.3J.sub.HH=6.2 Hz, 1H, .sup.6NH), 7.70 (s, 1H, .sup.11CH).

[0154] .sup.13C-NMR (125 MHz, DMSO-d.sub.6): δ (ppm)=17.08 (1C, .sup.4CH.sub.3), 41.36 (1C, .sup.3C.sub.q), 44.29 (1C, .sup.5CH.sub.2), 56.22 (1C, O.sup.15CH.sub.3), 56.32 (1C, O.sup.16CH.sub.3), 62.44 (1C, .sup.8CH.sub.2), 64.55 (2C, .sup.2CH.sub.2), 108.32 (1C, .sup.11CH), 110.64 (1C, .sup.12CH), 128.11 (1C, .sup.9C.sub.q), 139.43 (1C, .sup.10C.sub.q), 147.85 (1C, .sup.13C.sub.q), 153.48 (1C, .sup.14C.sub.q), 156.55 (1C, .sup.7C.sub.q).

[0155] ESI-ToF-MS (m/z): [M+Na].sup.+ calculated for C.sub.15H.sub.22N.sub.2O.sub.8, 381.1274. found, 381.1271.

Synthesis of 4,5-dimethoxy-2-nitrobenzyl ((5-methyl-2-oxo-1,3-dioxan-5-yl)methyl)-carbamate (7)

[0156] 4.132 g of 6 (11.53 mmol) was suspended in 30 mL anhydrous THF under Ar atmosphere and cooled to 0° C. Subsequently, 4.22 mL TEA (30.45 mmol) and 2.55 mL ethyl chloroformate (26.65 mmol) were added and the suspension was stirred at room temperature for 24 h. The precipitate was collected, washed with 100 mL THF, stirred in 100 mL dest. water for 30 min, filtered off, washed 2× with 100 mL water and 3× with 100 mL diethyl ether, and dried in vacuo to afford 4.011 g of an off-white solid. Yield: 73%

##STR00031##

[0157] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.93 (s, 3H, .sup.4CH.sub.3), 3.13 (d, .sup.3J.sub.HH=6.4 Hz, 2H, .sup.5CH.sub.2), 3.87 (s, 3H, O.sup.15CH.sub.3), 3.91 (s, 3H, O.sup.16CH.sub.3), 4.12 (d, .sup.2J.sub.HH=10.6 Hz, 2H, .sup.2CH.sub.2), 4.22 (d, .sup.2J.sub.HH=10.6 Hz, 2H, .sup.2CH.sub.2), 5.35 (s, 2H, .sup.8CH.sub.2), 7.20 (s, 1H, .sup.12CH), 7.67 (t, .sup.3J.sub.HH=6.3 Hz, 1H, .sup.6NH), 7.70 (s, 1H, .sup.11CH).

[0158] .sup.13C-NMR (125 MHz, DMSO-d.sub.6): δ (ppm)=16.70 (1C, .sup.4CH.sub.3), 32.70 (1C, .sup.3C.sub.q), 43.12 (1C, .sup.5CH.sub.2), 56.23 (1C, O.sup.15CH.sub.3), 56.35 (1C, O.sup.16CH.sub.3), 62.74 (1C, .sup.8CH.sub.2), 73.90 (2C, .sup.2CH.sub.2), 108.35 (1C, .sup.11CH), 111.01 (1C, .sup.12CH), 127.60 (1C, .sup.9C.sub.q), 139.60 (1C, .sup.10C.sub.q), 147.65 (1C, .sup.1C.sub.q), 147.95 (1C, .sup.13C.sub.q), 153.39 (1C, .sup.14C.sub.q), 156.53 (1C, .sup.7C.sub.q).

[0159] ESI-ToF-MS (m/z): [M+Na].sup.+ calculated for C.sub.16H.sub.20N.sub.2O.sub.9, 407.1066. found, 407.1067.

Synthesis of 4,5-dimethoxy-2-nitrobenzyl (4-nitrophenyl) carbonate (8)

[0160] ##STR00032##

[0161] 8 was synthesized according to the literature, see N. Fomina, C. McFearin, M. Sermsakdi, O. Edigin and A. Almutairi, UV and Near-IR Triggered Release from Polymeric Nanoparticles, J. Am. Chem. Soc., 2010, 132, 9540-9542.

[0162] To a mixture of 8.80 g 4,5-dimethoxy-2-nitrobenzyl alcohol (41.28 mmol), 15.55 g 4-nitrophenyl chloroformate (77.15 mmol), and 90 mL DCM was added dropwise 15 mL diisopropylethylamine (DIPEA) (86.11 mmol) under Ar atmosphere. The solution was stirred at room temperature for 1.5 h and diluted with 90 mL DCM. After stirring at room temperature overnight the solvent was removed under reduced pressure. The resulting crude product was heated at 85° C. in 290 mL ethanol for 45 min until a fine precipitate was formed. The precipitate was collected and dried in vacuo to afford 14.16 g of a yellowish solid. Yield: 91%

##STR00033##

[0163] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=3.89 (s, 3H, O.sup.13CH.sub.3), 3.92 (s, 3H, O.sup.14CH.sub.3), 5.61 (s, 2H, .sup.6CH.sub.2), 7.26 (s, 1H, .sup.11CH), 7.59 (d, .sup.3J.sub.HH=9.1 Hz, 2H, .sup.3CH), 7.73 (s, 1H, .sup.9CH), 8.33 (d, .sup.3J.sub.HH=9.1 Hz, 2H, .sup.2CH).

