MICELLAR COMPOSITION FROM AN AMPHIPHILIC COPOLYMER FOR TUMOR THERAPY

Abstract

An amphiphilic copolymer includes a first block, a second block and a linker covalently linking the first block with the second block, wherein the first block is a hydrophilic dendritic polyglycerol derivative having a polyglycerol backbone and carrying a plurality of sulfate or sulfonate residues substituting hydroxyl groups of the polyglycerol backbone, wherein the second block is a hydrophobic block comprising a polymer chosen from the group consisting of polycaprolactone, a polylactic acid polymer, and a copolymer of lactic acid and glycolic acid. The linker comprises a hydrocarbon having at least six consecutive methylene residues and a cleavable entity. The linker is devoid of a triazole-containing residue resulting from a reaction between an alkyne and an azide.

Claims

1. An amphiphilic copolymer comprising: a first block, a second block, and a linker covalently linking the first block with the second block, wherein: the first block is a hydrophilic dendritic polyglycerol derivative having a polyglycerol backbone and carrying a plurality of sulfate or sulfonate residues substituting hydroxyl groups of the polyglycerol backbone, the second block is a hydrophobic block comprising a polymer chosen from the group consisting of polycaprolactone, a polylactic acid polymer, and a copolymer of lactic acid and glycolic acid, the linker includes a hydrocarbon having at least six consecutive methylene residues and a cleavable entity, and the linker is devoid of a triazole-containing residue resulting from a reaction between an alkyne and an azide.

2. The amphiphilic copolymer according to claim 1, wherein the cleavable entity is a redox-sensitive entity or a pH-cleavable entity.

3. The amphiphilic copolymer according to claim Error! Reference source not found., wherein the redox-sensitive entity is a disulfide bridge.

4. The amphiphilic copolymer according to claim Error! Reference source not found., wherein the pH-cleavable entity is at least one an entity chosen from the group consisting of imines, oximes, hydrazones, and acetals.

5. The amphiphilic copolymer according to claim 1, wherein the polylactic acid polymer is chosen from the group consisting of poly-L-lactic acid, poly-D-lactic acid, and poly-D,L-lactic acid.

6. The amphiphilic copolymer according to claim 1, wherein the linker comprises 6 to 20 consecutive methylene residues.

7. The amphiphilic copolymer according to claim 1, wherein the polymer of the second block comprises 10 to 200 repeating units.

8. The amphiphilic copolymer according to claim 1, wherein the amphiphilic copolymer correspond to following general formula: ##STR00002## wherein: m=6 to 20, o=0 to 4, p=0 to 4, and q=0 to 4.

9. A micelle, comprising a plurality of molecules of at least one amphiphilic copolymer according to claim 1.

10. (canceled)

11. A medical method for administering a medicament to a subject, wherein the medicament comprises a micelle according to claim Error! Reference source not found. and an agent encapsulated in an interior of the micelle.

12. The medical method of claim 11, wherein an anti-tumor agent is encapsulated in the interior of the micelle.

13. A method for manufacturing an amphiphilic copolymer according to claim 1, comprising the following steps: a) providing an carboxylated polymer chosen from the group consisting of carboxylated polycaprolactone, a carboxylated polylactic acid polymer, and a carboxylated copolymer of lactic acid and glycolic acid, b) providing a polyglycerol derivative starting material comprising a polyglycerol backbone and an alkyl residue bonded to the polyglycerol backbone, the alkyl residue having at least six consecutive methylene residues, wherein the polyglycerol derivative starting material further comprises a thioamine residue being covalently bonded to the alkyl residue in a direct or indirect manner, c) conjugating the carboxylated polymer to the polyglycerol derivative starting material by an amide coupling, and d) sulfating or sulfonating at least some hydroxyl groups of the polyglycerol backbone to obtain an amphiphilic copolymer according to claim 1.

14. The method according to claim Error! Reference source not found., wherein the provided carboxylated polymer is obtained by reacting a polymer chosen from the group consisting of polycaprolactone, a polylactic acid polymer, and a copolymer of lactic acid and glycolic acid with an organic acid anhydride.

15. The method according to claim Error! Reference source not found., wherein the provided polyglycerol derivative starting material is obtained by: polymerizing an alkenol comprising at least six carbon atoms and glycidol to obtain a monofunctional polyglycerol allyl, adding a mercapto alkyl carboxylic acid and allowing a reaction between the monofunctional polyglycerol allyl and the mercapto alkyl carboxylic acid to obtain a carboxylated polyglycerol, and reacting a thioamine with the carboxylated polyglycerol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Further details of aspects of the solution will be explained with respect to exemplary embodiments and accompanying Figures.

[0091] FIG. 1 shows a general synthetic route for an amphiphilic copolymer.

