A combined drug-loaded nanomedicine delivery system and a method of making the same

Abstract

The application discloses a kind of combined drug-loaded nanomedicine delivery systems and its preparation method, it is by loading doxorubicin's surface is weakly negative charge nanoparticle DOX@HA-DA-BPEI-DMA with loading curcumin's nitrogen-doped carbon quantum dots N-CQDs-Cur through electrostatic interaction assembled and constructed nanomedicine delivery system, 3,4-dihydroxybenzoic acid DA is modified to HA surface, one step constructs carboxyl functionalized hydroxyapatite HA, then respectively using branched polyethyleneimine modified hydroxyapatite HA as doxorubicin carrier, nitrogen-doped carbon quantum dots as curcumin carrier, by electrostatic interaction and be assembled into a combined drug-loaded nanomedicine delivery system.The application constructs combined loading doxorubicin DOX and curcumin Cur nanomedicine delivery system, realizes the new drug-loading system of inorganic material on chemotherapy drug combined loading, and present obvious response to pH, and the induced apoptosis toxicity of specific tumor cell is enhanced.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials and pharmaceutical technology, and in particular to a combined drug-loaded nanomedicine delivery system and its preparation method. Background Technology

[0002] Hydroxyapatite (HA) is a calcium phosphate ceramic, typically modified with surface treatments to achieve various properties, such as folic acid-targeted carriers. As an inorganic material, HA requires suitable coupling agents for functionalization with specific organic compounds like folic acid. Currently, silane coupling agents are commonly used, employing silanes with different functional groups, such as aminopropyltrimethoxysilane, to obtain HA functionalized with different types of functional groups. However, achieving carboxyl functionalization on the HA surface often requires more reaction steps and cannot be accomplished in one step. For example, Chinese patent application number 201710066664.1 discloses a nitric oxide donor-type β-elemene derivative, its preparation method, and its uses. By introducing furazolidone nitrate containing a polar group into the β-elemene structure, it improves the water solubility of the β-elemene derivative; furthermore, furazolidone nitrate, as a nitric oxide releasing unit, releases NO, exhibiting a synergistic antitumor effect with β-elemene; and enriches the molecular library of antitumor drug β-elemene derivatives.

[0003] In nanomedicine delivery systems, traditional dosage forms such as liposomes, polymer nanoparticles, hydrogels, and micelles are commonly used as carriers for co-loading doxorubicin (DOX) and curcumin (Cur). Currently, there are no reports on constructing co-loading carriers for two chemotherapeutic drugs using inorganic materials. Therefore, this invention discloses a co-loaded nanomedicine delivery system. 3,4-Dihydroxybenzoic acid (DA) is modified onto the surface of hydroxyapatite (HA) to construct a carboxyl-functionalized hydroxyapatite (HA) in one step. Then, branched polyethyleneimine-modified hydroxyapatite (HA) is used as the doxorubicin carrier, and nitrogen-doped carbon quantum dots are used as the curcumin carrier. These components are assembled into a co-loaded nanomedicine delivery system via electrostatic interactions. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a combined drug-loaded nanomedicine delivery system and its preparation method. This invention constructs a nanomedicine delivery system that combines doxorubicin (DOX) and curcumin (Cur), loading them together into the same drug delivery system. This achieves a novel drug delivery system for the combined loading of chemotherapeutic drugs onto inorganic materials, realizing the combined delivery of chemotherapeutic drugs. Furthermore, it exhibits significant pH responsiveness and enhances apoptosis-inducing toxicity against specific tumor cells.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0006] A combined drug-loaded nanomedicine delivery system is constructed by electrostatically assembling doxorubicin-loaded nanoparticles DOX@HA-DA-BPEI-DMA with a weakly negatively charged surface and curcumin-loaded nitrogen-doped carbon quantum dots N-CQDs-Cur.

[0007] Preferably, the nitrogen-doped carbon quantum dots (N-CQDs-Cur) loaded with curcumin are prepared by the following method, including the following steps:

[0008] S11: Weighed citric acid and polyethyleneimine are dissolved in deionized water in a certain proportion. After hydrothermal reaction for a period of time, acetone is added to precipitate the mixture and nitrogen-doped carbon quantum dots (N-CQDs) are obtained.

