A fluorescently labeled hydroxypropyl-beta-cyclodextrin nanocapsule, and a preparation method and application thereof

Abstract

The application relates to the field of biological medicine, in particular to a fluorescently labeled hydroxypropyl-beta-cyclodextrin nanocapsule as well as a preparation method and application thereof. The nanocapsule is prepared from hydroxypropyl-beta-cyclodextrin through a reverse-phase miniemulsion polymerization method. The fluorescently labeled hydroxypropyl-beta-cyclodextrin nanocapsule provided by the application can load a hydrophobic antitumor drug, can stay at a tumor site for a long time, can release the antitumor drug, and has application prospects in tumor diagnosis and treatment integration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsule, its preparation method, and its application. Background Technology

[0002] Nanocarriers can exhibit different imaging phenomena through their own optical, electrical, magnetic, thermal, and radioactive properties, or by loading materials or molecules with these properties. They can be used to study the targeted delivery, distribution, enrichment, and metabolism of drug carriers in vivo, to perform targeted imaging of tumors, and to optimize treatment plans, thereby achieving the goal of integrated diagnosis and treatment.

[0003] Inorganic nanocarriers typically possess inherent imaging capabilities but suffer from significant biocompatibility issues. In contrast, organic nanocarriers can achieve better biocompatibility through the selection of construction materials. Organic nanocarriers often require the simultaneous loading of imaging reagents and therapeutic drugs to achieve integrated diagnosis and treatment. However, conventional organic nanocarriers, such as polymer micelles and polymer nanoparticles, have relatively simple structures. When therapeutic drugs and imaging reagents are simultaneously loaded into the hydrophobic microdomains of the nanocarrier, interactions occur, making it difficult to separately control the release of drugs and imaging reagents. Therefore, the key technical challenge of constructing novel organic nanocarriers that can separately load therapeutic drugs and imaging reagents while simultaneously achieving sustained release of therapeutic drugs and minimizing leakage of imaging reagents remains to be solved. Summary of the Invention

[0004] The first objective of this invention is to provide a fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsule that can be loaded with hydrophobic antitumor drugs, can remain at the tumor site for a long time, and can release antitumor drugs in a sustained manner, making it suitable for use as an integrated drug for tumor diagnosis and treatment.

[0005] The second objective of this invention is to provide a method for preparing fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules, the specific steps of which are as follows:

[0006] (1) Preparation of reverse fine emulsion

[0007] Hydroxypropyl-β-cyclodextrin was dissolved in ultrapure water, then a water-soluble fluorescent dye dissolved in dimethyl sulfoxide was added, followed by cyclohexane. The resulting mixture was then ultrasonically emulsified (30W power, 5 minutes) to prepare a reverse-phase fine emulsion.

[0008] The volume ratio of ultrapure water to cyclohexane is 1–2:10.

[0009] The mass ratio of ultrapure water, hydroxypropyl-β-cyclodextrin, and water-soluble fluorescent dye is 10:0.5~2:0.001~0.002;

[0010] Water-soluble fluorescent dyes include Cy series near-infrared fluorescent dyes such as Cy5, Cy5.5, Cy7, and Cy7.5.

[0011] (2) Preparation of nanocapsules

[0012] Toluene diisocyanate was dissolved in cyclohexane at a concentration of 25 mg / mL and added dropwise to the emulsion in step (1) with stirring. The reaction mixture was then stirred at 800 rpm for 24 hours at room temperature. The nanocapsules were separated by centrifugation (5000 rpm, 10 min) and washed once with cyclohexane.

[0013] The mass ratio of toluene diisocyanate to hydroxypropyl-β-cyclodextrin in step (1) is 0.5 to 1:1.

[0014] (3) The obtained nanocapsules were dispersed in a 0.1% sodium dodecyl sulfate aqueous solution and stirred overnight. Then, the dispersion was ultrasonically dispersed in a water bath for 10 minutes and centrifuged (8000 rpm, 20 minutes) to remove sodium dodecyl sulfate from the solution to obtain purified nanocapsules. The nanocapsules were redispersed in ultrapure water at a concentration of 10-15 mg / mL and stored in the dark at 4°C.

