CN115317461A

CN115317461A - Adriamycin delivery system Cu-GA-DOX NPs and preparation method thereof

Info

Publication number: CN115317461A
Application number: CN202210943843.XA
Authority: CN
Inventors: 王程; 常玉枫; 周正花; 沈悦; 崔朋飞; 周舒文; 邱琳; 蒋鹏举; 王建浩
Original assignee: Changzhou University
Current assignee: Changzhou University
Priority date: 2022-08-05
Filing date: 2022-08-05
Publication date: 2022-11-11

Abstract

The invention belongs to the technical field of nano materials, and particularly discloses Cu-GA-DOX NPs of a delivery system of adriamycin (DOX) and a preparation method thereof. The invention adopts Cu ²⁺ GA and DOX are spontaneously assembled in one step, and then ammonia water is added as a precipitating agent to obtain Cu-GA-DOX NPs with uniform particle size, relatively high drug loading capacity and good particle sizeBiocompatibility and anti-colorectal cancer (CT 26) activity in vitro and in vivo. The Cu-GA-DOX NPs prepared by the method are low in cost, simple and convenient to prepare, high in reproducibility, high in drug loading capacity and good in biocompatibility, and provide reference and guidance for subsequent development of a DOX-related Drug Delivery System (DDS).

Description

Adriamycin delivery system Cu-GA-DOX NPs and preparation method thereof

Technical Field

The invention belongs to the technical field of nano materials, and particularly relates to Cu-GA-DOX NPs of a delivery system of adriamycin (DOX) and a preparation method thereof.

Background

Doxorubicin (DOX), the first line drug of cancer chemotherapy, is clinically used in its free form and often has poor bioavailability and high side effects, such as severe cardiotoxicity, etc., and therefore, certain measures are needed to avoid or overcome these disadvantages.

Nano-scale Drug Delivery Systems (DDS) are one of the research hotspots. The DOX-loaded Drug Delivery System (DDS) has been recognized to have more advantages, such as higher bioavailability and relatively lower toxic and side effects, and more importantly, the DDS can protect the carried DOX to a certain extent, ensure the release of the DOX in the desired target tissue, and further avoid the problems of low bioavailability, high cardiotoxicity and the like of the free DOX in conventional treatment. And enrichment of the tumor site can be achieved using the enhanced osmotic retention effect of the tumor tissue.

Although DOX-loaded Drug Delivery Systems (DDS) have made unprecedented progress, DDS that can be successfully developed and marketed are still rare. Many DDS preparation processes are complex, generally involve chemical reactions, and have low reproducibility, on the other hand, the introduction of a large amount of toxic chemical reagents and solvents in the preparation process also leads to the fact that the finally obtained DDS has high toxic and side effects, and in addition, the further development of the DDS is limited by the relatively low DOX drug-loading rate (DLC) in the DDS, and the like. For example, doxil

As a successful product of DOX, its DLC is only around 10%, which means that 90% of the administered formulations are non-functional ingredients that increase the metabolic burden on the subject. Such factors limit the further development of DOX-loaded DDS in cancer therapy.

Disclosure of Invention

In order to overcome the defects of the existing DOX drug delivery system, the invention adopts a one-step self-assembly method to synthesize the DOX-loaded Cu-GA-DOX NPs. The Cu-GA-DOX NPs prepared by the invention have excellent drug loading capacity and biocompatibility, and can be further applied to the anti-tumor field.

The specific preparation method of the Cu-GA-DOX NPs comprises the following steps:

(1) Doxorubicin hydrochloride (DOX · HCl) was added to the vial, 3mL of distilled water was added, and the mixture was stirred at room temperature in the dark to dissolve DOX, yielding a DOX solution.

Wherein the concentration of the doxorubicin hydrochloride in the deionized water is 1-5mg/ml.

(2) Adding CuCl ₂ ·2H ₂ O (10 mg/mL) solution was quickly injected into the DOX solution while adding 3mL of absolute ethanol, and the solution was further stirred in the dark for 5min. Then, a tannic acid solution (GA, 10 mg/mL) was added thereto, and the mixture was stirred in the dark for 5min, and then ammonia was added thereto to obtain a purple-black precipitate, which was then centrifuged at 13000rpm for 15min to collect the precipitate.