[0164] .sup.13C-NMR (125 MHz, DMSO-d.sub.6): δ (ppm)=56.14 (1C, O.sup.13CH.sub.3), 56.32 (1C, O.sup.14CH.sub.3), 67.22 (1C, .sup.6CH.sub.2), 108.30 (1C, .sup.9CH), 112.00 (1C, .sup.11CH), 122.57 (2C, .sup.3CH), 124.37 (1C, .sup.7C.sub.q), 125.45 (2C, .sup.2CH), 139.04 (1C, .sup.8C.sub.q), 145.25 (1C, .sup.1C.sub.q), 148.40 (1C, .sup.12C.sub.q), 151.62 (1C, .sup.5C.sub.q), 153.17 (1C, .sup.10C.sub.q), 155.17 (1C, .sup.4C.sub.q).

Example 1b

Synthesis of Light-Sensitive Polycarbonate (PC01) by Ring-Opening Polymerization of 1,3-dioxan-2-one (TMC) and 4,5-dimethoxy-2-nitrobenzyl ((5-methyl-2-oxo-1,3-dioxan-5-yl)methyl)carbamate (7)

[0165] ##STR00034##

[0166] In a Schlenk tube, 0.461 g of 7 (1.20 mmol), 0.123 g TMC (1.20 mmol) and 4.16 μL BnOH (0.04 mmol) were suspended in 2 mL dry 1,4-dioxane under Ar atmosphere. After adding 9.06 μL DBU (0.06 mmol) the reaction mixture was stirred at 80° C. for 24 h and then quenched by adding 10 mg benzoic acid (0.07 mmol). The polymer was purified by precipitation into diethyl ether and dried in vacuo to give a yellow solid. Yield: 73%, M.sub.w=6500 g/mol, ÐM=1.47

##STR00035##

[0167] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.70-1.00 (m, 3H, .sup.4CH.sub.3), 1.80-2.00 (m, 2H, .sup.2CH.sub.2), 2.90-3.10 (m, 2H, .sup.5CH.sub.2), 3.75-4.00 (m, 3H, O.sup.10CH.sub.3; 3 H, O.sup.11CH.sub.3; 4H, .sup.3CH.sub.2), 4.00-4.25 (m, 4H, .sup.1CH.sub.2), 5.11 (s, 2H, .sup.bCH.sub.2), 5.25-5.40 (m, 2H, .sup.7CH.sub.2), 7.00-7.85 (m, 1H, .sup.6NH; 2H, .sup.8,9CH; 5H, .sup.aCH).

Example 1c

Synthesis of Light-Sensitive Sensitive PEGylated Polycarbonate (PC01-PEG) by Ring-Opening Polymerization of 1,3-dioxan-2-one (TMC) and 4,5-dimethoxy-2-nitrobenzyl ((5-methyl-2-oxo-1,3-dioxan-5-yl)methyl)carbamate (7) Using Poly(Ethylene Glycol) Methyl Ether (PEG) as Initiator

[0168] ##STR00036##

[0169] In a Schlenk tube 0.231 g of 7 (0.60 mmol), 0.061 g TMC (0.60 mmol) and 0.040 g PEG 2000 (0.02 mmol) were suspended in 2 mL dry 1,4-dioxane under Ar atmosphere. After adding 4.53 μL DBU (0.03 mmol) the reaction mixture was stirred at 80° C. for 24 h and then quenched by adding 5 mg benzoic acid (0.035 mmol). The polymer was purified by precipitation into diethyl ether and dried in vacuo to give a yellow solid. Yield: 72%, M.sub.w=5000 g/mol, Ð.sub.M=1.61

##STR00037##

[0170] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.70-1.00 (m, 3H, .sup.4CH.sub.3), 1.80-2.00 (m, 2H, .sup.2CH.sub.2), 2.90-3.10 (m, 2H, .sup.5CH.sub.2), 3.35-3.60 (m, 4H, .sup.aCH.sub.2; 3 H, O.sup.bCH.sub.3), 3.75-4.00 (m, 3H, O.sup.10CH.sub.3; 3 H, O.sup.11CH.sub.3; 4 H, .sup.3CH.sub.2), 4.00-4.25 (m, 4H, .sup.1CH.sub.2), 5.25-5.40 (m, 2H, .sup.7CH.sub.2), 7.00-7.85 (m, 1H, .sup.6NH; 2H, .sup.8,9CH).

Example 1d

Synthesis of Light-Sensitive Polycarbonate (PC02) by Polycondensation of 4,5-dimethoxy-2-nitrobenzyl (3-hydroxy-2-(hydroxymethyl)-2-methylpropyl)-carbamate (6) and a Diol with Diphenylcarbonate (DPC)

[0171] ##STR00038##

[0172] In a Schlenk flask 0.250 g of 6 (0.698 mmol), diol (0.698 mmol), 0.329 g of DPC (1.536 mmol) and 0.3 mg lithiumacetylacetonate (LiAcac) (0.0028 mmol) were added under Ar atmosphere. The used diols are listed in TABLE 1.

TABLE-US-00001 TABLE 1 Results of light degradable PCO2 synthesis M.sub.w Code Diols (g/mol) .sub.M.sup.1 Yields PCO2-BD 1,4-Butanediol (BD) 8,100 2.02 77% PCO2-PD 1,5-Pentanediol (PD) 9,300 2.27 77% PCO2-HD 1,6-Hexanediol (HD) 9,500 1.86 81% PCO2-CDM 1,4-Cyclohexanedimethanol (CDM) 6,600 1.58 70% PCO2-BDM 1,4-Benzenedimethanol (BDM) 7,200 1.66 74% .sup.1Determined using APC in THF/DMF (8/2) with PMMA standards, M.sub.w, being the molecular weight and .sub.M.sup.1 being the polydispersity index, PDI, of the polymer.

[0173] The reaction mixture was stirred at 120° C. for 1 h and then at 100° C. for an additional 2 h under reduced pressure. The mixture was then cooled to room temperature and dissolved in DCM. The polymer was purified by precipitation into diethyl ether and dried in vacuo to give a yellow solid. Yield: 70-81%, polymer characteristics were shown in TABLE 1.