[0009] S12: Nitrogen-doped carbon quantum dots (N-CQDs) and curcumin (Cur) are added to sodium hydroxide solution in a certain proportion. After stirring and reacting for a period of time, acetone is added to precipitate the mixture. After washing and drying, nitrogen-doped carbon quantum dots (N-CQDs) loaded with curcumin (Cur) are obtained.

[0010] Preferably, in step S11, the mass ratio of citric acid to polyethyleneimine is 1:0.1-0.5; the hydrothermal reaction temperature is 150-180℃ and the time is more than 3 hours.

[0011] Preferably, in step S12, the mass ratio of nitrogen-doped carbon quantum dots (N-CQDs) to curcumin (Cur) is 20:0.5-1; the stirring reaction is carried out at room temperature for more than 3 hours.

[0012] Preferably, the nanoparticles DOX@HA-DA-BPEI-DMA loaded with doxorubicin and having a weakly negatively charged surface are prepared by the following method, including the following steps:

[0013] S21: HA-DA-BPEI nanoparticles with positive surface charge were dispersed in dimethyl sulfoxide, and dimethyl maleic anhydride DMA, as well as the catalysts triethylamine and pyridine were added. After stirring vigorously for 12-24 hours, the precipitate was washed with dimethyl sulfoxide, ethanol and deionized water in sequence and then freeze-dried under vacuum to obtain HA-DA-BPEI-DMA nanoparticles with negative surface charge.

[0014] S22: HA-DA-BPEI-DMA nanoparticles with negative surface charge were dispersed in doxorubicin DOX solution and stirred at room temperature for a period of time. After loading was completed, the precipitate was collected by centrifugation to obtain DOX@HA-DA-BPEI-DMA nanoparticles loaded with doxorubicin and with a weakly negative surface charge.

[0015] Preferably, the amount of dimethylmaleic anhydride DMA is 1.5-3 times the molar amount of the primary amine group in the BPEI of the positively charged nanoparticles HA-DA-BPEI; the amount of the catalysts triethylamine and pyridine is 1-2 times the molar amount of dimethylmaleic anhydride DMA.

[0016] Preferably, the positively charged nanoparticles HA-DA-BPEI are prepared by the following method, including the following steps:

[0017] S31: 3,4-Dihydroxybenzoic acid (DA) and hydroxyapatite (HA) were ultrasonically dispersed in anhydrous ethanol at a certain ratio. After stirring at room temperature for 1-3 hours, the precipitate was collected by centrifugation and then vacuum dried to obtain carboxyl-functionalized hydroxyapatite HA-DA.

[0018] S32: Carboxyl-functionalized hydroxyapatite HA-DA was dispersed in dimethyl sulfoxide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. After stirring for a period of time, branched polyethyleneimine BPEI was added. After stirring and reacting for another 24 hours under nitrogen protection, the nanoparticles HA-DA-BPEI with positive surface charge were obtained after centrifugation, washing of the precipitate and vacuum drying.

[0019] Preferably, in step S31, the mass ratio of 3,4-dihydroxybenzoic acid (DA) to hydroxyapatite (HA) is 1:1-3; the stirring reaction temperature is room temperature and the time is more than 6 hours; in step S22, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1.2-2 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA; the amount of N-hydroxysuccinimide is 2-4 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA.

[0020] The present invention also discloses a method for preparing the aforementioned combined drug-loaded nanomedicine delivery system, comprising the following steps: ultrasonically dispersing nanoparticles DOX@HA-DA-BPEI-DMA with a weakly negatively charged surface loaded with doxorubicin into deionized water, adding nitrogen-doped carbon quantum dots N-CQDs-Cur loaded with curcumin, mixing and stirring for a period of time, centrifuging, collecting the precipitate, and obtaining the combined drug-loaded nanomedicine delivery system DOX@HA-DA-BPEI-DMA / N-CQDs-Cur.

[0021] Preferably, the weight ratio of the nanoparticles DOX@HA-DA-BPEI-DMA loaded with doxorubicin and having a weakly negatively charged surface to the nitrogen-doped carbon quantum dots N-CQDs-Cur loaded with curcumin is 1:2-4.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. In this invention, 3,4-dihydroxybenzoic acid (DA) is modified onto the surface of hydroxyapatite (HA) to construct carboxyl-functionalized hydroxyapatite HA in one step. Moreover, the ortho-dihydroxyl groups in 3,4-dihydroxybenzoic acid (DA) can not only form a phenolic hydroxy calcium structure with calcium ions in the hydroxyapatite HA lattice, but also form hydrogen bonds with hydroxyl groups on the surface of hydroxyapatite HA. Therefore, 3,4-dihydroxybenzoic acid (DA) can be modified onto the surface of hydroxyapatite HA. Subsequently, branched polyethyleneimine-modified hydroxyapatite HA is used as an doxorubicin carrier, and nitrogen-doped carbon quantum dots are used as a curcumin carrier. They are assembled into a co-loaded nanomedicine delivery system through electrostatic interaction.

