ß-Cyclodextrin-Based Star-Shaped Polymer, a Preparation Method Therefor and an Integrated Unimolecular Micelle System for Diagnosis and Treatment Thereof

Abstract

Disclosed is a -cyclodextrin-based star-shaped polymer and a preparation method therefor, and an integrated unimolecular micelle system for diagnosis and treatment based on the star-shaped polymer and the use thereof. The polymer has a structure represented by the following formula (I):

##STR00001## wherein x=4-15, y=3-20, and z=10-30.

Claims

1. An amphipathic pH-responsive -cyclodextrin-based star-shaped polymer with the structure represented by the following formula (I): ##STR00009## wherein x=4-15, y=3-20, z=10-30.

2. A method for preparing the amphipathic pH-responsive -cyclodextrin-based star-shaped polymer according to claim 1, comprising: (1) preparation of a pH-responsive monomer precursor (tBAM), wherein tert-butyl N-(2-hydroxyethyl)carbamate, N,N-diisopropylethylamine are mixed in a solvent, and methacryloyl chloride is added under ice bath condition; the reaction is carried out under ice bath condition and subsequently at room temperature to obtain the precursive pH-responsive monomer t-butyl methacrylate-2-carbamate (tBAM); (2) preparation of a polymer containing polycaprolactone -CD-(PCL-OH).sub.21, wherein -CD, -CL and a catalyst are mixed and heated to react, so as to obtain the polymer -CD-(PCL-OH).sub.21; (3) preparation of a macroinitiator -CD-(PCL-Br).sub.21, wherein the -CD-(PCL-OH).sub.21 prepared in step (2) is dissolved in a solvent; triethylamine (TEA) and initiator 2-bromoisobutyryl bromide (BIBB) are then added under ice bath; the reaction is carried out under ice bath condition and subsequently at room temperature to obtain the macroinitiator -CD-(PCL-Br).sub.21; (4) preparation of a pH-responsive polymer precursor -CD-(PCL-b-PtBAM).sub.21: the macroinitiator -CD-(PCL-Br).sub.21 prepared in step (3), the precursor tBAM prepared in step (1) and a catalyst are dissolved in a solvent, after which a ligand 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) is further added; then a reducing agent is introduced and the solution is heated to react to obtain the pH-responsive polymer -CD-(PCL-b-PtBAM).sub.21; (5) preparation of an amphipathic star-shaped polymer -CD-(PCL-b-tBAM-b-PPEGMA).sub.21, wherein the pH-responsive polymer precursor -CD-(PCL-b-PtBAM).sub.21 prepared in step (4), poly(ethylene glycol) methyl ether methacrylate (PEGMA), a ligand 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) and a catalyst are dissolved in a solvent; after mixing homogeneously, a reducing agent is added and the solution is heated to react to obtain the amphipathic star-shaped polymer -CD-(PCL-b-tBAM-b-PPEGMA).sub.21; (6) preparation of an amphipathic pH-responsive -cyclodextrin-based star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21, wherein the amphiphilic star-shaped polymer prepared in step (5) is dissolved in a solvent, and after addition of trifluoroacetic acid (TFA), the reaction is carried out under ice bath condition and subsequently at room temperature to obtain the amphipathic pH-responsive -cyclodextrin-based star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21.

3. The method for preparing the amphipathic pH-responsive -cyclodextrin-based star-shaped polymer according to claim 2, wherein the molar parts of the reactants in step (1) are: TABLE-US-00007 tert-butyl N-(2-hydroxyethyl)carbamate 1 N,N-diisopropylethylamine 1-3 methacryloyl chloride 1-3 the molar parts of the reactants in step (2) are: TABLE-US-00008 -CD 1 -CL 84-315 the molar parts of the reactants in step (3) are: TABLE-US-00009 -CD-(PCL-OH).sub.21 1 TEA 21-84 BIBB 21-84 the molar parts of the reactants in step (4) are: TABLE-US-00010 macroinitiator (-CD-(PCL-Br).sub.21) 1 tBAM 63-420 HMTETA 8-12 the molar parts of the reactants in step (5) are: TABLE-US-00011 pH-responsive polymer precursor 1 PEGMA 210-630 HMTETA 8-12 the molar parts of the reactants in step (6) are: TABLE-US-00012 amphipathic star-shaped polymer 1 TFA 30-60.

4. The method for preparing the amphipathic pH-responsive -cyclodextrin-based star-shaped polymer according to claim 2, wherein, in step (1), the reaction is carried out under ice bath condition for 0.5-4 h, and subsequently at room temperature for 24-48 h.

5. The method for preparing the amphipathic pH-responsive -cyclodextrin-based star-shaped polymer according to claim 2, wherein: in step (2) the solution is heated to 90-130 C. to react for 24-48 h; in step (3) the reaction is carried out under ice bath condition for 4-6 h, and subsequently at room temperature for 24-48 h; in step (4) the solution is heated to 60-90 C. to react for 24-48 h; in step (5) the solution is heated to 60-90 C. to react for 48-96 h; in step (6) the reaction is carried out under ice bath condition for 0.5-4 h, and subsequently at room temperature for 4-10 h.