[0015] The nanocapsules dispersed in ultrapure water have a hydration diameter of 0.5–1 μm and a negative potential.

[0016] The fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules prepared by the above method are used for drug loading, that is, the hydrophobic antitumor drug curcumin is loaded into the capsule wall of the nanocapsule.

[0017] The technical solution of this invention can achieve the following beneficial effects:

[0018] The fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules provided by this invention can load hydrophobic antitumor drugs, can remain at the tumor site for a long time, and can release antitumor drugs in a sustained manner, which has application prospects in the integrated diagnosis and treatment of tumors. Attached image description:

[0019] Figure 1 This is a transmission electron microscope image of the nanocapsules prepared in Example 1 of the present invention.

[0020] Figure 2 This is a transmission electron microscope image of the nanocapsules prepared in Example 5 of the present invention. Detailed Implementation

[0021] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0022] Example 1: Preparation of Cy5-labeled hydroxypropyl-β-cyclodextrin nanocapsules

[0023] Dissolve 150 mg of hydroxypropyl-β-cyclodextrin in 1.5 mL of ultrapure water, and add 50 μL of [the solution]. A 5 mg / mL solution of dimethyl sulfoxide (Cy5) was stirred, and 12 mL of cyclohexane was added. The resulting mixture was ultrasonically emulsified at 30 W for 5 minutes to obtain a reverse emulsion. Then, 100 mg of toluene diisocyanate was dissolved in 4 mL of cyclohexane and added dropwise to the emulsion while stirring at 800 rpm. The reaction mixture was then stirred at 800 rpm for 24 hours at room temperature. The nanocapsules were separated by centrifugation (5000 rpm, 10 minutes) and washed once with 16 mL of cyclohexane. The obtained nanocapsules were dispersed in 30 mL of 0.1% sodium dodecyl sulfate aqueous solution and stirred overnight. The dispersion was then ultrasonically dispersed in a water bath for 10 minutes and centrifuged (8000 rpm, 20 minutes) to remove sodium dodecyl sulfate from the solution, resulting in purified nanocapsules. The nanocapsules were redispersed in 12 mL of ultrapure water and stored at 4 °C in the dark for further use.

[0024] Example 2: Preparation of Cy5-labeled hydroxypropyl-β-cyclodextrin nanocapsules

[0025] Dissolve 100 mg of hydroxypropyl-β-cyclodextrin in 1.5 mL of ultrapure water, and add 50 μL of [the solution]. A 5 mg / mL solution of dimethyl sulfoxide (Cy5) was stirred, and 12 mL of cyclohexane was added. The resulting mixture was ultrasonically emulsified at 30 W for 5 minutes to obtain a reverse emulsion. Then, 100 mg of toluene diisocyanate was dissolved in 4 mL of cyclohexane and added dropwise to the emulsion while stirring at 800 rpm. The reaction mixture was then stirred at 800 rpm for 24 hours at room temperature. The nanocapsules were separated by centrifugation (5000 rpm, 10 minutes) and washed once with 16 mL of cyclohexane. The obtained nanocapsules were dispersed in 30 mL of 0.1% sodium dodecyl sulfate aqueous solution and stirred overnight. The dispersion was then ultrasonically dispersed in a water bath for 10 minutes and centrifuged (8000 rpm, 20 minutes) to remove sodium dodecyl sulfate from the solution, resulting in purified nanocapsules. The nanocapsules were redispersed in 12 mL of ultrapure water and stored at 4 °C in the dark for further use.