[0092] FIG. 2 shows a synthetic route for manufacturing monofunctional dPG-SS-NH.sub.2.

[0093] FIG. 3 shows an .sup.1H NMR spectrum of dPG-ether-C11-undecen measured in methanol-d4 and its corresponding structure.

[0094] FIG. 4 shows an IR spectrum of dPG-COOH and its structure.

[0095] FIG. 5 shows an .sup.1H NMR spectrum of mdPG-COOH measured in methanol-d4.

[0096] FIG. 6 shows an .sup.1H NMR spectrum of dPG-SS-NH.sub.2 measured in methanol-d4.

[0097] FIG. 7 shows an 1H-NMR of PCL-COOH in CDCl.sub.3.

[0098] FIG. 8 shows a synthetic route for the synthesis of dPGS-SS-PCL.

[0099] FIG. 9A shows an .sup.1H-NMR spectrum of sedimentation (PCL-COOH) in CDCl.sub.3.

[0100] FIG. 9B shows an .sup.1H-NMR spectrum of supernatant (dPGS-SS-PCL) in DMF-d7:D.sub.2O.

[0101] FIG. 10 shows a DLS spectrum of loaded/unloaded PCL-SS-dPGS micelles.

[0102] FIG. 11A shows a first set of DLS plots of Sunitinib-loaded PCL-SS-dPGS micelles directly after Sephadex and after resuspension from dry state.

[0103] FIG. 11B shows a second set of DLS plots of Sunitinib-loaded PCL-SS-dPGS micelles directly after Sephadex and after resuspension from dry state.

[0104] FIG. 12 shows a plot for CMC determination by light scattering intensity of SU-PCL-SS-dPGS and empty PCL-SS-dPGS micelles with varying concentration (dilution series).

[0105] FIG. 13 shows the results of a release study of sunitinib-loaded micelles in presence of 10 mM GSH in dialysis set up for 1 week, at 37° C., and the result of a leaching study of sunitinib-loaded micelles in MilliQ at 37° C.

[0106] FIG. 14 shows the results of a cell viability study of unloaded PCL-SS-dPGS micelles.

[0107] FIG. 15A shows the results of a cell viability study of free Sunitinib malate (hydrophilic).

[0108] FIG. 15B shows the results of a cell viability study of free Sunitinib (hydrophobic).

[0109] FIG. 15C shows the results of a cell viability study of PCL-SS-dPGS-micellar-encapsulated Sunitinib (hydrophobic).

[0110] FIG. 16 shows DLS plots of dPGS-SS-PCL micelles in PBS buffer and after incubation with 10 mM GSH.

[0111] FIG. 17 shows DLS plots of dPGS-SS-PLGA micelles in PBS buffer and after incubation with 10 mM GSH.

DETAILED DESCRIPTION

[0112] Description of the Synthesis and Physicochemical Properties

[0113] The synthesis of the amphiphilic copolymer was performed using a reactive coupling strategy that forms amide bonds. In brief, the polymerized hydrophobic second block (PCL, PLGA, or PLA) is reacted with succinic acid anhydride to provide the corresponding acid (e.g. PCL-COOH).

[0114] The hydrophilic first block consists of an 11-undecenyl-polyglycerol that is linked to thiopropionic acid and cysteamine to provide monofunctional dPG-SS-NH.sub.2. This reactive polymer was then conjugated to the hydrophobic second block (e.g. PCL-COOH) by an amide coupling. The final block copolymer was obtained after sulfation of the hydroxyl groups. This reaction scheme is depicted in FIG. 1.

[0115] Materials. 3,6-Dimethyl-1,4-dioxane-2,5-dione (Lactide), 1,4-Dioxane-2,5-dione (Glycolide), 1-Oxa-2-oxocycloheptane (Caprolactone), N-Ethyl-N-(propan-2-yl)propan-2-amine (DIPEA), 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 1,3,4,6,7,8-Hexahydro-2H-pyrimido[1,2-a]pyrimidine (TBD), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), Oxolane-2,5-dione (succinic anhydride) N,N′-Diisopropylcarbodiimide (DIC), N-Hydroxysuccinimide (NHS), sodium hydride (NaH), 1-Hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), Mercaptopropionic acid, and Sulfur trioxide pyridine complex (SO.sub.3*Py) were purchased from sigma Aldrich. Glycidol (96%, Acros) was dried over CaH.sub.2 overnight and freshly distilled prior to use. N,N-dimethylformamide, (DMF, 99.8%, extra dry, Acros), ethanol (99.8%, extra dry), THF (99.8%, extra dry) and DCM (99.8%, extra dry) were purchased from Acros. Cystamine dihydrochloride (Cystamine.2HCl, >98%, Alfa Aesar), dry triethylamine (Et.sub.3N, 99%, Alfa Aesar), 11-Bromo-1-undecene (Alfa Aesar), dichloromethane, pyridine, toluene, ethanol, methanol and tetrahydrofuran were used as received. Dialysis was performed in benzoylated cellulose tubing purchased from Sigma-Aldrich (MWCO 2 kDa) and standard regenerated cellulose tubing purchased from Spectrumlab (MWCO 1 kDa, 2 kDa, and 3.5-5 kDa).