6. A unimolecular micelle system comprising the amphipathic pH-responsive star-shaped polymer according to claim 1, wherein the micelle system is obtained by dissolving the amphiphilic pH-responsive -cyclodextrin-based star-shaped polymer as claimed in claim 1 in a solvent.

7. A method of loading water-insoluble drug(s) comprising the steps of: dissolving a water-insoluble drug in an organic solvent, mixing the unimolecular micelle system of claim 6 with the water-insoluble drug solution, stirring homogeneously, and dialyzing to obtain a water-insoluble-drug-loaded micelle system.

8. A gold-nanoparticles (AuNPs)-loaded composite material based on the amphipathic pH-responsive -cyclodextrin-based star-shaped polymer unimolecular micelle system according to claim 6, wherein after the amphiphilic pH-responsive -cyclodextrin-based star-shaped polymer and the water-soluble gold salt are respectively dissolved in the same solvent, the amphiphilic pH-responsive -cyclodextrin-based star-shaped polymer solution and the water-soluble gold salt solution are mixed and stirred to obtain an AuNPs-loaded composite material.

9. A method for CT imaging a patient in need thereof, comprising administering to the patient the AuNPs-loaded composite material of claim 8.

10. A method of loading water-insoluble drug(s) comprising the steps of: dissolving a water-insoluble drug in an organic solvent, mixing the AuNPs-loaded composite material of claim 8 with the water-insoluble drug solution, stirring homogeneously, and dialyzing to obtain a water-insoluble-drug-loaded micelle system.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is the .sup.1H NMR spectrum of pH-responsive precursor tBAM in Embodiment 1.

[0066] FIG. 2 is the .sup.13C NMR spectrum of pH-responsive precursor tBAM in Embodiment 1.

[0067] FIG. 3 shows the .sup.1H NMR spectra of -CD-(PCL-OH).sub.21 (A), macroinitiator -CD-(PCL-Br).sub.21 (B), -CD-(PCL-b-PtBAM).sub.21 (C), amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21 (D), and amphipathic pH-sensitive star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21 (E) in Embodiment 3.

[0068] FIG. 4 shows the GPC traces of -CD-(PCL-OH).sub.21 (A), macroinitiator -CD-(PCL-Br).sub.21 (B), -CD-(PCL-b-PtBAM).sub.21 (C), amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21 (D), and amphipathic pH-sensitive star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21 (E) in Embodiment 3.

[0069] FIG. 5 shows the FT-IR spectra of -CD-(PCL-OH).sub.21 (A), macroinitiator -CD-(PCL-Br).sub.21 (B), -CD-(PCL-b-PtBAM).sub.21 (C), amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21 (D), and amphipathic pH-sensitive star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21 (E) in Embodiment 3.

[0070] FIG. 6 shows the DLS plot of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21 unimolecular micelles in Embodiment 6.

[0071] FIG. 7 shows the TEM image of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21 unimolecular micelles in Embodiment 6.

[0072] FIG. 8 is the UV-vis absorption spectra of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au with different molar ratios of [AEMA]:[HAuCl.sub.4] in Embodiment 7.

[0073] FIG. 9 shows the TEM image of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au in Embodiment 7.

[0074] FIG. 10 shows the XRD plot of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au in Embodiment 7.

[0075] FIG. 11 shows the loading amount, entrapment efficiency, and particle size of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au/DOX in Embodiment 8.

[0076] FIG. 12 illustrates the in vitro drug release profiles of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au/DOX in Embodiment 9.

[0077] FIG. 13 illustrates the cytotoxicity of blank micelles and -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au in Embodiment 10.

[0078] FIG. 14 illustrates the cytotoxicity of drug-loaded micelles in Embodiment 11.

[0079] FIG. 15 shows the CT values of in vitro materials in Embodiment 11.

[0080] FIG. 16 shows the CT values of in vitro cells in Embodiment 11.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0081] The present invention will be further described in detail with reference to the embodiments and drawings, which form a part hereof. But the embodiments of the invention are not meant to be limiting thereto.

[0082] The sources of reagents used in the following embodiments are all commercially available.