Wherein, cuCl ₂ ·2H ₂ The mass ratio of the O to the tannic acid is 1:1-5:1;

CuCl ₂ ·2H ₂ the mass ratio of O, tannic acid (GA) and doxorubicin hydrochloride (DOX) is 1.

(3) The precipitate was dispersed in an appropriate volume of 5% glucose solution, and the mixture was sonicated at room temperature for 20min at 50% power (work 3s, rest 3 s) to give a uniformly dispersed solution of Cu-GA-DOX NPs.

The hydrated particle size of the Cu-GA-DOX NPs synthesized by the method is 58.98 +/-17.87 nm, the potential is-18.03 mV, and the polydispersity index is 0.188.

The Cu-GA-DOX NPs synthesized by the method have excellent drug loading capacity of 9.01-23.07% and encapsulation rate of 99.07-99.95%, show higher in vitro stability and more effective killing effect on CT26 cells, and can be used for preparing drugs for inhibiting in vivo and in vitro CT26 cell activity.

The Cu-GA-DOX NPs are conveniently stored in a variety of media (water, PBS at various pH, 0.9% NaCl).

The invention has the following beneficial effects:

the invention utilizes the high complexing ability of GA and DOX to metal ions, and uses Cu ²⁺ Are model ions. And a one-step self-assembly method is adopted, and Cu-GA-DOX NPs with high DOX load are obtained by optimizing a formula. In vitro experiments show that compared with free DOX, the Cu-GA-DOX NPs have stronger killing effect on CT26 cells, and in an ectopic colorectal cancer animal model, the Cu-GA-DOX NPs group has better inhibition effect on tumors than the free DOX group.

In addition, the invention finally adopts 5 percent glucose to disperse Cu-GA-DOX NPs, the used raw materials are low in price and convenient to obtain, the biological safety is higher, the preparation process disclosed by the invention is simple and convenient, the raw materials are wide in source, the preparation conditions are mild, and the preparation method is suitable for further popularization.

Description of the drawings:

FIG. 1 is a particle size distribution diagram of Cu-GA-DOX NPs;

FIG. 2 is a graph of the zeta potential of Cu-GA-DOX NPs;

FIG. 3 is a transmission electron micrograph of Cu-GA-DOX NPs;

FIG. 4 shows DOX, GA, and CuCl ₂ Ultraviolet absorption spectrograms of Cu-GA NPs and Cu-GA-DOX NPs;

FIG. 5 is a Fourier transform infrared spectrum of Cu-GA-DOX NPs;

FIG. 6 is a graph of UV absorption spectra of Cu-GA-DOX NPs in different media;

FIG. 7 is a graph (bar graph) showing the change in particle size of Cu-GA-DOX NPs in 5% glucose;

FIG. 8 is a graph of the change in polydispersity index of Cu-GA-DOX NPs in 5% glucose;

FIG. 9 is a fluorescence plot of uptake of DOX and Cu-GA-DOX NPs by CT26 cells;

FIG. 10 is a graph (bar graph) showing the effect of Cu-GA-DOX NPs on CT26 cell viability;

FIG. 11 is a graph showing the plasma kinetics of Cu-GA-DOX NPs in mice;

FIG. 12 is a graph of the change in body weight of mice during treatment;

FIG. 13 is a graph of mouse tumor growth during treatment;

figure 14 is a graph of H & E staining and Ki67 proliferation analysis of tumor tissue after treatment is complete;

FIG. 15 is a graph (bar graph) showing the effect of Cu-GA NPs on HUVEC cell viability;

FIG. 16 is a graph (bar graph) showing the hemolysis rate of Cu-GA NPs after co-incubation with erythrocytes;

FIG. 17 is a graph of H & E staining of major organs after treatment was completed.

Detailed Description

The present invention is further illustrated by the following examples.