[0024] 2. The preparation method of the combined drug-loaded nanomedicine delivery system of the present invention uses doxorubicin (DOX) and curcumin (Cur) to be jointly loaded into the same drug delivery system, realizing a novel drug loading system for the combined loading of chemotherapeutic drugs on inorganic materials.

[0025] 3. This invention achieves the co-delivery of chemotherapeutic drugs by electrostatically assembling nitrogen-doped carbon quantum dots (N-CQDs-Cur) loaded with curcumin and nanoparticles (DOX@HA-DA-BPEI-DMA) loaded with doxorubicin and having a weakly negatively charged surface. The system, DOX@HA-DA-BPEI-DMA / N-CQDs-Cur, exhibits significant pH responsiveness and enhances apoptosis-inducing toxicity against specific tumor cells.

[0026] 4. This invention uses 3,4-dihydroxybenzoic acid as a coupling agent to achieve the preparation of carboxyl-functionalized HA in one step, which shortens the reaction cycle, is simple and easy to implement, and avoids the cumbersome reaction process of obtaining carboxyl functional groups after modification with silane coupling agents. It realizes the rapid preparation of carboxyl-functionalized HA, saving process flow and cost.

[0027] The above is an overview of the invention's technical solution. The invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0028] Figure 1 The FT-IR spectrum of N-CQDs-Cur in this embodiment is shown.

[0029] Figure 2 This is a UV-vis absorption spectrum of N-CQDs-Cur in this embodiment;

[0030] Figure 3 The FT-IR spectrum of the DOX@HA-DA-BPEI-DMA / N-CQDs-Cur nanoparticles in this embodiment is shown.

[0031] Figure 4 Another FT-IR spectrum of the DOX@HA-DA-BPEI-DMA / N-CQDs-Cur nanoparticles in this embodiment;

[0032] Figure 5 This is a zeta potential diagram of the DOX@HA-DA-BPEI-DMA / N-CQDs-Cur nanoparticles in this embodiment;

[0033] Figure 6 Zeta potential diagrams of DOX@HA-DA-BPEI-DMA / N-CQDs-Cur nanoparticles with different ratios in this embodiment;

[0034] Figure 7 This image shows the cell viability of DOX@HA-DA-BPEI-DMA / N-CQDs-Cur nanoparticles (NPs) and CT26 cells after incubation at different pH values ​​in this embodiment. Detailed implementation method:

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0036] Example 1: This example provides a combined drug-loaded nanomedicine delivery system and its preparation method, including the following preparation route:

[0037] (I) The preparation process of N-CQDs-Cur includes the following steps: Citric acid (16g) and polyethyleneimine (2g) are dissolved in 240mL of deionized water and reacted at 180℃ for 5h. Acetone is added to precipitate N-CQDs. N-CQDs (500mg) are dissolved in 20mL of NaOH solution (0.2mol / L), 25mg of Cur is added, and the mixture is stirred at room temperature for 12h. Acetone is added to precipitate the precipitate, which is washed until the supernatant is colorless and dried under vacuum to obtain N-CQDs-Cur. The FT-IR and UV-Vis absorption spectra of the prepared N-CQDs-Cur are shown in the appendix. Figure 1-2 .

[0038] (ii) The DOX@HA-DA-BPEI-DMA process includes the following steps:

[0039] 1) 3,4-Dihydroxybenzoic acid (DA, 50 mg) and hydroxyapatite (HA, 100 mg) were ultrasonically dispersed in anhydrous ethanol and stirred at room temperature for 24 h. The precipitate was collected by centrifugation and dried under vacuum to obtain HA-DA.

[0040] 2) HA-DA (100 mg) was dispersed in 20 mL of dimethyl sulfoxide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg) and N-hydroxysuccinimide (120 mg) were added. The mixture was stirred at room temperature for 1 h, and branched polyethyleneimine (BPEI, 160 mg) was added. The mixture was stirred for another 24 h under nitrogen protection. After centrifugation, the precipitate was washed successively with dimethyl sulfoxide, ethanol, and deionized water, and then dried under vacuum to obtain HA-DA-BPEI.