##STR00039##

[0174] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.70-1.00 (m, 3H, .sup.4CH.sub.3), 1.50-1.80 (m, 4H, .sup.2CH.sub.2), 2.90-3.10 (m, 2H, .sup.5CH.sub.2), 3.75-4.00 (m, 3H, O.sup.10CH.sub.3; 3H, O.sup.11CH.sub.3; 4H, .sup.3CH.sub.2), 4.00-4.25 (m, 4H, .sup.1CH.sub.2), 5.25-5.40 (m, 2H, .sup.7CH.sub.2), 7.00-7.85 (m, 1H, .sup.6NH; 2H, .sup.8,9CH).

##STR00040##

[0175] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.70-1.00 (m, 3H, .sup.4CH.sub.3), 1.25-1.80 (m, 6H, .sup.2CH.sub.2), 2.90-3.20 (m, 2H, .sup.5CH.sub.2), 3.75-4.00 (m, 3H, O.sup.10CH.sub.3; 3H, O.sup.11CH.sub.3; 4H, .sup.3CH.sub.2), 4.00-4.25 (m, 4H, .sup.1CH.sub.2), 5.25-5.40 (m, 2H, .sup.7CH.sub.2), 7.00-7.80 (m, 1H, .sup.6NH; 2H, .sup.8,9CH).

##STR00041##

[0176] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.70-1.00 (m, 3H, .sup.4CH.sub.3), 1.25-1.80 (m, 8H, .sup.2CH.sub.2), 2.90-3.20 (m, 2H, .sup.5CH.sub.2), 3.75-4.00 (m, 3H, O.sup.10CH.sub.3; 3H, O.sup.11CH.sub.3; 4H, .sup.3CH.sub.2), 4.00-4.25 (m, 4H, .sup.1CH.sub.2), 5.25-5.40 (m, 2H, .sup.7CH.sub.2), 7.00-7.80 (m, 1H, .sup.6NH; 2H, .sup.8,9CH).

##STR00042##

[0177] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.75-1.85 (m, 3H, .sup.4CH.sub.3; 10H, .sup.2CH.sub.2), 2.90-3.20 (m, 2H, .sup.5CH.sub.2), 3.70-4.20 (m, 3H, O.sup.10CH.sub.3; 3H, O.sup.11CH.sub.3; 4H, .sup.3CH.sub.2; 4H, .sup.1CH.sub.2), 5.25-5.40 (m, 2H, .sup.7CH.sub.2), 7.00-7.80 (m, 1H, .sup.6NH; 2H, .sup.8,9CH).

##STR00043##

[0178] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=0.70-1.00 (m, 3H, .sup.4CH.sub.3), 2.90-3.20 (m, 2H, .sup.5CH.sub.2), 3.75-4.10 (m, 3H, O.sup.10CH.sub.3; 3H, O.sup.11CH.sub.3; 4H, .sup.3CH.sub.2), 4.90-5.25 (m, 4H, .sup.1CH.sub.2), 5.25-5.50 (m, 2H, .sup.7CH.sub.2), 7.00-7.80 (m, 4H, .sup.2CH; 1H, .sup.6NH; 2H, .sup.8,9CH).

Example 1e

Synthesis of 4,5-dimethoxy-2-nitrobenzyl (1,3-dihydroxypropan-2-yl)carbamate (9)

[0179] ##STR00044##

[0180] In a round bottom flask 2.94 g serinol (27.75 mmol), 7.00 g of 8 (18.50 mmol), 9.66 mL TEA (69.38 mmol) were suspended in 140 mL dry MeCN. The reaction mixture was stirred at room temperature overnight. The precipitate was collected by filtration, washed with 150 mL DCM and dried in vacuo to achieve 3.97 g of an off-white solid. Yield: 65%

##STR00045##

[0181] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=3.30-3.60 (m, 4H, .sup.2CH.sub.2; 1H, .sup.3CH), 3.87 (s, 3H, O.sup.14CH.sub.3), 3.91 (s, 3H, O.sup.13CH.sub.3), 4.59 (t, .sup.3J.sub.HH=5.4 Hz, 2H, .sup.1OH), 5.33 (s, 2H, .sup.6CH.sub.2), 7.14 (d, .sup.3J.sub.HH=8.0 Hz, 1H, .sup.4NH), 7.21 (s, 1H, .sup.8CH), 7.69 (s, 1H, .sup.11CH).

[0182] .sup.13C-NMR (125 MHz, DMSO-d.sub.6): δ (ppm)=55.49 (1C, .sup.3CH), 56.55 (1C, O.sup.14CH.sub.3), 56.73 (1C, O.sup.13CH.sub.3), 60.96 (2C, .sup.2CH.sub.2), 62.69 (1C, .sup.6CH.sub.2), 108.58 (1C, .sup.11CH), 110.73 (1C, .sup.8CH), 128.76 (1C, .sup.7C.sub.q), 139.53 (1C, .sup.12C.sub.q), 148.09 (1C, .sup.9C.sub.q), 153.96 (1C, .sup.10C.sub.q), 156.00 (1C, .sup.5C.sub.q).

[0183] ESI-ToF-MS (m/z): [M+Na].sup.+ calculated for C.sub.13H.sub.18N.sub.2O.sub.8, 353.0961. found, 353.0959.