[0116] Characterization. .sup.1HNMR spectra were recorded on a Bruker ECX 400 spectrometer operating at 400 MHz using Pyridine-d5, CDCl.sub.3, DMF-d7, CD.sub.3OD-d4, D.sub.2O, or DMSO-d6 as a solvent. The chemical shifts were calibrated against residual solvent signal. The molecular weight and polydispersity of the polymers were determined by a Waters 1515 gel permeation chromatograph (GPC) instrument equipped with two linear PLgel columns (Mixed-C) following a guard column and a differential refractive-index detector. The measurements were performed using THF (hydrophobic segments) or water (hydrophilic segments) as the eluent at a flow rate of 1.0 mL min.sup.−1 at 30° C. and a series of narrow polystyrene standards for the calibration of the columns. The size of micelles was determined using dynamic light scattering (DLS) at 25° C. using Zetasizer Nano-ZS (Malvern Instruments) equipped with a 633 nm He—Ne laser using back-scattering detection. Elemental analysis was performed with a VARIO EL III (Elementar). IR spectra were recorded with Nicolet AVATAR 320 FT-IR 5 SXC (Thermo Fisher Scientific, Waltham, Mass., USA) with a DTGS detector from 4000 to 650 cm.sup.−1. Sample measurement was done by dropping a solution of compound and letting the solvent evaporate for a few seconds.

[0117] Synthesis of Monofunctional Dendritic Polyglycerol (dPG-SS-NH.sub.2)

[0118] The hydrophilic first block consisting of an 11-undecenyl-polyglycerol linked to thiopropionic acid and cysteamine provides the dPG-SS-NH.sub.2 and was synthesized in a two-step reaction protocol by starting with 10-undecen-1-ol. The simple approach realizes the development of redox-sensitive dendritic polyglycerols with a monofunctional group as terminal unit (cf. FIG. 2). These monofunctional dendritic polyglycerols were used to couple different hydrophobic blocks via an amide coupling reaction. The 10-undecen-1-ol is used as inert starter for the anionic ring opening polymerization with glycidol to obtain monofunctional allylated polyglycerols, which provides a platform for a variety of functionalization reactions. Additionally, the unique branched architecture of the polyglycerol with the multivalent 1,2-diols of the terminal glycerol units can be further modified to generate a core-shell-type architecture (cf. references 1 and 2).

[0119] Here, the allyl group was further modified by a thiol-ene and amide coupling, to form monofunctional dPG-COOH and redox sensitive dPG-SS-NH.sub.2 respectively. After coupling the monofunctional dendritic polyglycerol with the hydrophobic counterpart (e.g. PCL-SA), the 1,2-diols were sulfated to provide a copolymer with active targeting to inflammation-related tumor tissues.

[0120] In the first step, the 10-undecen-1-ol was polymerized by an anionic ring opening polymerization to monofunctional dPG-allyl. For this purpose, the 10-undecen-1-ol (20.66 g, 0.12 mol) was deprotonated by potassium methoxide (KOH 0.31 g, 5 mL MeOH; MeOK, 15% deprotonation) and water was evaporated at 60° C. under vacuum. The synthesis reactor was heated to 100° C. and glycidol (200 g, 2.7 mol) was added over a period of 24 h. The success of reaction was evaluated by .sup.1H-NMR as shown in FIG. 3. After 26 h, the reaction temperature was reduced to 75° C. and 600 mL of dry Dimethylformamide (DMF) was added. Subsequently mercaptopropionic acid (29.50 g, 278 mmol) and azobisisobutyro-nitrile (AIBN) (4.56 g, 27.8 mmol) was admitted to the reaction mixture. The in-situ Thiol-ene reaction to the monofunctional dPG-ether-COOH occurred within 4 h. The crude product was precipitated in acetone and purified by TFF dialysis in a water/ethanol mixture 10:1 (MWCO: 1000 Da) for 3 d. The solvent was removed under reduced pressure to achieve the monofunctional dPG-COOH (150 g) as viscous yellow polymer. The polymer was analyzed via GPC and a number average molecular weight (Mn) of 3.85 kg mol.sup.−1 was obtained. The ratio of the weight average molecular weight Mw to Mn (Mw/Mn) was 1.63. The characteristic absorbance bond of carbonyl group at 1715 cm.sup.−1 in IR spectrum (cf. FIG. 4) and the absence of the allyl peak in the .sup.1H NMR proved the successful reaction of 3-Mercaptopropionic acid with the dPG-ether-C11-undecen as shown in FIG. 5.