Embodiment 1: pH-Responsive Monomer Precursor (tBAM)

[0083] A 250 mL Schlenk flask was dried by a spirit lamp for 10 min. After cooling down, a magnetic stirring bar and tert-butyl N-(2-hydroxyethyl)carbamate (5 g, 31 mmol) were added to the flask, which was sealed by a rubber stopper. The flask was evacuated to vacuum and filled with argon for three times. Then under Ar atmosphere, 100 mL dichloromethane (DCM) and N,N-diisopropylethylamine (16.2 mL, 93 mmol) were added in sequence. Methacryloyl chloride (3.3 mL, 31 mmol) was further added dropwise under ice bath condition. The reaction was carried out at 0 C. for 4 h and then at room temperature for another 24 h. The mixture was extracted successively with water, 400 mL 10% citric acid, 400 mL saturated NaHCO.sub.3 and 400 mL saturated NaCl. The organic phase was collected and dried through Na.sub.2SO.sub.4 followed by rotary evaporation to remove part of the solvent. The obtained white liquid was precipitated by n-hexane, filtered, and finally dried under vacuum (40 C., 35 mb) for 24 h to obtain the product.

[0084] The reaction formula is shown in formula (1). The structure and composition of the monomer were analyzed using .sup.1H NMR and .sup.13C NMR, and the results are shown in FIGS. 1 and 2.

##STR00003##

Embodiment 2: Precursor pH-Responsive Monomer (tBAM)

[0085] A 250 mL Schlenk flask was dried by a spirit lamp for 10 min. After cooling down, a magnetic stirring bar and tert-butyl N-(2-hydroxyethyl)carbamate (5 g, 31 mmol) were added to the flask, which was sealed by a rubber stopper. The flask was evacuated to vacuum and flushed with argon for three times. Then under Ar atmosphere, 100 mL dichloromethane (DCM) and N,N-diisopropylethylamine (5.4 mL, 31 mmol) were added in sequence. Methacryloyl chloride (9.9 mL, 93 mmol) was further added dropwise under ice bath condition. The reaction was carried out at 0 C. for 0.5 h and then at room temperature for another 48 h. The mixture was extracted successively with water, 400 mL 10% citric acid, 400 mL saturated NaHCO.sub.3 and 400 mL saturated NaCl. The organic phase was collected and dried through Na.sub.2SO.sub.4 followed by rotary evaporation to remove part of the solvent. The obtained white liquid was precipitated by n-hexane, filtered, and finally dried under vacuum (40 C., 35 mb) for 24 h to obtain the product.

Embodiment 3: Preparation of an Amphipathic pH-Sensitive Star-Shaped Polymer -CD-(PCL-b-PAEMA-b-PPEGMA).SUB.21 .(x:y:z=6:11:17)

[0086] (1) Preparation of a polycaprolactone polymer -CD-(PCL-OH).sub.21: A 50 mL Schlenk flask was dried by a spirit lamp for 10 min. After cooling down, a magnetic stirring bar and -CD (681 mg, 0.6 mmol) were added to the flask, which was sealed by a rubber stopper. The flask was evacuated to vacuum and flushed with argon for three times. Then under Ar atmosphere, the monomer -CL (8.0 mL, 75.6 mmol) and a required amount of Sn(Oct).sub.2 (0.1 wt. % of -CL, 86 mg) were added into the flask, after which three cycles of freeze-pump-heat with liquid nitrogen were performed. Reaction was then carried out under oil bath at 110 C. for 24 h and argon protection. After the reaction, the crude polymer was dissolved in approximately 50 mL THF followed by adding to 300 mL water/methanol (1:1, v/v) mixture for precipitation. -CD-(PCL-OH).sub.21 was collected and dried under vacuum (40 C., 35 mb).

[0087] The reaction formula is shown in formula (2). The structure and composition of -CD-(PCL-OH).sub.21 were analyzed using .sup.1H NMR, GPC and IR, and the results are shown in FIG. 3-5 (M.sub.n=15499, M.sub.w/M.sub.n=2.75).

##STR00004##

[0088] (2) Preparation of macroinitiator -CD-(PCL-Br).sub.21: A magnetic stirring bar, -CD-(PCL-OH).sub.21 (4650 mg, 0.3 mmol) and anhydrous THF (150 mL) were added to a dried 250 mL three-necked flask, and the flask was flushed with argon for 10 min. Then TEA (2.78 mL, 20 mmol) was injected into the flask after sealing the flask. After the solution was cooled to 0 C. with an ice/water bath, 2-bromoisobutyryl bromide (2.48 mL, 20 mmol) was added dropwise by an injector. The reaction was continued at 0 C. for 5 h and then at room temperature for another 24 h. After reaction, the mixture was passed through a neutral alumina column to remove quaternary ammonium salt. Most of the solvent was removed by rotary evaporation and water/methanol (1:1, v/v) mixture was added to precipitate the product twice, which was then filtered and dried under vacuum.

[0089] The reaction formula is shown in formula (3). The structure and composition were analyzed using .sup.1H NMR, GPC and FT IR, and the results are shown in FIG. 3-5 (M.sub.n=18628, M.sub.w/M.sub.n=1.79).