Example 1

1. Preparation of Cu-GA NPs

1) Five portions of 0.1mL CuCl ₂ ·2H ₂ The O (10 mg/mL) solution is added into 5 penicillin bottles respectively, and 3mL absolute ethyl alcohol is added simultaneously.

2) 0.1mL,0.2mL,0.3mL,0.4mL, and 0.5mL of GA (10 mg/mL) solutions were added to the above mixture, respectively, and stirred for 5min, 0.1mL of aqueous ammonia was added to each solution to obtain a tan precipitate, and the precipitate was collected by centrifugation at 13000rpm for 15min.

3) The precipitate was dispersed in 2mL of water, and the mixture was sonicated at room temperature for 20min at 50% power (working 3s, standing 3 s) to give a uniformly dispersed nano solution of Cu-GA NPs.

By preparing GA CuCl ₂ ·2H ₂ The O feed ratio is 1:1-5:1, and the result shows that the generated nano-particle precipitation is reduced along with the increase of the GA dosage, and the GA is CuCl ₂ ·2H ₂ The largest amount of precipitate is obtained when the O feed ratio is 1:1, and finally the carrier ratio is 1:1.

2. Preparation of Cu-GA-DOX NPs

1) Add 9mg doxorubicin hydrochloride (DOX. HCl) to the vial, add 3mL distilled water, stir the mixture at room temperature in the dark to dissolve DOX.

2) 1.5mL of CuCl ₂ ·2H ₂ O (10 mg/mL) solution was quickly injected into the DOX solution above while adding 3mL of absolute ethanol. The solution was stirred in the dark for a further 5min. Then, 1.5mL of tannic acid (GA, 10 mg/mL) was added, the mixture was stirred in the dark for 5min, 0.1mL of ammonia water was added to obtain a purple-black precipitate, and the precipitate was collected by centrifugation at 13000rpm for 15min.

3) The precipitate was dispersed in a volume of 2mL of 5% glucose solution, and the mixture was sonicated at 50% power for 20min at room temperature (working 3s, standing 3 s) to give a uniformly dispersed solution of Cu-GA-DOX NPs.

In order to screen out proper drug loading and encapsulation efficiency, 3,6 and 15mg of doxorubicin hydrochloride (DOX & HCl) are weighed respectively and added into three penicillin bottles, and Cu-GA-DOX NPs are synthesized according to the method.

The following characteristics are not specified for Cu-GA-DOX NPs obtained with an addition of 9mg of doxorubicin hydrochloride.

3. Characterization of Cu-GA-DOX NPs

1) Determination experiment of hydrated particle size and Zeta potential of Cu-GA-DOX NPs

Taking CuCl ₂ Adding 2mL of deionized water into a synthesized Cu-GA-DOX NPs into an EP (ethylene propylene glycol) tube when the feeding ratio of GA to DOX is 1.

The hydrated particle size shown in FIG. 1 and the Zeta potential result shown in FIG. 2 show that CuCl ₂ The Cu-GA-DOX NPs prepared when the feeding ratio of GA to DOX is 1 to 0.6 have good dispersibility, the average hydrated particle size is 58.98 +/-17.87nm, the Zeta potential is-18.03 mV, and the polydispersity index is 0.188. Hydrated particle size and Zeta potential, unless otherwise stated, will be used in the following studies.

2) Transmission electron microscopy experiment of Cu-GA-DOX NPs

Adding CuCl into deionized water ₂ And when the feeding ratio of GA to DOX is 1. The results are shown in FIG. 3, and the Cu-GA-DOX NPs are shown inA net structure is present.

3) UV-visible absorption Spectrum of Cu-GA-DOX NPs

To further verify the successful preparation of Cu-GA-DOX NPs, DOX solution, GA solution, cuCl were assayed ₂ UV-visible absorption spectra of the solutions, cu-GA NPs nano-solutions and Cu-GA-DOX NPs nano-solutions. Diluting the above materials to final concentration of 100 μ g/mL, mixing by vortex, and adding 150 μ L DOX, GA, and CuCl into 96-well plate ₂ Cu-GA NPs and Cu-GA-DOX NPs, and recording the ultraviolet-visible absorption spectrum at 400-800nm by using an enzyme-labeled instrument, wherein as shown in figure 4, a single DOX aqueous solution has a characteristic peak at 480nm, and the absorption peak is red-shifted after being loaded in the Cu-GA-DOX NPs, thereby indicating the success of the preparation of the Cu-GA-DOX NPs.