[0026] Example 3: Preparation of Cy5-labeled hydroxypropyl-β-cyclodextrin nanocapsules

[0027] Dissolve 150 mg of hydroxypropyl-β-cyclodextrin in 1.5 mL of ultrapure water, and add 50 μL of [the solution]. A dimethyl sulfoxide solution (5 mg / mL) of Cy5 was stirred, and 7.5 mL of cyclohexane was added. The resulting mixture was ultrasonically emulsified at 30 W for 5 minutes to obtain a reverse emulsion. Then, 100 mg of toluene diisocyanate was dissolved in 4 mL of cyclohexane and added dropwise to the emulsion while stirring at 800 rpm. The reaction mixture was then stirred at 800 rpm for 24 hours at room temperature. The nanocapsules were separated by centrifugation (5000 rpm, 10 min) and washed once with 16 mL of cyclohexane. The obtained nanocapsules were dispersed in 30 mL of 0.1% sodium dodecyl sulfate aqueous solution and stirred overnight. The dispersion was then ultrasonically dispersed in a water bath for 10 min and centrifuged (8000 rpm, 20 min) to remove sodium dodecyl sulfate from the solution, resulting in purified nanocapsules. The nanocapsules were redispersed in 12 mL of ultrapure water and stored at 4 °C in the dark for further use.

[0028] Example 4: Preparation of Cy7.5-labeled hydroxypropyl-β-cyclodextrin nanocapsules

[0029] Dissolve 150 mg of hydroxypropyl-β-cyclodextrin in 1.5 mL of ultrapure water, and add 50 μL of [the solution]. A 5 mg / mL solution of dimethyl sulfoxide (Cy7.5) was stirred, and 12 mL of cyclohexane was added. The resulting mixture was ultrasonically emulsified at 30 W for 5 minutes to obtain a reverse emulsion. Then, 100 mg of toluene diisocyanate was dissolved in 4 mL of cyclohexane and added dropwise to the emulsion while stirring at 800 rpm. The reaction mixture was then stirred at 800 rpm for 24 hours at room temperature. The nanocapsules were separated by centrifugation (5000 rpm, 10 min) and washed once with 16 mL of cyclohexane. The obtained nanocapsules were dispersed in 30 mL of 0.1% sodium dodecyl sulfate aqueous solution and stirred overnight. The dispersion was then ultrasonically dispersed in a water bath for 10 min and centrifuged (8000 rpm, 20 min) to remove sodium dodecyl sulfate from the solution, resulting in purified nanocapsules. The nanocapsules were redispersed in 12 mL of ultrapure water and stored at 4 °C in the dark for further use.

[0030] Comparative Example 1: Preparation of Cy5-labeled β-cyclodextrin nanocapsules

[0031] 150 mg β-cyclodextrin was dissolved in 1.5 mL of ultrapure water, and 50 μL of Cy5 dimethyl sulfoxide solution (5 mg / mL) was added and stirred. 12 mL of cyclohexane was added, and the resulting mixture was ultrasonically emulsified at 30 W for 5 minutes to obtain a reverse emulsion. Then, 100 mg toluene diisocyanate was dissolved in 4 mL of cyclohexane and added dropwise to the above emulsion while stirring at 800 rpm. The reaction mixture was then stirred at 800 rpm for 24 hours at room temperature. The nanocapsules were separated by centrifugation (5000 rpm, 10 min) and washed once with 16 mL of cyclohexane. The obtained nanocapsules were dispersed in 30 mL of 0.1% sodium dodecyl sulfate aqueous solution and stirred overnight. Then, the dispersion was ultrasonically dispersed in a water bath for 10 min and centrifuged (8000 rpm, 20 min) to remove sodium dodecyl sulfate from the solution, obtaining purified nanocapsules. The nanocapsules were redispersed in 12 mL of ultrapure water and stored at 4 °C in the dark for further use.

[0032] Example 5: Preparation of dimethylcurcumin-loaded nanocapsules

[0033] Dissolve 2 mg of dimethylcurcumin in 2 mL of tetrahydrofuran and add it dropwise to 8 mL of the nanocapsules prepared in Example 1 (2.5 mg / mL). After stirring at room temperature for 2 hours, evaporate the tetrahydrofuran under vacuum and centrifuge the resulting mixture (1000 rpm, 20 min) to remove unencapsulated dimethylcurcumin. Then, centrifuge the supernatant at 8000 rpm for 10 min to obtain nanocapsules loaded with dimethylcurcumin, redisperse them in 4 mL of ultrapure water, and store them at 4 °C in the dark for further use.