##STR00005##

[0090] (3) Preparation of pH-responsive polymer precursor -CD-(PCL-b-PtBAM).sub.21: a magnetic stirring bar, macroinitiator -CD-(PCL-Br).sub.21 (560 mg, 0.03 mmol) and CuBr.sub.2 (10 mg, 0.045 mmol) were added to a dried 100 mL eggplant-shaped flask, which was sealed by rubber stopper. The flask was evacuated and flushed with argon for three times. Solvent (20 mL), tBAM (1905 mg, 6.93 mmol) and ligand HMTETA (62 L, 0.24 mmol) were injected sequentially into the flask using syringes. The mixture was stirred for 10 min and Sn(Oct).sub.2 (78 L, 0.24 mmol) dissolved in toluene (1 mL) was added into the flask. After stirring for 5 min, the mixture was heated under 80 C. oil bath for 24 h. The mixture was then cooled to room temperature, and THF (50 mL) was added into the flask followed by removal of catalyst through a neutral alumina column. The concentrated mixture was slowly added to 10-fold volume of n-hexane for precipitation. Finally the mixture was filtered and dried under vacuum (40 C., 35 mb) to obtain the product.

[0091] The reaction formula is shown in formula (4). The structure and composition were analyzed using .sup.1H NMR, GPC and IR, and the results are shown in FIG. 3-5 (M.sub.n=76840, M.sub.w/M.sub.n=1.88).

##STR00006##

[0092] (4) Preparation of an amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21: a magnetic stirring bar, macroinitiator-CD-(PCL-b-PtBAM).sub.21 (2305 mg, 0.03 mmol) and CuBr.sub.2 (10 mg, 0.045 mmol) were added to a dried 100 mL eggplant-shaped flask, which was sealed by rubber stopper. The flask was evacuated and flushed with argon for three times. Solvent (30 mL), monomer PEGMA (5985 mg, 10.71 mmol) and ligand HMTETA (62 L, 0.24 mmol) were added sequentially into the flask using degassed syringes. The mixture was stirred for 10 min and Sn(Oct).sub.2 (78 L, 0.24 mmol) dissolved in toluene (1 mL) was added into the flask. After stirring for 5 min, the mixture was heated under 80 C. oil bath for 72 h. The mixture was then cooled to room temperature, and THF (50 mL) was added into the flask followed by removal of catalyst through a neutral alumina column. The concentrated mixture was slowly added to 10-fold volume of n-hexane for precipitation. Finally the mixture was filtered and dried under vacuum (40 C., 35 mb) to obtain the amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21.

[0093] The reaction formula is shown in formula (5). The structure and composition were analyzed using .sup.1H NMR, GPC and IR, and the results are shown in FIG. 3-5 (246415, M.sub.w/M.sub.n=2.42).

##STR00007##

[0094] (5) Preparation of amphipathic pH-responsive -cyclodextrin-based star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21: -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21 (1232 mg, 0.005 mmol) was dissolved in 20 mL DCM in a 100 mL round bottom flask. After cooling to 0 C. with ice/water bath, TFA (15 mL, 0.20 mmol) was slowly injected with vigorous stirring. The mixture was stirred at 0 C. for 2 h and then at room temperature for 6 h to remove tert-butyl ester. After removing all the solvent by rotary evaporation, THF (10 mL) was added into the flask and then the oil phase was precipitated by 200 mL n-hexane followed by drying overnight. Afterwards, the obtained polymer was washed with 0.5 M NaOH solution until pH=8.0, and immediately placed into a dialysis bag for three days. The final polymer was obtained by lyophilization (yield 98%).

[0095] The reaction formula is shown in formula (6). The structure and composition were analyzed using .sup.1H NMR, GPC and IR, and the results are shown in FIG. 3-5 (M.sub.n=218002, M.sub.w/M.sub.n=1.94).

##STR00008##

Embodiment 4: Amphipathic pH-Sensitive Star-Shaped Polymer -CD-(PCL-b-PAEMA-b-PPEGMA).SUB.21 .(x:y:z=4:20:30)

[0096] (1) Preparation of a polycaprolactone polymer -CD-(PCL-OH).sub.21: A 50 mL Schlenk flask was dried by a spirit lamp for 10 min. After cooling down, a magnetic stirring bar and -CD (681 mg, 0.6 mmol) were added to the flask, which was sealed by a rubber stopper. The flask was evacuated to vacuum and flushed with argon for three times. Then under Ar atmosphere, the monomer -CL (5.4 mL, 50.4 mmol) and a required amount of Sn(Oct).sub.2 (0.1 wt. % of -CL, 86 mg) were added into the flask, after which three cycles of freeze-pump-heat with liquid nitrogen were performed. Reaction was then carried out under oil bath at 90 C. for 48 h and argon protection. After the reaction, the crude polymer was dissolved in approximately 50 mL THF followed by adding to 300 mL water/methanol (1:1, v/v) mixture for precipitation. -CD-(PCL-OH).sub.21 was collected and dried under vacuum (40 C., 35 mb). The reaction formula is shown in formula (2). M.sub.n=9576, M.sub.w/M.sub.n=1.86.