Example 1f

Synthesis of Light-Sensitive Polycarbonate (PC03) by Polycondensation of 4,5-dimethoxy-2-nitrobenzyl (1,3-dihydroxypropan-2-yl)carbamate (9) and 1,4-butanediol (BD) with Triphosgene

[0184] ##STR00046##

[0185] In a Schlenk flask 0.180 g triphosgene (0.607 mmol) was dissolved in 0.6 mL DCM under Ar atmosphere. A solution of 0.300 g of 9 (0.909 mmol) and 0.086 g BD (0.909 mmol) in 0.9 mL pyridine (11 mmol) was added dropwise over 90 min. After complete addition, the mixture was allowed to stir for an additional 1 h. The DCM was removed under reduced pressure. The polymer was purified by precipitation into water/MeOH (v/v=1/1) mixture and dried in vacuo to give a yellow solid. Yield: 63%, M.sub.w=5300 g/mol, Ð.sub.M=1.57

##STR00047##

[0186] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=1.50-1.80 (m, 4H, .sup.2CH.sub.2), 3.50-4.30 (m, 4H, .sup.1CH.sub.2; 4H, .sup.3CH.sub.2; 1H, .sup.4CH; 3H, O.sup.9CH.sub.3; 3H, O.sup.10CH.sub.3), 5.20-5.40 (m, 2H, .sup.6CH.sub.2), 7.00-7.25 (m, 1H, .sup.7CH), 7.50-7.90 (m, 1H, .sup.5NH; 1H, .sup.8CH).

Example 1g

Synthesis of Light-Sensitive Polyester (PE) by Polycondensation of 4,5-dimethoxy-2-nitrobenzyl (1,3-dihydroxypropan-2-yl)carbamate (9) with Adipoyl Chloride (AC)

[0187] ##STR00048##

[0188] In a Schlenk flask 0.330 g of 9 (1.0 mmol), 0.18 mL pyridine (2.2 mmol) was dissolved in 5 mL DMF under Ar atmosphere. A solution of 0.192 g AC (1.05 mmol) in 2 mL DCM was added dropwise over 30 min. After complete addition, the mixture was allowed to stir overnight. The DCM was removed under reduced pressure. The polymer was purified by precipitation into water/MeOH (v/v=1/1) mixture and dried in vacuo to give a yellow solid. Yield: 80%, M.sub.w=3300 g/mol, Ð.sub.M=1.55

##STR00049##

[0189] .sup.1H-NMR (500 MHz, DMSO-d.sub.6): δ (ppm)=1.40-1.80 (m, 4H, .sup.2CH.sub.2), 2.15-2.35 (m, 4H, .sup.1CH.sub.2), 3.75-3.95 (m, 3H, O.sup.9CH.sub.3; 3H, O.sup.10CH.sub.3), 3.95-4.30 (m, 4H, .sup.3CH.sub.2; 1H, .sup.4CH), 5.25-5.40 (m, 2H, .sup.6CH.sub.2), 7.10-7.25 (m, 1H, .sup.7CH), 7.55-7.80 (m, 1H, .sup.5NH; 1H, .sup.8CH).

Example 1h

Light Degradation Investigation of PC01 with .SUP.1.H NMR Spectroscopy

[0190] PC01 solution was prepared in a vial at 15 mg/mL in DMSO-d.sub.6. The PC01 solution was irradiated directly in vial with UV light (0.607 W/cm.sup.2) for 15 min and .sup.1H NMR spectrum was taken. The .sup.1H NMR spectra of PC01 before and after light degradation are shown in FIG. 1 and the signal of .sup.2CH.sub.3 was used as reference, because the relative integral of .sup.2CH.sub.3 should remain unchanged. The result from .sup.1H NMR spectroscopy before irradiation (FIG. 1a) conformed fully to polymer structure as expected. Upon irradiation for 15 min (FIG. 1b) the relative integral of characteristic NMR peak corresponding to the .sup.3CH.sub.2 reduced from 1.69 to 0.13 indicating about 92% cleavage of the light-sensitive 4,5-dimethoxy-2-nitrobenzyl group. As a result of the subsequent intramolecular cyclization the peak at 3.94 ppm for polymer backbone (.sup.1CH.sub.2) reduced significantly and changed from broad peak to sharper small molecule peaks. The newly generated degradation products were observed between 3.50 and 3.80 ppm as well.

Example 1i

Light Degradation Investigation of PC01 with APC

[0191] Five polymer solutions of PC01 were prepared in APC vials at 2 mg/mL in THF. These samples were irradiated by UV light (0.607 W/cm.sup.2, 320-410 nm) for the specified times of 0, 1, 5, 15 and 30 min, and the molar masses were investigated by APC. FIG. 2 shows the APC traces of PC01 before and after light degradation. The degradation degree of PC was strongly affected by irradiation times. With increasing irradiation times, the peak maxima of APC traces for polymers shifted to low molar mass regions due to the loss of light sensitive protecting groups and subsequent polymer degradation caused by intramolecular cyclization, while more and more small molecules were observed in low molar mass region. More importantly, the polymer peak area, which is proportional to the number of polymer chains, consistently decreased upon irradiation, indicating strongly that the polymer chains were fragmented as a result of intramolecular cyclization. The small difference in APC traces for 15 and 30 min suggested that the rapid degradation process completed within 15 min of irradiation.

Example 1j

Light Degradation Investigation of PC01 with UV-VIS Spectrophotometer

[0192] A polymer solution of PC01 was prepared at 0.075 mg/mL in DCM. The PC01 solution was irradiated in quartz cuvette with UV light (0.607 W/cm.sup.2) for the specified periods of time up to 180 s (FIG. 3). UV-VIS absorbance spectra were recorded after each irradiation. Upon irradiation the absorbance at 346 nm decreased due to the cleavage of 4,5-dimethoxy-2-nitrobenzyl light-sensitive protecting group, while a new absorbance appeared at 430 nm, which could be assigned to the degradation product of 4,5-dimethoxy-2-nitrobenzaldehyde. The UV-VIS spectrum remained unchanged after irradiation for 180 s indicating complete cleavage of protecting group from polymer side chains.