4) Fourier infrared spectrogram of Cu-GA-DOX NPs

Cu-GA NPs and Cu-GA-DOX NPs were lyophilized separately and used together with DOX for infrared sample preparation, and the results are shown in FIG. 5, where the DOX molecule was at 2926cm ^-1 (C-H)、1617/1582cm ^-1 (N-H) has a characteristic absorption peak. 2924cm of characteristic absorption peak of DOX is also shown in the Cu-GA-DOX infrared spectrogram ^-1 (C-H)、1577cm ^-1 (N-H), confirming the successful synthesis of Cu-GA-DOX NPs.

5) Screening of Cu-GA-DOX NPs drug loading rate and encapsulation rate

The drug loading and encapsulation efficiency of the Cu-GA-DOX NPs at different feeding ratios are researched by using a fluorescence method. Firstly, cuCl is added ₂ The mass ratio of GA to GA is fixed to 1:1, and the adding mass of DOX is changed, so that the result is shown in table 1, the drug loading and encapsulation efficiency of Cu-GA-DOX NPs are in positive correlation with the mass of the added DOX, and when the mass ratio of the Cu-GA-DOX NPs to the GA is 1. And finally, when the mass ratio of the three components is 1.

Table 1 shows the drug loading and encapsulation efficiency of Cu-GA-DOX NPs at different ratios.

6) UV-VIS absorption spectra of Cu-GA-DOX NPs in different media

Cu-GA-DOX NPs were diluted to a final concentration of 50 μ g/mL in deionized water, PBS with pH =7.4, PBS with pH =6.5, PBS with pH =5.0, 0.9% NaCl solution, respectively, and incubated for 2h after a volume of 2mL, and then 150 μ L each was put into a 96-well plate, and the UV-VIS absorption spectra at 400-800nm were recorded, and as a result, as shown in FIG. 6, the absorption spectra of Cu-GA-DOX NPs in the above three media were substantially identical, indicating that Cu-GA-DOX NPs were not substantially affected in water, PBS with different pH values, and 0.9% NaCl.

7) Particle size stability of Cu-GA-DOX NPs

Taking 2mLCu-GA-DOX NPs in an EP tube, taking 20 mu L of each 2mLCu-GA-DOX NPs at 0,2,4,6,9 and 12 days, diluting the 20 mu L of each 2mLCu-GA-DOX NPs to 1mL of glucose for determining hydrated particle size, and obtaining the result as shown in figure 7,8, wherein the average particle size of the Cu-GA-DOX NPs fluctuates little up and down during the whole measurement period, and the polydispersity index PDI is basically kept below 0.4, which indicates that the Cu-GA-DOX NPs have better stability

4. CT26 cell Cu-GA-DOX NPs uptake experiment

2.5. Mu.g/mL of Cu-GA-DOX NPs were prepared with deionized water, while DOX solution of the same concentration was prepared as a control. Making CT26 cells into 5x10 ⁴ cell/ml concentration of cell suspension, 1ml per well in 24-well plates, placed at 37 ℃ C. And 5% CO ₂ The culture box is used for culturing for 16 hours. Primary medium was discarded and DOX, cu-GA-DOX NPs were added for co-incubation with CT26 cells, 3 wells for each sample. And sucking the upper culture medium at 2h,6h and 8h after incubation, adding a PBS solution for washing for 2-3 times, and adding 4% paraformaldehyde to fix the cells for 30min. Then washed again with PBS and stained for 30min with Hoechst33342 solution at a concentration of 10. Mu.g/ml and the nuclei are marked green (Hoechst 33342, E) _x ＝346nm)，DOX(E _x =485 nm), stained with a red color, the staining solution was aspirated, washed with PBS 3 times, and photographed by observation under a fluorescent inverted microscope. As shown in FIG. 9, the intracellular DOX signal was more significant at all time points tested in the case of the group of Cu-GA-DOX NPs than in the case of the group of DOX, indicating a higher uptake of Cu-GA-DOX NPs by CT26 cells.