[0097] (2) Preparation of macroinitiator -CD-(PCL-Br).sub.21: A magnetic stirring bar, -CD-(PCL-OH).sub.21 (2872 mg, 0.3 mmol) and anhydrous THF (150 mL) were added to a dried 250 mL three-necked flask, and the flask was flushed with argon for 10 min. Then TEA (0.93 mL, 6.67 mmol) was injected into the flask after sealing the flask. After the solution was cooled to 0 C. with an ice/water bath, 2-bromoisobutyryl bromide (0.83 mL, 6.67 mmol) was added dropwise by an injector. The reaction was continued at 0 C. for 6 h and then at room temperature for another 24 h. After reaction, the mixture was passed through a neutral alumina column to remove quaternary ammonium salt. Most of the solvent was removed by rotary evaporation and water/methanol (1:1, v/v) mixture was added to precipitate the product twice, which was then filtered and dried under vacuum. M.sub.n=10711, M.sub.w/M.sub.n=1.93

[0098] (3) Preparation of pH-responsive polymer precursor -CD-(PCL-b-PtBAM).sub.21: a magnetic stirring bar, macroinitiator -CD-(PCL-Br).sub.21 (322 mg, 0.03 mmol) and CuBr.sub.2 (10 mg, 0.045 mmol) were added to a dried 100 mL eggplant-shaped flask, which was sealed by rubber stopper. The flask was evacuated and flushed with argon for three times. Solvent (20 mL), tBAM (3463 mg, 12.6 mmol) and ligand HMTETA (78 L, 0.3 mmol) were injected sequentially into the flask using syringes. The mixture was stirred for 10 min and Sn(Oct).sub.2 (97.5 L, 0.3 mmol) dissolved in toluene (1 mL) was added into the flask. After stirring for 5 min, the mixture was heated under 60 C. oil bath for 48 h. The mixture was then cooled to room temperature, and THF (50 mL) was added into the flask followed by removal of catalyst through a neutral alumina column. The concentrated mixture was slowly added to 10-fold volume of n-hexane for precipitation. Finally the mixture was filtered and dried under vacuum (45 C., 35 mb) to obtain the product. M.sub.n=116551, M.sub.w/M.sub.n=1.69

[0099] (4) Preparation of an amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21: a magnetic stirring bar, macroinitiator -CD-(PCL-b-PtBAM).sub.21 (3495 mg, 0.03 mmol) and CuBr.sub.2 (10 mg, 0.045 mmol) were added to a dried 100 mL eggplant-shaped flask, which was sealed by rubber stopper. The flask was evacuated and flushed with argon for three times. Solvent (30 mL), monomer PEGMA (10561 mg, 10.71 mmol) and ligand HMTETA (78 L, 0.3 mmol) were added sequentially into the flask using degassed syringes. The mixture was stirred for 10 min and Sn(Oct).sub.2 (97.5 L, 0.3 mmol) dissolved in toluene (1 mL) was added into the flask. After stirring for 5 min, the mixture was heated under 90 C. oil bath for 48 h. The mixture was then cooled to room temperature, and THF (50 mL) was added into the flask followed by removal of catalyst through a neutral alumina column. The concentrated mixture was slowly added to 10-fold volume of n-hexane for precipitation. Finally the mixture was filtered and dried under vacuum (45 C., 35 mb) to obtain the amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21. M.sub.n=415801, M.sub.w/M.sub.n=2.21

[0100] (5) Preparation of amphipathic pH-responsive -cyclodextrin-based star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21: -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21 (2079 mg, 0.005 mmol) was dissolved in 20 mL DCM in a 100 mL round bottom flask. After cooling to 0 C. with ice/water bath, TFA (22.5 mL, 0.30 mmol) was slowly injected with vigorous stirring. The mixture was stirred at 0 C. for 2 h and then at room temperature for 10 h to remove tert-butyl ester. After removing all the solvent by rotary evaporation, THF (10 mL) was added into the flask and then the oil phase was precipitated by 200 mL n-hexane followed by drying overnight. Afterwards, the obtained polymer was washed with 0.5 M NaOH solution until pH=8.0, and immediately placed into a dialysis bag for three days. The final polymer was obtained by lyophilization (yield 98%). M.sub.n=364141, M.sub.w/M.sub.n=1.77

Embodiment 5: Amphipathic pH-Sensitive Star-Shaped Polymer -CD-(PCL-b-PAEMA-b-PPEGMA).SUB.21 .(x:y:z=15:3:10)