Example 2a

Preparation and Characterization of Light-Cleavable Nanoparticles (NP) Based on Polycarbonate (PC) and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC) in a Size Range of about 100 nm (Further Abbreviated as mTHPC-PC-PLGA-NP100)

[0193] The light-cleavable composition was prepared by a solvent displacement preparation technique. In brief, 7.5 mg light-cleavable polymer (PC) and 22.5 mg of the FDA-approved polymer PLGA were dissolved in 1 mL acetone, respectively. After combining both organic solutions, 3 mg of the PS mTHPC was added and dissolved. The organic solution was injected into 4 mL of an aqueous PVA solution (2% (w/v)). The nanoparticle suspension was stirred overnight, whereby evaporation of the organic solvent led to the final particle formation. Afterwards, purification of the nanoparticle suspension was conducted three times via centrifugation (30,000 g, 1.5 h) and redispersion in water. To obtain unloaded nanoparticles the preparation process was performed in the absence of PS.

[0194] For physicochemical particle characterization, photon correlation spectroscopy (PCS) measurements of diluted nanoparticle suspensions were performed. Therefore, a Malvern Zetasizer Nano ZS system was used (Malvern Instruments Ltd., Malvern, UK). The hydrodynamic diameter and polydispersity index (PDI) were measured at a temperature of 22° C. and a backscatter angle of 173°. The surface charge of the nanoparticles was determined by laser Doppler microelectrophoresis using the zeta potential mode.

[0195] The amount of the incorporated PS (mTHPC) was determined by HPLC. The nanoparticles were dissolved in acetone and subsequently, the dissolved PS was quantified via high performance liquid chromatography (HPLC) with the aid of a calibration curve of pure mTHPC (concentration range from 10 to 100 μg/mL). An HPLC-DAD system (Agilent Technologies 1200 series) with a reversed phase column (Gemini RP 18; 250×4.6 mm, particle diameter 5 μm (Phenomenex, Aschaffenburg, Germany)) was used. The mobile phase consisting of 42.5% water with 0.1% (w/v) trifluoroacetic acid and 57.5% acetonitrile was isocratic eluted at a flow rate of 1.0 mL/min. The PS was detected at a wavelength of 415 nm. TABLE 2 shows the obtained nanoparticle characteristics.

TABLE-US-00002 TABLE 2 shows the physicochemical characteristics of unloaded and mTHPC-loaded light- cleavable nanoparticles (mean ± S.D.; n = 3) prepared using the described Examples 2a-d. Drug load Hydrodynamic Zetapotential (μg mTHPC/ Ex. Nanoparticle system diameter (nm) PDI (mV) mg NP) 2a PC-PLGA-NP100 116.0 0.10 −22.0 — mTHPC-PC-PLGA-NP100 109.3 ± 4.1  0.08 ± 0.01 −29.2 ± 1.2 67.4 ± 13.7 PC-(PLA-PEG)-PLGA- 84.8 ± 3.2 0.16 ± 0.04 −16.8 ± 3.0 — NP100 2b mTHPC-PC-(PLA-PEG)- 98.3 ± 7.7 0.09 ± 0.01 −18.9 ± 2.7 96.1 ± 13.7 PLGA-NP100 (PC-PEG)-PLGA-NP100 104.9 0.10 −22.9 — 2c mTHPC-(PC-PEG)-PLGA- 103.4 ± 10.8 0.09 ± 0.02 −24.5 ± 4.3 94.5 ± 10.1 NP100 2d PC-PLGA-NP200 231.4 ± 10.0 0.06 ± 0.04 −25.3 ± 0.4 — mTHPC-PC-PLGA-NP200 229.2 0.09 −26.9 121.2

Investigation of Light-Induced Nanoparticle Degradation

[0196] Light-depending particle degradation was investigated after irradiation of a diluted nanoparticle suspension (0.1 mg NP/mL suspension) with light of a wavelength of 365 nm for 5 min. Variations in the count rate of the nanoparticle suspensions were observed over 24 h via PCS measurements at a temperature of 22° C. and a backscatter angle of 173°. FIG. 4 shows the light-induced particle degradation in comparison to other compositions. As illustrated, the tested formulations retained degradation properties after irradiation of the particle suspensions for 5 min, reflected by a decrease of the particle count rate. In contrast, standard PLGA-NP without a light-cleavable polymer showed no light-depending degradation.

Analysis of the Light-Dependent Drug Release Kinetics

[0197] Lyophilized nanoparticles were dispersed in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). Afterwards, aliquots of 1 mL were irradiated for 5 min with light of a wavelength of 365 nm and afterwards incubated at 37° C. for defined times (15 min, 30 min, 60 min, 2 h, 4 h, 6 h, 24 h). The collected samples were centrifuged (20,000 g, 15 min) and the supernatants were collected. 150 μL of each supernatant was mixed with 450 μL of acetone to precipitate remaining serum protein and to dissolve the drug. In a further centrifugation step (20,000 g, 10 min), the precipitated proteins were separated and the PS content of the supernatants was determined via HPLC-FLD analysis. The HPLC-parameters were the same as described above, except that released mTHPC was detected with a fluorescence detector at 421 nm excitation and 653 nm emission wavelength. For the non-irradiated control, the experimental setup was performed as described above, except the irradiation for 5 min. The conducted in vitro release studies confirmed the hypothesis, that irradiation of the manufactured systems leads to increased drug release (FIG. 5). Non-irradiated nanoparticles showed lower mTHPC release than irradiated particles. In contrast, nanoparticles including no light-cleavable polymer showed a significant reduced release of the incorporated PS (FIG. 5D).