[0041] 3) HA-DA-BPEI was dispersed in 15 mL of dimethyl sulfoxide, and dimethyl maleic anhydride (DMA, 52 mg), 57 μL of triethylamine, and 57 μL of pyridine were added. The mixture was stirred vigorously for 24 h. The precipitate was washed sequentially with dimethyl sulfoxide, ethanol, and deionized water, and then freeze-dried under vacuum to obtain HA-DA-BPEI-DMA. Next, HA-DA-BPEI-DMA (20 mg) was dispersed in 2 mL of DOX solution (1 mg / mL) and stirred at room temperature for 24 h. After loading was complete, the precipitate was collected by centrifugation (DOX@HA-DA-BPEI-DMA).

[0042] (III) The preparation process of the combined drug-loaded nanomedicine delivery system includes the following steps:

[0043] DOX@HA-DA-BPEI-DMA (10 mg) was ultrasonically dispersed in deionized water, and N-CQDs-Cur (20 mg, in a 1:2 ratio) was added. The mixture was stirred for 6 h, centrifuged, and the precipitate was collected to obtain DOX@HA-DA-BPEI-DMA / N-CQDs-Cur. The structural changes of the prepared DOX@HA-DA-BPEI-DMA / N-CQDs-Cur were analyzed by FT-IR spectroscopy and changes in Zeta potential (see Appendix). Figure 2-6 Furthermore, the cytotoxicity of the drug delivery system was analyzed using the CCK-8 assay. This system reduced the toxic side effects of chemotherapeutic drugs in the physiological environment (pH 7.4) and improved their antitumor activity in the tumor microenvironment (pH 6.5). (See Appendix) Figure 7 .

[0044] Example 2: This example provides a combined drug-loaded nanomedicine delivery system and its preparation method, which is basically the same as in Example 1, except that: the amount of DOX@HA-DA-BPEI-DMA is kept constant, and the amount of N-CQDs-Cur is increased, with the ratio of the two reaching 1:4, and finally nanoparticles are assembled; the preparation route is as follows:

[0045] (I) The preparation process of N-CQDs-Cur includes the following steps: Citric acid (16g) and polyethyleneimine (2g) are dissolved in 240mL of deionized water and reacted at 180℃ for 5h. Acetone is added to precipitate N-CQDs. N-CQDs (500mg) are dissolved in 20mL of NaOH solution (0.2mol / L), and 25mg of Cur is added. The mixture is stirred at room temperature for 12h. Acetone is added to precipitate the precipitate, which is washed until the supernatant is colorless and dried under vacuum to obtain N-CQDs-Cur.

[0046] (ii) The DOX@HA-DA-BPEI-DMA process includes the following steps:

[0047] 1) 3,4-Dihydroxybenzoic acid (DA, 50 mg) and hydroxyapatite (HA, 100 mg) were ultrasonically dispersed in anhydrous ethanol and stirred at room temperature for 24 h. The precipitate was collected by centrifugation and dried under vacuum to obtain HA-DA.

[0048] 2) HA-DA (100 mg) was dispersed in 20 mL of dimethyl sulfoxide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg) and N-hydroxysuccinimide (120 mg) were added. The mixture was stirred at room temperature for 1 h, and branched polyethyleneimine (BPEI, 160 mg) was added. The mixture was stirred for another 24 h under nitrogen protection. After centrifugation, the precipitate was washed successively with dimethyl sulfoxide, ethanol, and deionized water, and then dried under vacuum to obtain HA-DA-BPEI.

[0049] 3) HA-DA-BPEI was dispersed in 15 mL of dimethyl sulfoxide, and dimethyl maleic anhydride (DMA, 52 mg), 57 μL of triethylamine, and 57 μL of pyridine were added. The mixture was stirred vigorously for 24 h. The precipitate was washed sequentially with dimethyl sulfoxide, ethanol, and deionized water, and then freeze-dried under vacuum to obtain HA-DA-BPEI-DMA. Next, HA-DA-BPEI-DMA (20 mg) was dispersed in 2 mL of DOX solution (1 mg / mL) and stirred at room temperature for 24 h. After loading was complete, the precipitate was collected by centrifugation (DOX@HA-DA-BPEI-DMA).