[0101] (1) Preparation of a polycaprolactone polymer -CD-(PCL-OH).sub.21: A 50 mL Schlenk flask was dried by a spirit lamp for 10 min. After cooling down, a magnetic stirring bar and -CD (681 mg, 0.6 mmol) were added to the flask, which was sealed by a rubber stopper. The flask was evacuated to vacuum and flushed with argon for three times. Then under Ar atmosphere, the monomer -CL (20.25 mL, 189 mmol) and a required amount of Sn(Oct).sub.2 (0.1 wt. % of -CL, 86 mg) were added into the flask, after which three cycles of freeze-pump-heat with liquid nitrogen were performed. Reaction was then carried out under oil bath at 130 C. for 24 h and argon protection. After the reaction, the crude polymer was dissolved in approximately 50 mL THF followed by adding to 300 mL water/methanol (1:1, v/v) mixture for precipitation. -CD-(PCL-OH).sub.21 was collected and dried under vacuum (45 C., 35 mb). The reaction formula is shown in formula (2). M.sub.n=35910, M.sub.w/M.sub.n=2.16.

[0102] (2) Preparation of macroinitiator -CD-(PCL-Br).sub.21: A magnetic stirring bar, -CD-(PCL-OH).sub.21 (10773 mg, 0.3 mmol) and anhydrous THF (150 mL) were added to a dried 250 mL three-necked flask, and the flask was flushed with argon for 10 min. Then TEA (3.72 mL, 27 mmol) was injected into the flask after sealing the flask. After the solution was cooled to 0 C. with an ice/water bath, 2-bromoisobutyryl bromide (3.32 mL, 27 mmol) was added dropwise by an injector. The reaction was continued at 0 C. for 4 h and then at room temperature for another 48 h. After reaction, the mixture was passed through a neutral alumina column to remove quaternary ammonium salt. Most of the solvent was removed by rotary evaporation and water/methanol (1:1, v/v) mixture was added to precipitate the product twice, which was then filtered and dried under vacuum. M.sub.n=37045, M.sub.w/M.sub.n=1.76

[0103] (3) Preparation of pH-responsive polymer precursor -CD-(PCL-b-PtBAM).sub.21: a magnetic stirring bar, macroinitiator -CD-(PCL-Br).sub.21 (1112 mg, 0.03 mmol) and CuBr.sub.2 (10 mg, 0.045 mmol) were added to a dried 100 mL eggplant-shaped flask, which was sealed by rubber stopper. The flask was evacuated and flushed with argon for three times. Solvent (20 mL), tBAM (520 mg, 1.89 mmol) and ligand HMTETA (93 L, 0.36 mmol) were injected sequentially into the flask using syringes. The mixture was stirred for 10 min and Sn(Oct).sub.2 (117 L, 0.36 mmol) dissolved in toluene (1 mL) was added into the flask. After stirring for 5 min, the mixture was heated under 80 C. oil bath for 24 h. The mixture was then cooled to room temperature, and THF (50 mL) was added into the flask followed by removal of catalyst through a neutral alumina column. The concentrated mixture was slowly added to 10-fold volume of n-hexane for precipitation. Finally the mixture was filtered and dried under vacuum (45 C., 35 mb) to obtain the product. M.sub.n=52921, M.sub.w/M.sub.n=1.88

[0104] (4) Preparation of an amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21: a magnetic stirring bar, macroinitiator -CD-(PCL-b-PtBAM).sub.21 (1588 mg, 0.03 mmol) and CuBr.sub.2 (10 mg, 0.045 mmol) were added to a dried 100 mL eggplant-shaped flask, which was sealed by rubber stopper. The flask was evacuated and flushed with argon for three times. Solvent (30 mL), monomer PEGMA (3520 mg, 6.3 mmol) and ligand HMTETA (93 L, 0.36 mmol) were added sequentially into the flask using degassed syringes. The mixture was stirred for 10 min and Sn(Oct).sub.2 (117 L, 0.36 mmol) dissolved in toluene (1 mL) was added into the flask. After stirring for 5 min, the mixture was heated under 60 C. oil bath for 96 h. The mixture was then cooled to room temperature, and THF (50 mL) was added into the flask followed by removal of catalyst through a neutral alumina column. The concentrated mixture was slowly added to 10-fold volume of n-hexane for precipitation. Finally the mixture was filtered and dried under vacuum (45 C., 35 mb) to obtain the amphipathic star-shaped polymer -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21.M.sub.n=152641, M.sub.w/M.sub.n=2.03

[0105] (5) Preparation of amphipathic pH-responsive -cyclodextrin-based star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21: -CD-(PCL-b-PtBAM-b-PPEGMA).sub.21 (764 mg, 0.005 mmol) was dissolved in 20 mL DCM in a round bottom flask. After cooling to 0 C. with ice/water bath, TFA (7.5 mL, 0.10 mmol) was slowly injected with vigorous stirring. The mixture was stirred at 0 C. for 4 h and then at room temperature for 4 h to remove tert-butyl ester. After removing all the solvent by rotary evaporation, THF (10 mL) was added into the flask and then the oil phase was precipitated by 200 mL n-hexane followed by drying overnight. Afterwards, the obtained polymer was washed with 0.5 M NaOH solution until pH=8.0, and immediately placed into a dialysis bag for three days. The final polymer was obtained by lyophilization (yield 98%). M.sub.n=144922, M.sub.w/M.sub.n=1.92