5. Experiment on influence of Cu-GA-DOX NPs on CT26 cell survival rate

The effect of Cu-GA-DOX NPs on the survival of CT26 cells was examined by MTT method, CT26 cells were assayed at 37 ℃ in DMEM medium containing 10% FBS and 5% CO ₂ Culturing in medium. CT26 cells were plated at 8X 10 ³ cells/well Density into 96-well plates, place at 37 ℃ 5% CO ₂ The culture box is used for culturing for 16h, primary culture medium is discarded, cu-GA-DOX NPs and DOX are diluted by a DMEM high-sugar medium until the final concentration is 0.05,0.1,0.25,0.5,1 mu g/mL, and 100 mu L of Cu-GA-DOX NPs and DOX solution with different concentrations are added into each hole for further culturing for 24h. The cells were then co-cultured with fresh medium containing MTT (20. Mu.L, 5 mg/mL) for 4h. After that, the supernatant was discarded, 150. Mu.L of DMSO was added to the wells, and the 96-well plate was incubated for 15min at 250rpm in a shaker at 37 ℃. The absorbance of the solution at 570nm in each well was recorded by a microplate reader to determine the relative cell viability, with 5 replicate wells set at each concentration. As shown in FIG. 10, the cell viability was inversely correlated with the concentration of Cu-GA-DOX NPs. Under the concentration of 1 mu g/mL, the death rate of the tumor cells can reach more than 50 percent, and the effect is obviously superior to that of DOX.

6. Pharmacokinetic experiments of Cu-GA-DOX NPs in KM mice

The pharmacokinetic characteristics of the Cu-GA-DOX NPs in mice were analyzed by fluorescence method. Plasma drug content was determined according to a calibration curve of DOX. First, 1mg/mL of Cu-GA-DOX NPs and DOX are prepared, 100 μ L of each mouse is injected by tail vein injection, blood is collected from the orbital vein into an anticoagulation tube at 5, 15, 30min, 1, 2,4, 8, 24h after injection, blood samples are processed by centrifugation at 3000rpm for 10min at 4 ℃, plasma is collected, 1mL of a hydrochloric acid/isopropanol mixed solution (75 mM hydrochloric acid/isopropanol =1/1,v/v) is added, the mixture is processed at 37 ℃,250rpm in a shaker for 4h,13000rpm for 10min, supernatant is obtained, and the fluorescence intensity of DOX in the supernatant is measured by a fluorescence spectrophotometer. Determining DOX amount in supernatant according to corresponding standard curve, drawing drug-time curve, and calculating half-life t _1/2 . The results are shown in FIG. 11, where DOX cleared faster in vivo than Cu-GA-DOX NPs, whereas Cu-GA-DOX NPs absorbed faster, cleared slower, and the cycle time was significantly longer, the t of Cu-GA-DOX NPs _1/2 Is 12.64hT of DOX _1/2 The time is 3.76h. These results are consistent with the conclusions obtained in previous studies, suggesting that Cu-GA-DOX NPs can significantly prolong the half-life of DOX, not only can increase the bioavailability of the drug, but also can contribute to the accumulation and retention effects of DOX in tumor tissues through enhanced permeability.

7. In vivo antitumor assay of Cu-GA-DOX NPs

1) Establishment of tumor-bearing mouse animal model

In order to research the in vivo anti-tumor efficiency of Cu-GA-DOX NPs, we firstly establish an ectopic colorectal cancer animal model with CT26 cells, and subcutaneously inject the CT26 cells into the right hind limb of a female Balb/c mouse with the age of 4-6 weeks by 2X 10 ⁷ mL, wait for tumor growth, which is about 2 weeks. When some bulges start at the injection site, the long diameter L and the short diameter W of the tumor at the bulge sites and the weight of the mouse are measured every other day, and the size of the tumor is calculated by adopting the following formula: v = L × W ² /2. When the tumor volume reaches 50mm ³ At time, mice were grouped (3 per group).