Example 2b

Preparation and Characterization of Light-Cleavable PEGylated Nanoparticles (NP) Based on Polycarbonate (PC), Poly(Ethylene Glycol) Methyl Ether-Block-Poly(Lactic Acid) (PLA-PEG)-, and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC) in a Size Range of about 100 nm (Further Abbreviated as mTHPC-PC-(PLA-PEG)-PLGA-NP100)

[0198] To obtain PEGylated light-cleavable nanoparticles 7.5 mg of a polycarbonate-based light-cleavable polymer (PC) was dissolved in 1 mL acetone. A second organic solution was prepared by dissolving 13.5 mg PLA-PEG and 9 mg PLGA in 1 mL acetone. Both organic solutions were combined and 3 mg of the model PS mTHPC were added and dissolved. The organic solution was injected into 4 mL of an aqueous PVA solution (2% (w/v)). The nanoparticle suspension was stirred overnight, whereby evaporation of the organic solvent led to the final particle formation. Afterwards, purification of the nanoparticle suspension was conducted three times via centrifugation (30,000 g, 1.5 h) and redispersion in water. To obtain unloaded nanoparticles the preparation process was performed in the absence of PS. The nanoparticles were characterized as described within Example 2a. TABLE 2 shows the obtained nanoparticle characteristics.

Example 2c

Preparation and Characterization of Light-Cleavable PEGylated Nanoparticles (NP) Based on Polycarbonate-PEG (PC-PEG) and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC) in a Size Range of about 100 nm (Further Abbreviated as mTHPC-(PC-PEG)-PLGA-NP100)

[0199] For the preparation of PEGylated light-cleavable nanoparticles, 10.7 mg PC-PEG and 19.3 mg PLGA were dissolved in 1 mL acetone, respectively. Afterwards, both organic solutions were combined and 3.0 mg PS (mTHPC) was added and dissolved. The organic solution was injected into 4 mL of an aqueous PVA solution (2% (w/v)). The nanoparticle suspension was stirred overnight, whereby evaporation of the organic solvent led to the final particle formation. Afterwards, purification of the nanoparticle suspension was conducted three times via centrifugation (30,000 g, 1.5 h) and redispersion in water. To obtain unloaded nanoparticles the preparation process was performed in the absence of PS. The nanoparticles were characterized as described within Example 2a. TABLE 2 shows the obtained nanoparticle characteristics.

Example 2d

Preparation and Characterization of Light-Cleavable Nanoparticles (NP) Based on Polycarbonate (PC) and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC) in a Size Range of about 200 nm (Further Abbreviated as mTHPC-PC-PLGA-NP200)

[0200] For the preparation of nanoparticles in a size range of about 200 nm, an emulsion diffusion method was used. Briefly, 5 mg of the light-cleavable polymer (PC) and 15 mg PLGA were dissolved in a mixture of 1 mL dichloromethane and 1 mL ethyl acetate. Afterwards, 3 mg of the model PS mTHPC was added and dissolved. The organic solution and 2 mL of an aqueous PVA solution (1% (w/v)) were emulsified using an Ultra-Turrax® at 24,000 rpm for 30 min. Afterwards, the pre-emulsion was poured into 8 mL of an aqueous PVA solution (1% (w/v)). The organic solvents were evaporated over night by stirring at 500 rpm. The manufactured nanoparticles were purified in three centrifugation steps (30,000 g, 1 h) with subsequent resuspension in ultrapure water. To obtain unloaded nanoparticles the preparation process was performed in the absence of PS. The nanoparticles were characterized as described within Example 2a. TABLE 2 shows the obtained nanoparticle characteristics.

Example 2e

Preparation and Characterization of Light-Cleavable PEGylated Nanoparticles (NP) with Increased Amounts of Light-Cleavable Polymer Based on Polycarbonate-PEG (PC-PEG) and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC) in a Size Range of about 100 nm (Further Abbreviated as mTHPC-(PC-PEG).SUB.50%.-PLGA.SUB.50%.-NP100 and mTHPC-(PC-PEG).SUB.100%.-NP100)

[0201] For the preparation of PEGylated light-cleavable nanoparticles with increased (50% and 100%) amounts of light-cleavable polymer, 21 mg PC-PEG and 9 mg PLGA (for mTHPC-(PC-PEG).sub.50%-PLGA.sub.50%-NP100) or 30 mg (for mTHPC-(PC-PEG).sub.100%-NP100) were dissolved in 2 mL acetone. Afterwards 3.0 mg PS (mTHPC) was added and dissolved. The organic solution was injected into 4 mL of an aqueous PVA solution (2% (w/v)). The nanoparticle suspension was stirred overnight, whereby evaporation of the organic solvent led to the final particle formation. Afterwards, purification of the nanoparticle suspension was conducted three times via centrifugation (30,000 g, 1.5 h) and redispersion in water. The nanoparticles were characterized as described within Example 2a. TABLE 3 shows the obtained nanoparticle characteristics.

TABLE-US-00003 TABLE 3 shows the physicochemical characteristics of mTHPC-loaded light-cleavable nanoparticles (mean ± S.D.; n = 3) prepared using the described Examples 2e. Drug load Hydrodynamic Zetapotential (μg mTHPC/ Nanoparticle system diameter (nm) PDI (mV) mg NP) mTHPC-(PC-PEG)-PLGA-NP100 103.4 ± 10.8 0.09 ± 0.02 −24.5 ± 4.3   94.5 ± 10.1 (Example 2c, 25% PC-PEG) mTHPC-(PC-PEG).sub.50%-PLGA.sub.50%-  93.0 ± 18.3 0.10 ± 0.03 −20.2 ± 11.9 123.7 ± 4.6  NP100 mTHPC-(PC-PEG).sub.100%-NP100 95.1 ± 4.6 0.10 ± 0.03 −0.2 ± 3.6 74.8 ± 2.5

Investigation of Light-Induced Nanoparticle Degradation

[0202] FIG. 6 shows the light-induced particle degradation of nanoparticles with 50% and 100% light-responsive polymer. As illustrated, the tested formulations retained degradation properties after irradiation of the particle suspensions for 5 min, reflected by a decrease of the particle count rate. In comparison to nanoparticles with 50% (PC-PEG), nanoparticles with 100% (PC-PEG) showed increased light-induced nanoparticle degradation, due to the increased number of light-responsive groups in the particle matrix.