Embodiment 6: Preparation of Amphipathic pH-Responsive Star-Shaped Polymers Unimolecular Micelles

[0106] Unimolecular micelles were prepared by dialysis. Briefly, amphipathic the pH-sensitive star-shaped polymer -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21 (100 mg) obtained in embodiment 3 was dissolved in DMSO (40 mL) and stirred for 4 h. The solution was then dialyzed against water for 24 h using a dialysis bag, wherein the fresh deionized water was replaced every 2 h during the first 12 h and then every 6 h during the following 12 h. After dialysis, the dialyzed solution were filtered by a membrane filter (0.45 m pore) and lyophilized to obtain white powdery blank unimolecular micelles.

[0107] Particle size (Dh), distribution (PDI), and zeta potential of the blank unimolecular micelles were measured by dynamic light scattering (DLS). The particle size is 18 nm (FIG. 6), PDI is 0.41 and zeta potential is 15.6 mV. The particle size determined by TEM image was around 10.5 nm (FIG. 7).

Embodiment 7: Preparation of Unimolecular Micelle-Stabilized AuNPs

[0108] Briefly, -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21 obtained in embodiment 5 (100 mg, [PAEMA]=2.4 mM) and 20 mL deionized water were added to a 50 mL beaker with stirring to dissolve for 2 h. HAuCl.sub.4 (24 mM, [DMAEMA]:[HAuCl.sub.4]=1/5/10) solution was further added dropwise to the polymer solution with stirring to mix together. Then NaBH.sub.4 ([NaBH.sub.4]: [HAuCl.sub.4]=3:1) was added dropwise to the mixture solution, after which the reaction was carried out at room temperature in darkness for 48 h to obtain gold-nanoparticle-loaded composite material -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au. With increasing concentrations of HAuCl.sub.4, the maximum wavelength (.sub.max) of AuNPs shown in UV-vis spectra were 522 nm, 524 nm, and 540 nm, which were induced by the plasma resonance excitation or interband transition of gold nanoparticle and were characteristic surface plasma resonance absorption peak of spherical gold nanoparticle (FIG. 8). The AuNP size under TEM was 3.5 nm when [AEMA]:[HAuCl.sub.4]=5 (FIG. 9). X-ray diffraction spectrum in FIG. 10 shows peaks at 38.5, 44.8, 64.2 and 78.0 corresponding to (111), (200), (220) and (311) crystal faces of standard gold diffraction spectrum, respectively, which proves the face-centered-cubic (fcc) crystal structure of gold nanoparticle.

Embodiment 8: Preparation of Amphipathic pH-Responsive Star-Shaped Polymer Drug-Loaded Micelles

[0109] The amphipathic pH-responsive star-shaped polymer drug-loaded micelles were prepared by dialysis. DOXHCl (1050 mg) was first mixed with 2 times molar equivalent of triethylamine in 20 mL DMSO followed by stirring overnight to obtain DOX base. 100 mg -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au obtained in Embodiment 7 was mixed with another 20 mL DMSO and a micelle system was obtained after complete dissolvation. The micelle system was mixed with the DOX solution. After that, the mixture was stirred for 4 h and then dialyzed against deionized water using a dialysis bag, wherein the fresh deionized water was replaced every 2 h during the first 12 h and then every 6 h during the following 12 h. After dialysis, the dialyzed solution was filtered by a membrane filter (0.45 m pore) and lyophilized to obtain red powdery DOX-loaded micelles.

[0110] The DOX-loaded micelles were examined by UV-vis spectrophotometer. The calculated LC was 2.1-17.1%, EE was 21.0-62.5% and particle size was 18.9-30.5 nm (FIG. 11).

Embodiment 9: In Vitro Release of Drug-Loaded Micelles

[0111] The release profiles of DOX under different pH value were measured by dissolution Tester. 5 mg of DOX-loaded micelles obtained in Embodiment 8 were dispersed in 5 mL of PBS buffer whose pH=5.0, 6.5 and 7.4, respectively. Then the solutions were placed in dialysis bags. The whole bags were placed into 46 mL of PBS and then in dissolution tester with constant shaking (100 rpm) at 37 C. to promote in vitro release. At each predetermined time interval, a 3-mL sample was collected for UV analysis, meantime an equal volume of fresh buffer solution was added. The amounts of released DOX in the buffer solution at different time were measured by UV-vis spectrophotometer, as shown in FIG. 12.