[0121] For analysis, a sample was taken after the first polymerization step, diluted with methanol and stirred over ion exchange resin (Dowex® Monosphere® 650C UPW) overnight. After filtering off the resin, the crude product was purified by dialysis in distilled water (MWCO: 1000 Da) for 3 d. The compound was obtained as a viscous yellow polymer after lyophilization.

[0122] To couple the monofunctional dPG-COOH with the hydrophobic second block (e.g. PCL-SA), it has to be modified via an amidation coupling with cystamine (second step). Therefore, monofunctional dPG-COOH (25 g, 6.58 mmol) was dissolved in 500 mL MES-buffer (pH 5.0, 50 mM) and NHS (3.79 g, 32.89 mmol) and EDC.HCl (6.31 g, 32.89 mmol) was added to the polymer solution, and the mixture was stirred for 30 min at room temperature (r.t.) to form the active ester. Separately, cystamine (7.41 g, 32.89 mmol) was dissolved in 1 L PB-buffer (pH 7.4, 100 mM) and added to the reaction flak. The reaction was stirred for 16 h at room temperature. The raw product was precipitated in acetone and purified by TFF dialysis in a water (MWCO: 1000 Da) for 3 d. Finally, the solvent was removed under reduced pressure to achieve the monofunctional dPG-SS-NH.sub.2 (16.8 g). The corresponding structure and .sup.1H-NMR are shown in FIG. 6.

[0123] Overall, it took two reaction steps to achieve a monofunctional dendritic polyglycerol with a redox-sensitive dithiol group for the further coupling of various hydrophobic blocks. The initial reaction of the 10-undecen-1-ol is performed in a solvent free procedure in a bulk polymerization process followed by an in-situ modification to the monofunctional dPG-COOH. The second modification to the dPG-SS-NH.sub.2 is performed in aqueous media.

[0124] Synthesis of the Hydrophobic Second Block (PLA-COOH, PLGA-COOH, PCL-COOH)

[0125] All three variants of the hydrophobic second block (i.e., a block containing polycaprolactone (PCL), poly(lactic acid) (PLA), or poly(lactic-co-glycolic acid) (PLGA)) are generally synthesized by ring opening polymerization of F-caprolactone, lactide and glycolide at room temperature, using ethanol as initiator and DBU or TBD as catalyst. The reaction is performed in dry solvents (DCM, THF or toluene) at room temperature in several hours. The polymerization is quenched by addition of succinic anhydride. Thus, the reaction can be performed in only one day with one purification step leading to high yields. The solvent is evaporated by rotary evaporator and the crude polymer is dissolved in low amount of THF and precipitated three times in cold methanol to remove small oligomers and unreacted monomers. The solvent was evaporated, and the acid-functionalized polymer is obtained as a white powder after precipitation in cold methanol and drying under vacuum.

[0126] Synthesis of Random PLGA-COOH

[0127] Lactide (12.00 g, 83.26 mmol) was placed in a flame-dried 200 mL Schlenk flask equipped with a magnetic stir bar and a rubber septum. Subsequently, lactide was dried while stirring in vacuum over 30 min. Then, dry DCM (80 mL) was added to dissolve lactide to obtain homogenous solution, that was degassed over 15 min. For initiation of reaction, ethanol (111 μL, 1.89 mmol) and DBU (282 μL, 1.89 mmol) were added to the lactide solution and the polymerization mixture was stirred at room temperature. As fast as possible, dried glycolide (3.2 g, 3.22 mmol) dissolved in degassed dry THF (0.5 M) was added under use of a syringe pump (rate 0.1 mL/min) to the polymerization mixture. After addition of glycolide, the reaction was further stirred for 30 min at room temperature. The reaction mixture was terminated by addition of succinic anhydride (757 mg, 7.57 mmol) dissolved in dry THF (0.5 M) and stirred overnight at room temperature. The reaction mixture was concentrated in vacuum with following precipitation in cold methanol for three times. The polymer was obtained as a white solid with a yield of 76%. The polymer was analyzed via GPC and a Mn: 10.4 kg mol−1, Mw/Mn=1.43 was obtained. .sup.1H-NMR in CDCl.sub.3 confirmed that polymer with molecular weight of 9922 g/mol was obtained.