2) In vivo treatment of ectopic colorectal cancer with Cu-GA-DOX NPs

First, cu-GA-DOX NPs and DOX with the same concentration of 1mg/mL are prepared, PBS is prepared as a reference substance, the three different substances are injected into the tail vein for treatment, the administration dosage is calculated according to DOX (5 mg/kg,100 mu L/mouse), the injection administration is carried out on the 0 th, 2 th, 4 th, 6 th, 8 th and 10 th days, the administration is carried out for 6 days, the whole treatment period lasts for 14 days, the long diameter and the short diameter of the tumor are measured by a vernier caliper on the alternate days of administration, the weight of the mouse is weighed, and the growth curve and the weight change curve of the tumor are drawn after the administration is finished. The results are shown in FIGS. 12 and 13, and the tumor volume of the control mice continued to increase compared to the PBS control group, while the tumor growth of the other treatment groups was significantly inhibited. More surprisingly, the group of Cu-GA-DOX NPs showed the best tumor suppression efficiency. The body weight of each group of mice remained essentially constant throughout the treatment period.

2) H & E staining and Ki67 proliferation assay of tumor tissues

Tumor tissues were harvested for imaging at day 14 after the end of dosing, and the results are shown in FIG. 14. Then, the tumor tissues were subjected to tissue analysis, and the results are shown in FIG. 15, and the results of H & E staining showed that the degree of necrosis of tumor cells was the highest in the Cu-GA-DOX NPs group, and the results of Ki67 showed that the degree of necrosis, proliferation and apoptosis were the highest in the Cu-GA-DOX NPs group, as compared with those in the other two groups. The Cu-GA-DOX NPs are proved to have good in-vivo anti-tumor effect.

8. In vivo biosafety analysis of Cu-GA-DOX NPs

1) Effect of Cu-GA-DOX NPs on HUVEC cell viability

The effect of Cu-GA-DOX NPs on HUVEC cell viability was determined by MTT method, HUVEC cells were assayed in DMEM medium containing 10% FBS at 37 ℃ and 5% CO ₂ And (4) medium culture. HUVEC cells were cultured at 8X 10 ³ cells/well Density into 96-well plates, place at 37 ℃ 5% CO ₂ The culture box is cultured for 16h, primary culture medium is discarded, cu-GANPs are diluted to the final concentration of 0.1,0.5,1,5, 10 and 20 mu g/mL by using DMEM high-sugar medium, and 100 mu L of Cu-GA NPs with different concentrations are added into each hole for continuous culture for 24h. The cells were then co-cultured with fresh medium containing MTT (20. Mu.L, 5 mg/mL) for 4h. After that, the supernatant was discarded, 150. Mu.L of DMSO was added to the wells, and the 96-well plate was incubated for 15min at 250rpm in a shaker at 37 ℃. The absorbance of the solution at 570nm in each well was recorded by a microplate reader to determine the relative cell viability, with 5 replicate wells set at each concentration. When the viability of HUVEC cells co-cultured with Cu-GANPs exceeded 80%, the cytotoxicity of this material was considered negligible, and the results are shown in fig. 15, with the survival of HUVEC cells co-cultured with Cu-GANPs exceeding 80% in all groups (fig. 8A), indicating that there was no significant cytotoxicity of Cu-GA NPs over the tested concentration range.