[0112] DOX release rate increased obviously as pH decreased from 7.4 to 5.0. At pH 7.4, only 25% of DOX was released after 120 h owing to compact structure of micelles. In the slightly acidic extracellular space of the tumor cell (pH 6.6), approximately 46% of DOX was released at 120 h. In the more acidic (pH 5.0) environment of tumor cell, the release rate of DOX significantly increased, wherein approximately 45% and 81% of DOX was released after 24 h and 120 h, respectively.

Embodiment 10: Cytotoxicity Assay

[0113] 100 L DMEM mediums were added to the surrounding wells of a 96-well plate as control groups, and other wells were seeded with 100 L HepG2 cell culture media (American Type Culture Collection ATCC, HB-8065) with a concentration of 110.sup.4 cells/well. The plate was placed at 37 C. and saturated humidity in a CO.sub.2 (5%) incubator for 24 h. During this process, cells adhered to the bottom of 96-well plates and began to resume growth.

[0114] Subsequently, the blank micelles (product of embodiment 6), -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au obtained in Embodiment 7, DOX-loaded micelles obtained in Embodiment 8, and free DOX were diluted by cell culture medium to different concentration gradients. After removing the cell culture medium from wells 2 to 11 in a 96-well plate, 100 L of the above solutions were added separately as the experimental group. 100 L of fresh cell culture medium was added to column 11 as control groups. Parallel experiments were done six times for each concentration.

[0115] After 48 h incubation, the supernatant from all wells with cells was aspirated, and the cells were rinsed with 200 L PBS, which was aspirated subsequently. From column 2 to column 11, 20 L of MTT solution and 180 L of fresh medium were added to each well, and the 96-well plate was placed in an incubator for 4 h.

[0116] The unreduced MTT solution and medium were aspirated, each well was washed with 200 pt of PBS, which was aspirated subsequently. From column 2 to column 11, 200 L DMSO was added to each well to dissolve the MTT crystals. The 96-well plate was shaken in a 37 C. shaker for 15 min, and then the absorbance of each well at 570 nm was measured using a microplate reader. The relative cell viability (%) was calculated using the equation as follows:

Cell viability (%)=(OD.sub.test/OD.sub.control)100%.

[0117] FIG. 13 shows the results of the cytotoxicity test of blank micelles (Embodiment 6) and -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au (Embodiment 7). As the concentration increases from 0 to 400 g/mL, blank micelles and -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au still exhibit more than 80% cell viability, indicating that the polymer has low cytotoxicity.

[0118] FIG. 14 shows the results of the cytotoxicity test of DOX-loaded micelles in Embodiment 8. Low-dose (0.1 mg/L) DOX-loaded micelles had a lethal effect on cancer cells; DOX-loaded micelles and free doxorubicin were similar in cytotoxicity with more than 77% of the cell killed at high concentrations (20 mg/L), indicating that the loaded DOX still has good anti-cancer activity.

Embodiment 11: CT Imaging Test

[0119] (1) In Vitro CT Imaging Test of the Material

[0120] 29.88 mg of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au/DOX (product of Embodiment 8) was dissolved in 580 L of PBS buffer to prepare a solution with 0.1M gold and then diluted to obtain solutions with different concentration of gold, the volume of which is 200 L respectively. The solutions were then loaded into 1.5 mL centrifuge tubes. The same concentrations of Omnipaque solutions were used as a comparison. CT scanning was then performed to measure the CT signal of each picture.

[0121] (2) In Vitro CT Imaging Test of HepG2

[0122] Six-well plates were used to culture HepG2 cells with a certain amount of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au/DOX material (product of Embodiment 8) for CT scan at a density of 210.sup.6 cells/well. After being cultured over night, the old medium was removed, then 2.35 mL of fresh medium and 250 L of material were added, after which the plate was placed in the incubator and cultivated for 4 h. Finally, the remaining material was washed out with PBS, and the cells were collected and placed in 1.5-mL centrifuge tubes. After centrifugation at 1000 r/min for 5 min, the supernatant was discarded, the cells at the bottom were retained, and the solutions were homogenized with the addition of 0.2 mL of PBS. By comparing the X-ray absorption coefficients, the CT imaging effect of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au/DOX on HepG2 cells was analyzed using an in vitro CT scan.

[0123] CT imaging results for the product of Embodiment 8 were shown in FIG. 15 (in vitro materials CT imaging) and FIG. 16 (in vitro cell CT imaging). With increasing concentrations, the HU values of -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au/DOX and Omnipaque increased, but -CD-(PCL-b-PAEMA-b-PPEGMA).sub.21/Au/DOX had higher HU values than Omnipaque at the same concentrations, indicating that the gold nanoparticles provide high CT imaging efficiency for mice implanted with tumor cells.

[0124] The above described embodiments are preferred embodiments of the present invention, but the embodiments of the invention are not limited by the foregoing embodiments. Other changes, modifications, substitutions, combinations, and simplification made without departing from the spirit or score of the present invention are also possible, all of which are explicitly contemplated and made part of this disclosure.