[0034] The drug loading and encapsulation efficiency of dimethylcurcumin in the dimethylcurcumin-loaded nanocapsules were determined by high performance liquid chromatography and were 8.3% and 86%, respectively.

[0035] Example 6: Preparation of dimethylcurcumin-loaded nanocapsules

[0036] Dissolve 2 mg of dimethylcurcumin in 2 mL of tetrahydrofuran and add it dropwise to 8 mL of the nanocapsules prepared in Example 2 (2.5 mg / mL). After stirring at room temperature for 2 hours, evaporate the tetrahydrofuran under vacuum and centrifuge the resulting mixture (1000 rpm, 20 min) to remove unencapsulated dimethylcurcumin. Then, centrifuge the supernatant at 8000 rpm for 10 min to obtain dimethylcurcumin-loaded nanocapsules, redisperse them in 4 mL of ultrapure water, and store them at 4 °C in the dark for further use.

[0037] The drug loading and encapsulation efficiency of dimethylcurcumin in the dimethylcurcumin-loaded nanocapsules were determined by high performance liquid chromatography and were 6.1% and 63%, respectively.

[0038] Example 7: Preparation of dimethylcurcumin-loaded nanocapsules

[0039] Dissolve 2 mg of dimethylcurcumin in 2 mL of tetrahydrofuran and add it dropwise to 8 mL of the nanocapsules prepared in Example 3 (2.5 mg / mL). After stirring at room temperature for 2 hours, evaporate the tetrahydrofuran under vacuum and centrifuge the resulting mixture (1000 rpm, 20 min) to remove unencapsulated dimethylcurcumin. Then, centrifuge the supernatant at 8000 rpm for 10 min to obtain dimethylcurcumin-loaded nanocapsules, redisperse them in 4 mL of ultrapure water, and store them at 4 °C in the dark for further use.

[0040] The drug loading and encapsulation efficiency of dimethylcurcumin in the dimethylcurcumin-loaded nanocapsules were determined by high performance liquid chromatography and were 6.8% and 70.4%, respectively.

[0041] Example 8: Preparation of dimethylcurcumin-loaded nanocapsules

[0042] Dissolve 2 mg of dimethylcurcumin in 2 mL of tetrahydrofuran and add it dropwise to 8 mL of the nanocapsules prepared in Example 4 (2.5 mg / mL). After stirring at room temperature for 2 hours, evaporate the tetrahydrofuran under vacuum and centrifuge the resulting mixture (1000 rpm, 20 min) to remove unencapsulated dimethylcurcumin. Then, centrifuge the supernatant at 8000 rpm for 10 min to obtain dimethylcurcumin-loaded nanocapsules, redisperse them in 4 mL of ultrapure water, and store them at 4 °C in the dark for further use.

[0043] The drug loading and encapsulation efficiency of dimethylcurcumin in the dimethylcurcumin-loaded nanocapsules were determined by high performance liquid chromatography and were 8.2% and 85%, respectively.

[0044] Comparative Example 2: Preparation of dimethylcurcumin-loaded nanocapsules

[0045] Dissolve 2 mg of dimethylcurcumin in 2 mL of tetrahydrofuran and add it dropwise to 8 mL of nanocapsules prepared in Example 1 (2.5 mg / mL). Stir at room temperature for 2 hours, then evaporate the tetrahydrofuran under vacuum. Centrifuge the resulting mixture (1000 rpm, 20 min) to remove unencapsulated dimethylcurcumin. Then, centrifuge the supernatant at 8000 rpm for 10 min to obtain dimethylcurcumin-loaded nanocapsules, redisperse them in 4 mL of ultrapure water, and store them at 4 °C in the dark for further use.

[0046] The drug loading and encapsulation efficiency of dimethylcurcumin in the dimethylcurcumin-loaded nanocapsules were determined by high performance liquid chromatography and were 5.6% and 57%, respectively.