[0050] (III) The preparation process of the combined drug-loaded nanomedicine delivery system includes the following steps:

[0051] DOX@HA-DA-BPEI-DMA (10 mg) was ultrasonically dispersed in deionized water, and N-CQDs-Cur (40 mg, in a ratio of 1:4) was added. The mixture was stirred for 6 hours, centrifuged, and the precipitate was collected to obtain DOX@HA-DA-BPEI-DMA / N-CQDs-Cur.

[0052] Example 3: This example provides a combined drug-loaded nanomedicine delivery system and its preparation method, which is basically the same as in Example 1, except that: the amount of DOX@HA-DA-BPEI-DMA is kept constant, while the amount of N-CQDs-Cur is increased, with a ratio of 1:6, to finally assemble nanoparticles. The preparation route includes the following:

[0053] (I) The preparation process of N-CQDs-Cur includes the following steps:

[0054] Citric acid (16 g) and polyethyleneimine (2 g) were dissolved in 240 mL of deionized water and reacted at 180 °C for 5 h. Acetone was added to precipitate N-CQDs. N-CQDs (500 mg) were dissolved in 20 mL of NaOH solution (0.2 mol / L), and 25 mg of Cur was added. The mixture was stirred at room temperature for 12 h. Acetone was added to precipitate the precipitate, which was washed until the supernatant was colorless and dried under vacuum to obtain N-CQDs-Cur.

[0055] (ii) The DOX@HA-DA-BPEI-DMA process includes the following steps:

[0056] 1) 3,4-Dihydroxybenzoic acid (DA, 50 mg) and hydroxyapatite (HA, 100 mg) were ultrasonically dispersed in anhydrous ethanol and stirred at room temperature for 24 h. The precipitate was collected by centrifugation and dried under vacuum to obtain HA-DA.

[0057] 2) HA-DA (100 mg) was dispersed in 20 mL of dimethyl sulfoxide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg) and N-hydroxysuccinimide (120 mg) were added. The mixture was stirred at room temperature for 1 h, and branched polyethyleneimine (BPEI, 160 mg) was added. The mixture was stirred for another 24 h under nitrogen protection. After centrifugation, the precipitate was washed successively with dimethyl sulfoxide, ethanol, and deionized water, and then dried under vacuum to obtain HA-DA-BPEI.

[0058] 3) HA-DA-BPEI was dispersed in 15 mL of dimethyl sulfoxide, and dimethyl maleic anhydride (DMA, 52 mg), 57 μL of triethylamine, and 57 μL of pyridine were added. The mixture was stirred vigorously for 24 h. The precipitate was washed sequentially with dimethyl sulfoxide, ethanol, and deionized water, and then freeze-dried under vacuum to obtain HA-DA-BPEI-DMA. Next, HA-DA-BPEI-DMA (20 mg) was dispersed in 2 mL of DOX solution (1 mg / mL) and stirred at room temperature for 24 h. After loading was complete, the precipitate was collected by centrifugation (DOX@HA-DA-BPEI-DMA).

[0059] (III) The preparation process of the combined drug-loaded nanomedicine delivery system includes the following steps:

[0060] DOX@HA-DA-BPEI-DMA (10 mg) was ultrasonically dispersed in deionized water, and N-CQDs-Cur (60 mg, in a ratio of 1:6) was added. The mixture was stirred for 6 hours, centrifuged, and the precipitate was collected to obtain DOX@HA-DA-BPEI-DMA / N-CQDs-Cur.

[0061] Example 4: This example provides a combined drug-loaded nanomedicine delivery system and its preparation method, which is basically the same as that in Example 1, except that:

[0062] (I) In the preparation process of N-CQDs-Cur: the mass ratio of citric acid to polyethyleneimine is 1:0.1; the hydrothermal reaction temperature is 160℃ and the time is more than 3 hours.

[0063] The mass ratio of nitrogen-doped carbon quantum dots (N-CQDs) to curcumin (Cur) was 20:1; the stirring reaction was carried out at room temperature for more than 3 hours.

[0064] (II) In the preparation process of DOX@HA-DA-BPEI-DMA: the mass ratio of 3,4-dihydroxybenzoic acid (DA) to hydroxyapatite (HA) is 1:2; the stirring reaction temperature is room temperature and the time is more than 6 hours;

[0065] The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1.2 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA; the amount of N-hydroxysuccinimide is 3 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA.