[0128] Synthesis of PLA-COOH

[0129] Lactide (15.00 g, 104.07 mmol) was placed in a flame-dried 200 mL Schlenk flask equipped with a magnetic stir bar and a rubber septum. Subsequently, lactide was dried while stirring in vacuum over 30 min. Then, dry DCM (100 mL) was added to dissolve lactide to obtain homogenous solution, that was degassed over 15 min. For initiation of reaction, ethanol (113 μL, 1.93 mmol) and DBU (345 μL, 1.89 mmol) were added to the lactide solution and the polymerization mixture was stirred at room temperature. The reaction mixture was terminated by addition of succinic anhydride (757 mg, 7.57 mmol) dissolved in dry THF (0.5 M) and stirred overnight at room temperature. The reaction mixture was concentrated in vacuum with following precipitation in cold methanol for three times. The polymer was obtained as a white solid with a yield of 76%. The polymer was analyzed via GPC and a Mn: 12.1 kg mol−1, Mw/Mn=1.29 was obtained. .sup.1H-NMR in CDCl.sub.3 confirmed that polymer with molecular weight of 8736 g/mol was obtained.

[0130] Synthesis of PCL-COOH

[0131] Caprolactone (15.00 g, 13.89 mL, 131.42 mmol) was placed in a flame-dried 250 mL Schlenk flask equipped with a magnetic stir bar and a rubber septum. Then, toluene (100 mL) was added to dissolve caprolactone to obtain homogenous solution, that was degassed over 15 min. For initiation of reaction, ethanol (110 μL, 1.88 mmol) and TBD (523 mg, 3.75 mmol) were added to the caprolactone-solution and the polymerization mixture was stirred at room temperature for 5 h. The reaction mixture was terminated by addition of succinic anhydride (751 mg, 7.51 mol) dissolved in dry THF and stirred overnight. The reaction mixture was concentrated in vacuum with following precipitation in cold methanol (centrifugation, 7000 rpm, 30 min, two times). The polymer was obtained as a white solid with a yield of 90%. The polymer was analyzed via GPC and a Mn: 8.6 kg mol.sup.−1, Mw/Mn=1.22 was obtained. .sup.1H-NMR in pyridine-d5 showed that polymer with molecular weight of 6175 g/mol was obtained. FIG. 7 shows an .sup.1H-NMR spectrum in CDCl.sub.3 to prove the purity of polymer.

[0132] Coupling of the Monofunctional dPG-SS-NH.sub.2 with Hydrophobic Block

[0133] In a flame-dried Schlenk flask equipped with a rubber septum and a magnetic stir bar PCL-COOH (3 g, 0.484 mmol) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.302 g, 0.581 mmol) were dissolved in dry DMF (20 mL). Subsequently, N,N-diisopropylethylamine (DIPEA) (0.17 mL, 0.968 mmol) was added and the solution was stirred for 2 h at rt to active the acid. Then, dPG-SS-NH2 (1.16 g, 0.323 mmol) dissolved in dry DMF (10 mL) was added dropwise to the solution and the reaction mixture was further stirred overnight. The success of amide coupling was checked using ninhydrin test and could be confirmed by absence of color formation indication full conversion of all free amines. The solution was then transferred in a dialysis tubing (MWCO: 1 kDa) and first dialyzed against MeOH for one day and then dialyzed against water for several days under intensive changing of water to remove the organic solvents. The product was then freeze-dried using lyophilization. For a detailed overview, the reaction scheme of the amide coupling and sulfation to finally obtain dPGS-SS-PCL is shown in FIG. 8. This reaction can be carried out in the same way if PLA or PGLA are present in the second block.

[0134] Sulfation of the Amphiphilic Copolymer (dPG-SS-Hydrophobic Block)

[0135] Sulfation of the amphiphilic copolymer was performed using a previously established protocol (cf. Reference 3). To a stirred solution of dPG-SS hydrophobic block in N,N-dimethylformamide (DMF), a solution of SO3/pyridine complex was added dropwise at 60° C. under argon atmosphere. After addition, the mixture was reacted for 2 h at 60° C. and 24 h at r.t. Then, the pH of the solution was adjusted to pH 8.0 by 1M NaOH solution. Distilled water was added, and the product was obtained after dialysis with a NaCl solution (MWCO=2 kDa), using an ever-decreasing NaCl concentration, until the medium was changed with distilled water. The dialysis process was performed for 96 h. Then the amphiphilic copolymer and remained hydrophobic polymer was separated by sedimentation overnight, wherein the amphiphilic copolymer stays in solution (supernatant). The supernatant was collected and was dried. The precipitation was treated the same. The copolymer (e.g. dPGS-SS-PCL) was obtained after lyophilization. According to the calculations, the sulfur content of the dPGS-SS-PCL measured by elemental analysis showed almost complete sulfation of the hydroxyl groups (% N: 1.18, % C: 24.16, % H: 5.69, % S: 13:98). The .sup.1HNMR spectrum (cf. FIG. 9B) of sulfated di-block copolymer shows the signals of the hydrophobic block (4.78; 2.43; 1.74; 1.50; 1.39 ppm) and the hydrophilic block (3.60-4.4 ppm). The appearance of new signals at 4.33-4.17 ppm which are assigned to the methylene protons adjacent to the sulfate groups proves the sulfation reaction. The .sup.1H NMR spectra of the sedimentation containing the non-sulfated starting material (cf. FIG. 9A) and the product of the coupling reaction (cf. FIG. 9B) show the successful conjugation and formation of amphiphilic di-block copolymer. In summary, it was possible to synthesize an amphiphilic copolymer with a redox-sensitive moiety.