[0047] Comparing Examples 5-8 and Comparative Example 2, it can be seen that the drug loading capacity of the nanocapsules prepared in Examples 1 and 4 is better than that of the nanocapsules prepared in Examples 2 and 3, indicating that the amount of hydroxypropyl-β-cyclodextrin has a certain influence on the drug loading capacity. The water-soluble fluorescent probe has no significant effect on the drug loading capacity of the nanocapsules, because dimethylcurcumin is loaded in the nanocapsule wall, while the water-soluble probe is encapsulated in the cavity inside the nanocapsule. The drug loading capacity of the nanocapsules prepared in Example 1 is better than that of Control Example 1, indicating that hydroxypropyl-β-cyclodextrin is more advantageous than β-cyclodextrin as a capsule wall material.

[0048] Example 9 Characterization of particle size and morphology

[0049] Preparation of test solution: The nanocapsules prepared in Examples 1-8 and Comparative Examples 1 and 2 of this invention were diluted with ultrapure water, ultrasonically dispersed in a water bath for 5 min, and prepared into an aqueous solution of 0.2 mg / mL.

[0050] Particle size and morphology determination: Take 1 mL of each of the above-prepared test solutions and measure the hydrated particle size and potential by dynamic light scattering. Take 50 μL of the above-prepared test solutions of Example 1 and Example 5, drop them onto a copper grid covered with a carbon support film, let them air dry naturally, and observe the morphology using a transmission electron microscope.

[0051] As shown in Table 1, the hydrated diameters of the nanocapsules prepared in Examples 1-8 and Comparative Examples 1 and 2 of this invention range from 500 to 900 nm. All prepared nanocapsules have a negative charge, which is beneficial for increasing stability through electrostatic repulsion. The polydispersity index of the nanocapsules prepared in Examples 1-8 is less than 0.3, indicating that these nanocapsules have a narrow particle size distribution. The nanocapsules prepared in Example 3 have a larger particle size than those in Examples 1, 2, and 4. This is because the ratio of organic phase to aqueous phase was reduced in Example 3, resulting in larger reverse emulsion particles. The nanocapsules prepared in Comparative Example 1 have a significantly larger particle size than those prepared in Examples 1-4. This is because the emulsifying ability of β-cyclodextrin is weaker than that of hydroxypropyl β-cyclodextrin, resulting in larger and less uniform reverse emulsion particles, ultimately leading to larger nanocapsule particle size and a higher polydispersity index. The loading of dimethylcurcumin has little effect on the particle size of the nanocapsules.

[0052] like Figure 1 and Figure 2 As shown, the nanocapsules prepared in Examples 1 and 5 of this invention are spherical and have a typical capsule structure, consisting of an internal cavity and an outer capsule wall. The capsule wall is formed by the polymerization of hydroxypropyl-β-cyclodextrin, the internal cavity is loaded with the water-soluble fluorescent molecule Cy5, and dimethylcurcumin is loaded in the outer capsule wall through host-guest interaction with polyhydroxypropyl-β-cyclodextrin.

[0053] Table 1. Particle size and potential of nanocapsules prepared in Examples 1-8 and Comparative Examples 1 and 2

[0054] Example 1 537.9±6.6 -11.50±0.70 0.278±0.016 Example 2 527.4±23.4 -10.23±0.53 0.260±0.025 Example 3 634.6±12.2 -9.82±1.33 0.288±0.036 Example 4 539.3±7.2 -10.92±0.63 0.264±0.016 Compare with Example 1 843±43.4 -15.32±2.35 0.382±0.043 Example 5 528.4±5.3 -12.73±0.54 0.275±0.031 Example 6 521.2±12.8 -11.32±0.31 0.273±0.023 Example 7 626.4±7.4 -10.34±1.65 0.264±0.063 Example 8 528.3±4.5 -12.02±0.73 0.277±0.018 Comparative Example 2 864±53.7 -13.64±4.05 0.462±0.061

[0055] Example 10 Drug Release

[0056] Preparation of drug solution: The dimethylcurcumin-loaded nanocapsules prepared in Example 5 and Comparative Example 2 of this invention were diluted with ultrapure water, ultrasonically dispersed in a water bath for 5 min, and prepared into an aqueous solution of 1 mg / mL.