2) Determination of hemolysis rate of Cu-GA-DOX NPs

KM mouse blood is taken in an anticoagulation tube by the way of orbital venous blood collection, centrifuged at 2000rpm for 10min at 4 ℃, and the supernatant is discarded. The erythrocytes were collected and washed 3 times with PBS. The final red blood cells were diluted to 20% with PBS. 0.02mL of the diluted red blood cell suspension was mixed with 1mL of Cu-GA NPs solutions of different concentrations (Cu-GA NPs concentration: 2.5,5,8, 10, 20. Mu.g/mL). For positive and negative controls, 0.02mL of diluted erythrocyte suspension was mixed with 1mL of 1% triton and PBS. After incubation at 37 ℃ for 2h, centrifugation was carried out at 2000rpm for 10min, and 120. Mu.L of the supernatant was transferred to a 96-well plate at each concentration. The absorbance at 540nm was recorded with a microplate reader. The hemolysis rate is calculated as follows: hemolysis rate (%) = (sample absorbance-negative control absorbance)/(positive control absorbance-negative control absorbance) × 100%. As a result, as shown in FIG. 16, the hemolysis value of the concentration of all tested Cu-GA NPs was similar to that of the negative control group (PBS), indicating that Cu-GA NPs have good blood compatibility.

3) H & E staining analysis of major organs

The major organs (heart, liver, spleen, lung, kidney) were harvested on day 14 after the end of the administration and analyzed for H & E staining, the results of which are shown in fig. 17. Compared with the control group, the main organs of the other two groups of mice treated by the traditional Chinese medicine composition have no obvious abnormality or damage and are basically consistent with the control group. The Cu-GA-DOX NPs have good biological safety.

Claims

1. A preparation method of Cu-GA-DOX NPs of an adriamycin delivery system is characterized by comprising the following steps:

(1) Adding doxorubicin hydrochloride (DOX & HCl) into a penicillin bottle, adding 3mL of deionized water, and stirring the mixture at room temperature in a dark place to dissolve DOX to obtain a DOX solution;

(2) Adding CuCl ₂ ·2H ₂ Quickly injecting the O solution into the DOX solution obtained in the step (1), simultaneously adding 3mL of absolute ethyl alcohol, further stirring the mixed solution for 5min in the dark, then adding a tannic acid (GA) solution, stirring for 5min in a dark place, adding ammonia water to obtain a purple black precipitate, centrifuging at 13000rpm for 15min, and collecting the precipitate;

(3) And (3) dispersing the precipitate in the step (2) in a 5% glucose solution, and carrying out ultrasonic treatment for 20min at room temperature by using an ultrasonic cell crusher at a power of 50% to obtain a uniformly dispersed Cu-GA-DOX NPs solution.

2. The method for preparing Cu-GA-DOX NPs as delivery system of adriamycin according to claim 1, wherein the concentration of adriamycin hydrochloride in deionized water in step (1) is 1-5mg/ml.

3. The method for preparing Cu-GA-DOX NPs as delivery system of adriamycin according to claim 1, wherein the CuCl in step (2) ₂ ·2H ₂ The concentration of the O solution is 10mg/mL, and the concentration of the tannic acid solution is 10mg/mL.

4. The method for preparing Cu-GA-DOX NPs as delivery system of adriamycin according to claim 1, wherein the CuCl in step (2) ₂ ·2H ₂ The mass ratio of O to tannic acid is 1:1-5:1.

5. The method for preparing Cu-GA-DOX NPs as delivery system of adriamycin according to claim 1, wherein the CuCl in step (2) ₂ ·2H ₂ The mass ratio of O, tannic acid and doxorubicin hydrochloride is 1.

6. Delivery system for doxorubicin Cu-GA-DOX NPs prepared according to the method of any one of claims 1-5.

7. The doxorubicin delivery system Cu-GA-DOX NPs according to claim 6, wherein said Cu-GA-DOX NPs has a drug loading of 9.01-23.07% and an encapsulation efficiency of 99.07-99.95%.

8. The doxorubicin delivery system Cu-GA-DOX NPs according to claim 6 wherein said Cu-GA-DOX NPs have a hydrated particle size of 58.98 ± 17.87nm, a zeta potential of-18.03 mV, and a polydispersity index of 0.188.

9. Use of Cu-GA-DOX NPs as a delivery system for anticancer agent DOX prepared according to the method of any of claims 1-5 for the preparation of a medicament for inhibiting the activity of CT26 cells in vitro and in vivo.