[0136] Preparation of Micelles (dPGS-SS-Hydrophobic Block)

[0137] The micelles were prepared using an evaporation method. Briefly, the polymer was dissolved in a mixture of acetone, which is Class 3 solvent, and water, wherein the organic solvent hinders the formation of micelles. Then, the homogeneous solution of polymer or polymer/drug solution was dropwise added to a stirred solution of MilliQ. Subsequently, the organic solvent could be removed using a rotary evaporator.

[0138] Preparation of Micelles (c=1 mg/mL)

[0139] Micelles were prepared by dropwise addition of ultrasonicated acetone solution (100 μL) of dPGS-SS-PCL (1 mg, 10 mg mL.sup.−1)+10 μL of MiliQ or PB (pH 7.4, 10 mM) to 990 μL MiliQ or phosphate buffer (PB, pH 7.4, 10 mM) under stirring (550 rpm) at r.t. for 1-2 min, followed by removal of acetone using rotary evaporation.

[0140] Loading of the Drug (Loading 20 wt %, c=1 mg/mL).

[0141] Drug-loaded micelles were prepared by dropwise addition of a mixed solution of copolymer (10 mg, Acetone: 1000 μL, 10 mg mL.sup.−1)+100 μL Mili-Q or PB and the drug (2 mg) 9900 μL MiliQ/PB (10 mM, pH 7.4) under stirring (550 rpm) at r.t. for 1-2 min, followed by removal of acetone using rotary evaporation. The drug-loaded micelle solution was passed over sephadex column (G25) to remove the non-encapsulated drug. Besides sephadex work up, the drug-loaded micelles can be purified by filtering the carrier with a syringe filter (200 nm regenerated cellulose, Sartorius). The drug loading efficiency and capacity was evaluated by UV-VIS measurement.

[0142] Determination of Critical Micelle Concentration (CMC)

[0143] The critical micelle concentration (CMC) of the loaded and unloaded micelles was determined by measuring the light scattering intensity with a Zetasizer. Briefly, the light scattering intensity of the micelles was measured at various concentrations (μg/mL) prepared in deionized water by serial dilution at 25° C. By plotting the light scattering intensity against the log concentration of the carrier, the CMC was determined as the intersection of the best fit lines drawn through the data points.

[0144] Drug Loading Using UV

[0145] The loaded micelles were dissolved in a mixture of methanol and Milli-Q. Then, the amount of Sunitinib in the carrier was determined by measuring the absorbance at 431 nm. Besides a calibration curve was prepared constructed with different concentrations of Sunitinib in methanol/Milli-Q. The drug loading content and drug loading efficiency were calculated according to the following formulas.

[00001]$\mathrm{Drug}\mathrm{loading}\mathrm{content}\left(\%\right)=\frac{w\mathrm{eight}\mathrm{of}\mathrm{Sunitinib}\mathrm{in}\mathrm{carrier}}{\mathrm{total}\mathrm{weight}\mathrm{of}\mathrm{Sunitinib}\mathrm{in}\mathrm{carrier}}*100\%$$\mathrm{Drug}\mathrm{loading}\mathrm{effiency}\left(\%\right)=\frac{w\mathrm{eight}\mathrm{of}\mathrm{Sunitinib}\mathrm{in}\mathrm{carrier}}{w\mathrm{eight}\mathrm{of}\mathrm{Sunitinib}\mathrm{in}\mathrm{feed}}*100\%$

[0146] Drug Leaching/Release Study

[0147] To evaluate the release/leaching of the described carrier system a dialysis setup was used. To hinder sink conditions a small volume of loaded micelles were dialyzed against big excess of dialysis medium (cut-off 3.5 kDa). The medium was MilliQ in case of leaching study. For release study to the medium 10 mM of GSH were added. Also, the MilliQ water was degassed to hinder further oxidation of GSH due to oxygen. The samples were placed on a shaking plate and were incubated for 7 days at 37° C. At different time intervals the drug content in the sample was evaluated using the above described UV protocol.