[0066] The amount of dimethylmaleic anhydride (DMA) used is 1.5 times the molar amount of the primary amine group in the branched polyethyleneimine (BPEI) of the positively charged nanoparticles HA-DA-BPEI; the amount of the catalysts triethylamine and pyridine is 1 molar amount of dimethylmaleic anhydride (DMA).

[0067] (III) In the preparation of the combined drug-loaded nanomedicine delivery system: the weight ratio of the nanoparticles DOX@HA-DA-BPEI-DMA loaded with doxorubicin and the nitrogen-doped carbon quantum dots N-CQDs-Cur loaded with curcumin is 1:2.

[0068] Example 5: This example provides a combined drug-loaded nanomedicine delivery system and its preparation method, which is basically the same as that in Example 1, except that:

[0069] (I) In the preparation process of N-CQDs-Cur: the mass ratio of citric acid to polyethyleneimine is 1:0.4; the hydrothermal reaction temperature is 180℃ and the time is more than 3 hours.

[0070] The mass ratio of nitrogen-doped carbon quantum dots (N-CQDs) to curcumin (Cur) was 20:0.5; the stirring reaction was carried out at room temperature for more than 3 hours.

[0071] (II) In the preparation process of DOX@HA-DA-BPEI-DMA: the mass ratio of 3,4-dihydroxybenzoic acid (DA) to hydroxyapatite (HA) is 1:3; the stirring reaction temperature is room temperature and the time is more than 6 hours;

[0072] The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is twice the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA; the amount of N-hydroxysuccinimide is twice the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA.

[0073] The amount of dimethylmaleic anhydride (DMA) used is 2.5 times the molar amount of the primary amine group in the branched polyethyleneimine (BPEI) of the positively charged nanoparticles HA-DA-BPEI; the amount of the catalysts triethylamine and pyridine is 2 times the molar amount of dimethylmaleic anhydride (DMA).

[0074] (III) In the preparation of the combined drug-loaded nanomedicine delivery system: the weight ratio of the nanoparticles DOX@HA-DA-BPEI-DMA loaded with doxorubicin and the nitrogen-doped carbon quantum dots N-CQDs-Cur loaded with curcumin is 1:2.

[0075] Example 6: This example provides a combined drug-loaded nanomedicine delivery system and its preparation method, which is basically the same as that in Example 1, except that:

[0076] (I) In the preparation process of N-CQDs-Cur: the mass ratio of citric acid to polyethyleneimine is 1:0.3; the hydrothermal reaction temperature is 170℃ and the time is more than 3 hours.

[0077] The mass ratio of nitrogen-doped carbon quantum dots (N-CQDs) to curcumin (Cur) was 20:0.8; the stirring reaction was carried out at room temperature for more than 3 hours.

[0078] (II) In the preparation process of DOX@HA-DA-BPEI-DMA: the mass ratio of 3,4-dihydroxybenzoic acid (DA) to hydroxyapatite (HA) is 1:2; the stirring reaction temperature is room temperature and the time is more than 6 hours;

[0079] The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1.2 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA; the amount of N-hydroxysuccinimide is 4 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA.

[0080] The amount of dimethylmaleic anhydride (DMA) used is three times the molar amount of the primary amine groups in the branched polyethyleneimine (BPEI) of the positively charged nanoparticles HA-DA-BPEI; the amount of the catalysts triethylamine and pyridine is one molar amount of dimethylmaleic anhydride (DMA).

[0081] (III) In the preparation of the combined drug-loaded nanomedicine delivery system: the weight ratio of the nanoparticles DOX@HA-DA-BPEI-DMA loaded with doxorubicin and having a weakly negatively charged surface to the nitrogen-doped carbon quantum dots N-CQDs-Cur loaded with curcumin is 1:3.