Analysis of the Light-Dependent Drug Release Kinetics

[0203] The conducted in vitro release studies with (mTHPC-(PC-PEG)-PLGA-NP100) (FIGS. 5C and 7) showed that nanoparticles with 25% (PC-PEG) exhibited a high burst release, probably caused by the mixture of PLGA and (PC-PEG). To reduce the burst release, nanoparticles with 100% of (PC-PEG) were prepared. Unfortunately, the non-irradiated nanoparticles showed similar low mTHPC release as irradiated particles. Therefore, the light-triggered drug release of mTHPC-(PC-PEG).sub.100%-NP100 did not lead to the desired results (FIG. 7).

[0204] In contrast, nanoparticles including 50% light-cleavable polymer (mTHPC-(PC-PEG).sub.50%-PLGA.sub.50%-NP100 showed a small burst release when not irradiated and a significant increased drug release when irradiated. Hence, the increased amount of 50% (PC-PEG) in combination with 50% PLGA leads to a satisfying light-induced mTHPC-release characteristics (FIG. 7).

Example 3a

Biological Safety of Light-Cleavable Nanoparticles (NP) Based on Polycarbonate (PC) and Poly(DL-Lactide-Co-Glycolide) (PLGA) Before and after Irradiation with Light of a Wavelength of 365 nm

Biological Safety

[0205] Investigations of biological safety were performed using mucus producing HT-29-MTX cells. The used cells were cultivated in full supplemented DMEM. For investigations the following formulations were tested:

TABLE-US-00004 1. PC-PLGA-NP100 see Example 2a 2. PC-(PLA-PEG)-PLGA-NP100 see Example 2b 3. (PC-PEG)-PLGA-NP100 see Example 2c

[0206] HT-29-MTX cells were incubated with a nanoparticle concentration ranging from 10 μg NP/mL to 500 μg NP/mL in DMEM (serum free), over a period of 24 h. After removal of the incubation medium, WST-1 reagent was added and absorbance was measured immediately at 460 nm with a Synergy MX multi-well spectrophotometer (BioTek Instruments GmbH, Bad Friedrichshall, Germany). Blank measurement, positive and negative control were used for calculation of total cell viability.

[0207] FIG. 8A illustrates the biological safety of all unloaded nanoparticles. The used cells show a viability of almost 100%, indicating no cytotoxicity for the used concentrations. The experiment was done in triplicate and for each experiment three measurements (n=3) were performed. Error bars represent the standard deviation of three experiments.

Biological Safety after Light Irradiation

[0208] For examination of cytotoxic effects after irradiation, the different formulations were irradiated for 5 min at 365 nm prior to cell incubation. Afterwards the HT-29-MTX cells were treated with the same concentration of irradiated formulations as described above.

[0209] FIG. 8B depicts biological safety for formulations after irradiation at 365 nm using HT-29-MTX cells. Cell viability shows no toxic effects of products after irradiation at 365 nm on the cells. The experiment was done in triplicate and for each experiment three measurements (n=3) were performed. Error bars represent the standard deviation of three experiments.

Example 3b

Analysis of Intracellular Uptake of Light-Cleavable Nanoparticles Based on Polycarbonate (PC) and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC)

Intracellular Uptake Quantified by HPLC-FLD

[0210] Experiments to quantify the intracellular uptake in HT-29-MTX cells were performed for the following photosensitizer-loaded samples:

TABLE-US-00005 1. mTHPC-PC-PLGA-NP100 see Example 2a 2. mTHPC-PC-(PLA-PEG)-PLGA-NP100 see Example 2b 3. mTHPC-(PC-PEG)-PLGA-NP100 see Example 2c 4. mTHPP-PLGA-NP100 5. mTHPC

[0211] The used cells, cultivated in DMEM, were incubated with nanoparticle concentrations corresponding to 1 μM mTHPC (mTHPP) for 24 h. At certain time points (1 h, 2 h, 4 h, 6 h and 24 h), incubation medium was removed, cells were washed (PBS, 2×) and cell amount and cell volume were determined using CASY® TT Cell Counter. After incubation, cells were transferred to DMSO for cell lysis and extraction of photosensitizer. Quantification of photosensitizer was performed by HPLC-FLD system (Agilent Technologies 1200 series). In samples dissolved in DMSO, cell debris was removed before HPLC analysis via centrifugation (30,000 g, 30 min). A reversed phase column (Gemini RP 18; 250×4.6 mm, particle diameter 5 μm (Phenomenex, Aschaffenburg, Germany) was used for isocratic elution with a mobile phase consisting of 57.7% acetonitrile and 42.5% water containing 0.1% (w/v) trifluoroacetic acid (flow rate: 1.0 mL/min). The photosensitizer was detected at 421 nm excitation and 653 nm emission wavelength.

[0212] FIG. 9 shows the results of the intracellular uptake of mTHPC and photosensitizer-loaded nanoparticles by HT-29-MTX cells over 24 h. Non-light-cleavable mTHPP-PLGA-NP100 and free mTHPC served as control. The experiment was done in triplicate and for each experiment three wells were incubated with the same concentration. Error bars represent the standard deviation of three experiments. The used nanoparticles were accumulated by a factor of 8 to 17 from the initial concentration. mTHPC-PC-PLGA-NP100 and mTHPC-PC-(PLA-PEG)-PLGA-NP100 were superior to non-light cleavable mTHPP-PLGA-NP100 and free mTHPC.