[0057] Preparation of pH 7.4 phosphate release solution: Weigh 0.2g potassium dihydrogen phosphate, 2.16g disodium hydrogen phosphate dodecahydrate, and 5g Tween-80, add them to ultrapure water to dissolve, and bring the volume to 1000mL. Then adjust the pH to 7.4 with dilute hydrochloric acid.

[0058] Drug release experiment: 1 mL of the prepared drug solution was added to a dialysis bag and immersed in 50 mL of the prepared pH 7.4 phosphate release solution (pH 7.4, 10 mmol / L, 0.5% Tween 80). The bag was shaken at 150 rpm at 37°C. 3 mL of the release solution was collected at 2, 6, 24, 48, 72, 96, 120, 144, and 168 hours, and 3 mL of fresh release solution was added. The entire experiment was conducted under light-protected conditions. The release percentages of dimethylcurcumin and Cy5 were determined by high-performance liquid chromatography and ultraviolet spectrophotometry, respectively. The release experiment was repeated three times, and the average release amount was calculated.

[0059] As shown in Table 2, the nanocapsules prepared in Example 5 of this invention release dimethylcurcumin at a much higher rate than Cy5. This is because dimethylcurcumin is loaded in the outer wall of the nanocapsule, while Cy5 is loaded in the cavity of the nanocapsule. The nanocapsules prepared in Example 5 can continuously release dimethylcurcumin, with a cumulative release of 77.9% after 168 hours, thus it can be used for the sustained release of dimethylcurcumin. The nanocapsules prepared in Example 5 only have a cumulative release of 27.0% of Cy5 after 168 hours, which is beneficial for retaining the fluorescent probe inside the nanocapsule for tracking and imaging. As shown in Table 3, the nanocapsules prepared in Comparative Example 2 of this invention release both dimethylcurcumin and Cy5 at faster rates than those prepared in Example 5, which is not conducive to the sustained release of dimethylcurcumin and the retention of the fluorescent probe. This is mainly because β-cyclodextrin has a weaker emulsifying ability than hydroxypropyl-β-cyclodextrin, resulting in an incomplete nanocapsule structure and easy drug leakage.

[0060] Table 2. Cumulative release of dimethylcurcumin-loaded nanocapsules prepared in Example 5

[0061]

[0062]

[0063] Table 3. Cumulative release of dimethylcurcumin-loaded nanocapsules prepared in Comparative Example 2

[0064] 2h 25.3％ 5.1％ 6h 40.5％ 7.2％ 24h 65.2％ 10.2％ 48h 80.4％ 15.3％ 72h 84.6％ 25.3％ 96h 86.4％ 32.6％ 120h 90.0％ 40.8％ 144h 92.1％ 46.3％ 168h 93.9％ 53.2％

[0065] Example 11 Biocompatibility

[0066] The nanocapsules prepared in Example 1 of this invention were diluted to concentrations of 1, 5, 10, 100 and 500 μg / mL using 1640 medium and added to 96-well plates to incubate with mouse fibroblast NIH3T3 cells for 48 hours. The number of cells per well was approximately 5000, and each concentration was replicated in 5 wells. Cell viability was detected by the MTT assay, with cells incubated in blank medium as 100% viability, and the cell viability of each group was calculated.

[0067] As shown in Table 4, the nanocapsules prepared in Example 1 of the present invention, when incubated with NIH3T3 cells for 48 hours in a concentration range of 1 to 500 μg / mL, showed a cell survival rate of over 80%, indicating that the nanocapsules prepared in Example 1 of the present invention have good biocompatibility.