[0148] Physiochemical Characterization

[0149] The synthesized micelles (empty, and drug loaded) were characterized by several techniques. Their size in solution was analyzed via dynamic light scattering (DLS). Here, native micelles and drug loaded micelles were analyzed in Milli-Q water. Interestingly, both, empty and loaded micelles showed similar size ranges of 52 nm (d.nm, intensity) with a defined polydispersity of 0.19 (cf. Table 1 and FIG. 10). Furthermore, the surface charge of the micelles was measured by zeta potential measurements (ZP) and showed negatively charged particles (−66 mV, MilliQ-water) due to their sulfated groups at the micellar surface. Besides that, also loaded micelles showed a similar surface charge, and it can be assumed that the drug is located inside the hydrophobic parts of the micellar arrangement. By Cryo-TEM, slightly decreased micellar sizes were measured (21 nm) which can be explained by the methodical differences of the two measurements. In the Cryo-TEM, only the “naked” particles size was measured, whereas in the DLS measurements the hydrodynamic diameter is measured, which also takes the hydrate shell of the micelles into account and results in bigger diameter values. Micelles based on dPGS-SS-PLGA showed similar physiochemical properties.

TABLE-US-00001 TABLE 1 Characterization of unloaded dPGS-SS-PCL/PLGA micelles with DLS (c = 1 mg/mL). Molecular Diameter weight [nm, CMC CMC micelle (g/mol) intensity] PDI (μg/mL) (mM) dPGS-SS-PCL 17000 52 0.19 5.4 3.18E−04 dPGS-SS-PLGA 17000 52 0.15 1.82 1.07E−04

[0150] Drug Loading of the Micelles (CMC/DLC)/Cryo-Protection

[0151] The micelles were loaded with the model drug sunitinib (20 wt %). Here, decent loading efficiencies of 13 wt % for dPGS-SS-PCL and 14.2% for dPGS-SS-PLGA were observed. Interestingly, freeze dried loaded micelles (purified) showed similar physiochemical properties after resuspension in Milli-Q water, compared to the initial ones (52 nm, d.nm, intensity; 13 wt % DLC) (FIGS. 11A and 11B).

TABLE-US-00002 TABLE 2 Characterization of Sunitinib-loaded dPGS-SS-PCL/PLGA micelles with DLS. Release, Diameter DLC.sub.theo DLC.sub.UV DLE Leaching GSH CMC CMC micelle [nm, int.] PDI [wt %] [wt %] [%] [%].sup.a [%].sup.b (μg/mL) (mM) dPGS-SS-PCL 52.2 0.33 20 13 65 below 1 99 0.50 2.94E−05 dPGS-SS-PLGA 59.2 0.25 20 14.2 71 below 1 99 1.14 6.71E−05 .sup.aafter 7 days, 37° C., MilliQ. .sup.bafter 7 days, 37° C., MilliQ, 10 mM GSH.

TABLE-US-00003 TABLE 3 Comparison of original/resuspended PCL-micelles in respect to obtained micellar size PDI. Sephadex Freeze-dried, filtered Size (nm, intensity) PDI Size (nm, intensity) PDI 52 0.31 96 0.24

[0152] In addition, the influence of encapsulated drug in respect to the CMC was investigated. Surprisingly, the CMC of loaded PCL-micelles (2.94×10.sup.−5 mM) decreased by factor 11 compared to the unloaded micelles, shown in FIG. 12. The same phenomenon was observed for the PLGA analogues with factor 0.6. The low CMC values underline the high stability of the described micellar system. Interestingly, sunitinib stabilized the micellar system in the dry state via intermolecular interactions with the hydrophobic core, which allows a freeze drying/resuspension protocol without any cryo-protection agents.

[0153] In combination with the high loading values, the micelles were promising candidates as drug carrier system (DDS). The stability was further investigated by drug release/leaching studies in a physiological dialysis setup.

[0154] Drug Release/Leaching

[0155] The loaded micelles and their properties to stabilize and release the hydrophobic drug was analyzed in a dialysis setup. Here, no drug leaching was observed within 1 week for the dPGS-SS-PCL/PLGA micelles in aqueous media (FIG. 13). However, in the presence of 10 mM glutathione (level in cytosol of cancerous cells) the disulfide linkage between the dPGS and the hydrophobic core units can be cleaved and triggers a micellar destabilization and associated drug release. The release study revealed a 50% drug release for the dPGS-SS-PCL after 3.5 days, and 50% drug releases for the PLGA analog after 2 days. The measurements of the dialysis setup confirm the findings from the physiochemical property measurements and underline the stability of the micellar system which can release the drug in a controlled manner und reductive environment. In comparison to that, micelles with a smaller molecular weight showed higher leaching and faster drug release characteristics.

[0156] Cellular Studies

[0157] Cell Viability Assay.