Cellular Association Quantified by IncuCyte® Live-Cell Imaging

[0213] Cellular association was investigated by IncuCyte® Live-Cell Imaging (Essen Bioscience, Inc., Michigan, USA) over a period of 24 h for the following samples:

TABLE-US-00006 1. mTHPC-PC-PLGA-NP100 see Example 2a 2. mTHPC-PC-(PLA-PEG)-PLGA-NP100 see Example 2b 3. mTHPP-PLGA-NP100 4. mTHPC

[0214] Cell incubations were performed using nanoparticle concentrations corresponding to 1 μM mTHPC (mTHPP).

[0215] Nine images were taken of each well and time point (every hour) over a period of 24 h. The red channel (excitation: 565-605 nm/emission: 625-705 nm) was used to quantify the total red object area representing the photosensitizer. Blank measurements were performed in wells containing cells but without nanoparticle incubation (medium).

[0216] FIG. 10 shows the results of the cellular association of mTHPC and photosensitizer-loaded nanoparticles by HT-29-MTX cells over 24 h. Non-light-cleavable mTHPP-PLGA-NP100 and free mTHPC served as control. The experiment was done in triplicate and for each experiment three wells were incubated with the same concentration. Error bars represent the standard deviation of three experiments. Light-cleavable nanoparticles showed the higher intensity of red fluorescence after 24 h compared to mTHPP-PLGA-NP.

Example 3c

Dark Toxicity and Photodynamic Activity of Light-Cleavable Nanoparticles Based on Polycarbonate (PC) and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC)

Dark Toxicity

[0217] Investigation of dark toxicity of all mTHPC-loaded formulations was performed using WST-1 assay as described in Example 3a. HT-29-MTX cells were incubated with the following samples in a photosensitizer concentration ranging from 0.001 μM to 5 μM in DMEM over a period of 24 h:

TABLE-US-00007 1. mTHPC-PC-PLGA-NP100 see Example 2a 2. mTHPC-PC-(PLA-PEG)-PLGA-NP100 see Example 2b 3. mTHPC-(PC-PEG)-PLGA-NP100 see Example 2c 4. mTHPP-PLGA-NP100 5. mTHPC

[0218] After removal of the incubation medium, WST-1 reagent was added and absorbance was measured immediately at 460 nm by a Synergy MX multi-well spectrophotometer. Blank measurement, positive and negative control were used for calculation of total cell viability.

[0219] FIG. 11A shows the dark toxicity of all tested samples. With a viability of almost 100% no dark toxicity was detected in HT-29-MTX cells. Non-light-cleavable mTHPP-PLGA-NP100 and free mTHPC served as control. The experiment was done in triplicate and for each experiment three measurements (n=3) were performed. Error bars represent the standard deviation of three experiments.

Phototoxicity

[0220] For examination of photodynamic activity, after 24 h of incubation with tested formulations, cells were irradiated at 652 nm for 30 min at a light dose of 5 J/cm.sup.2. Afterwards, WST-1 assay was performed as described above for evaluation of dark toxicity.

[0221] FIG. 11B demonstrates the phototoxicity effects on HT-29-MTX cells, indicating a comparable efficacy of the formulations of present invention to the pure photosensitizer mTHPC and the non-light cleavable control mTHPP-PLGA-NP100. The experiment was done in triplicate and for each experiment six measurements (n=6) were performed. Error bars represent the standard deviation of three experiments.

[0222] Having described preferred embodiments of the invention with reference to the accompanying examples, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by skilled in the art without departing from the scope of the invention as defined in the appended claims.

Example 3d

Photodynamic Activity after Nanoparticle Degradation of Light-Cleavable Nanoparticles Based on Polycarbonate (PC) and Poly(DL-Lactide-Co-Glycolide) (PLGA) with the Photosensitizer (PS) 5,10,15,20-tetrakis(3-hydroxyphenyl)-chlorin (mTHPC)

[0223] A detailed investigation of toxicity after a two-wavelength irradiation (365 nm for polymer/nanoparticle degradation; 652 nm for photosensitizer activation) was performed using WST-1 assay as described in Example 3a. HT-29-MTX cells were incubated with the following formulation in mTHPC concentrations ranging from 0.001 μM to 5 μM in DMEM over a period of 24 h.

TABLE-US-00008 1. mTHPC-(PC-PEG).sub.50%-PLGA.sub.50%-NP100 see Example 2e

[0224] For examination of photodynamic activity in combination with a light induced nanoparticle degradation, WST-1 assay was performed after three different ways of irradiation: [0225] 1. 365 nm (10 min) [0226] 2. 652 nm (30 min) [0227] 3. 365 nm (10 min) followed by 652 nm (30 min)

[0228] After irradiation, cells were stored in an incubator for 1 h. WST-1 reagent was added and absorbance was measured immediately at 460 nm by a Synergy MX multi-well spectrophotometer. Blank measurement, positive and negative control were used for calculation of total cell viability.

[0229] FIG. 12 shows the toxicity of mTHPC-(PC-PEG).sub.50%-PLGA.sub.50%-NP100 after irradiation at different wavelengths. The highest toxicity was observed after irradiation at both wavelengths, indicating a beneficial effect of nanoparticle degradation at 365 nm prior to activation of mTHPC at 652 nm. The irradiation with either 365 nm for nanoparticle degradation or 652 nm for photosensitizer activation led to a significantly reduced toxicity. The experiment was done in triplicate and for each experiment three measurements (n=3) were performed. Error bars represent the standard deviation of three experiments.

[0230] The work leading to this invention has received funding from BMBF under grant agreement n° BMBF 13N13423.