[0068] Table 4. Biocompatibility of the nanocapsules prepared in Example 1

[0069] 1 98.9±9.2 10 97.4±3.1 50 94.3±2.1 100 86.7±4.4 500 82.7±1.7

[0070] Example 12 Tumor Retention

[0071] Mouse breast cancer cells 4T1 were injected at a dose of 1×10 7 To establish a mouse 4T1 subcutaneous mammary tumor model, 100 μL of cells per milliliter was seeded subcutaneously into the right posterior back of each mouse. The tumors were cultured until they reached a volume of approximately 80 mm². 3 At that time, tumor-bearing mice were randomly divided into two experimental groups, with 3 mice in each group. The nanocapsules prepared in Example 1 of this invention and free Cy5 with the same fluorescence intensity were injected into the corresponding experimental groups at a dose of 50 μL via intratumoral injection. The mice were sacrificed 5 days after intratumoral injection. During this period, in vivo fluorescence imaging was performed daily using a small animal in vivo imaging system to analyze the change in the average radiation efficiency of the tumor over time.

[0072] Table 5. Tumor residency of nanocapsules and free Cy5 prepared in Example 1

[0073]

[0074]

[0075] As shown in Table 5, the fluorescence intensity of the nanocapsules prepared in Example 1 of the present invention decreased slowly at the tumor site after intratumoral injection, while the fluorescence intensity of free Cy5 decreased rapidly at the tumor site after intratumoral injection, indicating that the nanocapsules prepared in Example 1 of the present invention have good tumor retention ability.

[0076] The fluorescently labeled nanocapsules provided by this invention can load hydrophobic antitumor drugs, and have good tumor retention ability and sustained release of hydrophobic antitumor drugs. Therefore, they have potential application value in the integrated diagnosis and treatment of tumors.

Claims

1. A fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsule, characterized in that, The nanocapsules were prepared by reverse-phase microemulsion polymerization of hydroxypropyl-β-cyclodextrin; the preparation steps of the fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules are as follows: (1) Hydroxypropyl-β-cyclodextrin was dissolved in ultrapure water, then a water-soluble fluorescent dye dissolved in dimethyl sulfoxide was added, followed by cyclohexane, and the resulting mixture was ultrasonically emulsified to prepare a reverse-phase fine emulsion; the water-soluble fluorescent dyes were Cy5, Cy5.5, Cy7, and Cy7.

5. (2) Toluene diisocyanate was dissolved in cyclohexane and added dropwise to the emulsion in step (1) with stirring. Then, the reaction mixture was stirred vigorously at room temperature for 24 hours. The nanocapsules were separated by centrifugation and washed once with cyclohexane. (3) The obtained nanocapsules were dispersed in a 0.1% sodium dodecyl sulfate aqueous solution and stirred overnight. Then, the dispersion was ultrasonically dispersed in a water bath for 10 minutes and centrifuged to remove sodium dodecyl sulfate from the solution to obtain purified nanocapsules. The nanocapsules were redispersed in ultrapure water and stored in the dark at 4°C.

2. The fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules according to claim 1, characterized in that, In step (1), the volume ratio of ultrapure water to cyclohexane is 1-2:10, and the mass ratio of ultrapure water, hydroxypropyl-β-cyclodextrin, and water-soluble fluorescent dye is 10:0.5-2:0.001-0.

002.

3. The fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules according to claim 1, characterized in that, In step (1), the ultrasonic emulsification power is 30W and the time is 5 minutes.

4. The fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules according to claim 1, characterized in that, The mass ratio of toluene diisocyanate in step (2) to hydroxypropyl-β-cyclodextrin in step (1) is 0.5 to 1:

1.

5. The fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules according to claim 1, characterized in that, In step (3), the nanocapsules dispersed in ultrapure water have a hydration diameter of 0.5–1 μm and a negative potential.

6. The application of the fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules according to claim 1 in the preparation of an antitumor drug delivery carrier.

7. The application of the fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules according to claim 6 in the preparation of antitumor drug delivery carriers, characterized in that, The antitumor drug is a hydrophobic antitumor drug, loaded in the cell wall of the nanocapsule.

8. The application of the fluorescently labeled hydroxypropyl-β-cyclodextrin nanocapsules according to claim 7 in the preparation of antitumor drug delivery carriers, characterized in that, The hydrophobic antitumor drug is dimethylcurcumin.

Images