[0158] HeLa cells were seeded in a transparent 96 well plate with a density of 10 000 cells per well and cultured for 24 hours. The medium (DMEM) was removed and replaced with medium containing PCL-SS-dPGS micelles (empty), followed by 48 hours of incubation. Subsequently, 10 μl of the pre-mixed Cell counting kit-8 (CCK-8) solution (Dojindo Molecular Technologies, Inc., Rockville, USA), containing the proprietary WST-8 tetrazolium salt, were added to each well. Viable cells reduce this salt to a formazan dye whose absorbance can be measured in the medium. Absorbance was measured at 450 nm using a Tecan Infinite 200 Pro microplate reader after two hours. Three independent experimental runs with triplicates each were performed (n=3).

[0159] In vitro experiments were carried out to study the ability of the micelles to deliver drugs. In order to evaluate the optimal micellar concentration for the delivery of pharmaceutical active compounds, the highest non-toxic concentration was investigated using a CCK-8 cell viability assay during the period of 48 hours. The output of these measurements was that the empty carrier is non-toxic up to a concentration of 1.25 mg/mL (FIG. 14). The IC-50 values of encapsulated and free drug are in the same order of magnitude and are in good agreement to the stability, leaching and release studies (FIGS. 15A to 15C). Thus, the micelles appear to have high potency for the delivery of hydrophobic active drugs in-vitro and in-vivo.

[0160] In the following, additional information on the surface-potential and biodegradation of the micellar polymer systems of dPGS-SS-PCL and dPGS-SS-PLGA is provided. The formed micelles of dPGS-SS-PCL and dPGS-SS-PLGA were further characterized by their negative surface charge of the micellar outer identity and their responsiveness to GSH. As explained above, the polymer architecture comprises a hydrophobic segment, either PCL or PLGA, and a hydrophilic block, here dPGS, connected by a disulfide bridge (—S—S—) forming the amphiphilic copolymer. This structural segment is aimed to be cleaved under tumor conditions such as in the presence of GSH. Due to the sulfation (—ROSO.sub.3.sup.−Na.sup.+) of the dendritic polyglycerol moieties (R—OH), the micelles possess a negative surface potential.

[0161] Biodegradation of dPGS-SS-PCL and dPGS-SS-PLG by GSH

[0162] The biodegradation of the dPGS micelles was studied by DLS and cryo-TEM measurements in more detail. The results are presented in Table 4. Upon incubation with 10 mM GSH, the PCL/PLGA-micelles changed their size, indicating that the disulfide bridge in the polymer structure got cleaved (FIGS. 16 and 17). The micelles formed by dPGS-SS-PCL revealed a size of 77 nm and micelles formed by dPGS-SS-PLGA of 131 nm in PBS, respectively. Upon the addition of reducing agents, the micelles shrank in their size, ending up in sizes of 52 nm and 117 nm for the PCL and PLGA amphiphilic copolymers.

TABLE-US-00004 TABLE 4 Characteristics of dPGS-SS-PCL and dPGS-SS-PLGA micelles regarding their size, biodegradation upon incubation with 10 mM GSH, and zeta-potential. Diameter Diameter (int. nm) Zeta-potential micelle [nm, int.].sup.a 10 mM GSH, 24 h.sup.b [mV].sup.c dPGS-SS-PCL 77 52 −44.7 dPGS-SS-PLGA 131 117 −43.0 .sup.ameasured in PBS buffer at 25° C., pH 7.4 .sup.bmeasured in PBS buffer, pH 5.5 .sup.cmeasured in PB buffer at 25° C., pH 7.4

[0163] By evaluating cryo-TEM micrographs of dPGS-SS-PCL micelles in PBS and after 24 h treatment with 10 mM GSH it could be shown that mot only a size shrinkage of the micelles occurs. Rather, the micelles fall apart when the disulfide bridge is cleaved by GSH. As observed by cryo-TEM, the micelles have a size of 60 nm in their native states, whereas the micelle size shrank to 40 nm in the presence of GSH. The TEM micrographs showed the spherical character of the formed micelles. In addition, the detected sizes by TEM matched the sizes determined by DLS.

[0164] Furthermore, a decreased number of particles was observed, indicating the disruption of micelles under reductive conductions supporting precise drug-release under tumor mirroring conditions. This observation matches with the previously performed release study where a promoted drug-release was detected in the presence of 10 mM GSH. Interestingly, several micelles were stable in the first 24 h treatment with GSH. However, this result correlates with the sustainable release profile of the discussed drug delivery system. The cleavage of the disulfide bridge breaks down the micelles in a steady process leading to a prolonged drug release.

CONCLUSION

[0165] In summary, the additional measurements proof the anionic character of the present amphiphilic copolymers and their respective micelles as shown by the zeta potentials. Furthermore, biodegradation studies confirmed that GSH causes a disruption of the micelles, thus promoting a selectively drug-release at the desired site of action, e.g., cancerous tissue. The disulfide bridge in the polymer structure is sensitive against reducing agents.

LIST OF REFERENCES

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