[0082] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A nanomedicine delivery system with combined drug loading, characterized in that, It is a nanomedicine delivery system constructed by electrostatically assembling DOX@HA-DA-BPEI-DMA nanoparticles loaded with doxorubicin and having a weakly negatively charged surface with nitrogen-doped carbon quantum dots N-CQDs-Cur loaded with curcumin. The nitrogen-doped carbon quantum dots (N-CQDs-Cur) loaded with curcumin are prepared by the following method, including the following steps: S11: Weighed citric acid and polyethyleneimine are dissolved in deionized water in a certain proportion. After hydrothermal reaction for a period of time, acetone is added to precipitate the mixture and nitrogen-doped carbon quantum dots (N-CQDs) are obtained. S12: Nitrogen-doped carbon quantum dots (N-CQDs) and curcumin (Cur) are added to sodium hydroxide solution in a certain proportion. After stirring and reacting for a period of time, acetone is added to precipitate the mixture. After washing and drying, nitrogen-doped carbon quantum dots (N-CQDs) loaded with curcumin (Cur) are obtained. In step S12, the mass ratio of nitrogen-doped carbon quantum dots (N-CQDs) to curcumin (Cur) is 20:0.5-1. The doxorubicin-loaded nanoparticles DOX@HA-DA-BPEI-DMA with a weakly negatively charged surface are prepared by the following method, including the following steps: S21: HA-DA-BPEI nanoparticles with positive surface charge were dispersed in dimethyl sulfoxide, and dimethyl maleic anhydride DMA, as well as the catalysts triethylamine and pyridine were added. After stirring vigorously for 12-24 hours, the precipitate was washed with dimethyl sulfoxide, ethanol and deionized water in sequence and then freeze-dried under vacuum to obtain HA-DA-BPEI-DMA nanoparticles with negative surface charge. S22: HA-DA-BPEI-DMA nanoparticles with negative surface charge were dispersed in doxorubicin DOX solution and stirred at room temperature for a period of time. After loading was completed, the precipitate was collected by centrifugation to obtain DOX@HA-DA-BPEI-DMA nanoparticles loaded with doxorubicin and with a weakly negative surface charge. The amount of dimethylmaleic anhydride (DMA) used is 1.5-3 times the molar amount of the primary amine groups in the BPEI of the positively charged HA-DA-BPEI nanoparticles; the positively charged HA-DA-BPEI nanoparticles are prepared by the following method, including the following steps: S31: 3,4-Dihydroxybenzoic acid (DA) and hydroxyapatite (HA) were ultrasonically dispersed in anhydrous ethanol at a certain ratio. After stirring at room temperature for 1-3 hours, the precipitate was collected by centrifugation and then vacuum dried to obtain carboxyl-functionalized hydroxyapatite HA-DA. S32: Carboxyl-functionalized hydroxyapatite HA-DA was dispersed in dimethyl sulfoxide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. After stirring for a period of time, branched polyethyleneimine BPEI was added. After stirring and reacting for another 24 hours under nitrogen protection, the nanoparticles HA-DA-BPEI with positive surface charge were obtained after centrifugation, washing of the precipitate and vacuum drying.

2. The nanomedicine delivery system with combined drug loading according to claim 1, characterized in that, In step S11, the mass ratio of citric acid to polyethyleneimine is 1:0.1-0.5; the hydrothermal reaction temperature is 150-180℃ and the time is more than 3 hours; in step S12, the stirring reaction temperature is room temperature and the time is more than 3 hours.

3. The nanomedicine delivery system with combined drug loading according to claim 1, characterized in that, In step S21, the amount of the catalyst triethylamine and pyridine is 1-2 times the molar amount of dimethylmaleic anhydride DMA.

4. The nanomedicine delivery system with combined drug loading according to claim 1, characterized in that, In step S31, the mass ratio of 3,4-dihydroxybenzoic acid (DA) to hydroxyapatite (HA) is 1:1-3.

5. The nanomedicine delivery system with combined drug loading according to claim 1, characterized in that, In step S32, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1.2-2 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA; the amount of N-hydroxysuccinimide is 2-4 times the molar amount of the carboxyl group in the DA of carboxyl-functionalized hydroxyapatite HA-DA.

6. A method for preparing a nanomedicine delivery system with co-loaded drug delivery as described in any one of claims 1-5, characterized in that, The process includes the following steps: DOX@HA-DA-BPEI-DMA nanoparticles loaded with doxorubicin and having a weakly negatively charged surface are ultrasonically dispersed in deionized water. Nitrogen-doped carbon quantum dots (N-CQDs-Cur) loaded with curcumin are added and mixed and stirred for a period of time. After centrifugation and collection of the precipitate, the combined drug-loaded nanodrug delivery system DOX@HA-DA-BPEI-DMA / N-CQDs-Cur is obtained.

7. The method for preparing the nanomedicine delivery system with co-loaded drug as described in claim 6, characterized in that, The weight ratio of the nanoparticles DOX@HA-DA-BPEI-DMA loaded with doxorubicin and having a weakly negatively charged surface to the nitrogen-doped carbon quantum dots N-CQDs-Cur loaded with curcumin is 1:2-4.

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