CN109260480A - A kind of chitosan nano meter level acoustic contrast agent and the preparation method and application thereof carrying adriamycin - Google Patents

Abstract

The present invention relates to a kind of chitosan nano meter level acoustic contrast agents and the preparation method and application thereof for carrying adriamycin, the chitosan nano meter level acoustic contrast agent prepared by the present invention for carrying adriamycin, it is using chitosan as shell membrane, adriamycin (DOX) and perfluoropropane gas are wrapped in inside chitosan shell membrane, partial size is 300~900nm, average grain diameter is 641nm (polymer dispersity index PDI:0.256), in nano-scale range, tumor blood vessels wall gap can be passed through, there is targeting and high efficiency for the treatment of tumour.The nanoscale ultrasound contrast agents have stronger enhancing visualization capabilities, and it can maintain to develop in a longer period of time, entrapment efficiency and drugloading rate also with higher, biological safety is high, combining ultrasonic targeting destroys microvesicle (UTMD) and can significantly improve the recovery rate of DOX and reduce tumour cell survival rate, is conducive to the treatment of tumor disease.

Description

Chitosan nanometer ultrasonic contrast agent carrying doxorubicin and preparation method and application thereof

Technical field

The invention relates to a chitosan nano-level ultrasonic contrast agent carrying doxorubicin and a preparation method and application thereof, and belongs to the technical field of ultrasonic molecular imaging.

Background technique

With the rapid development of ultrasonic molecular imaging technology and biological nanotechnology, nano-scale ultrasound contrast agents have developed rapidly. Nano-scale ultrasound contrast agent is one of the research hotspots in the field of molecular imaging in recent years, and it is also one of the important research directions of Targeted drug delivery system (TDDS), which can effectively overcome the poor targeting of traditional tumor drug therapy. , systemic toxic side effects and other shortcomings. Unlike traditional micron-sized ultrasound contrast agents (blood pool imaging agents), nano-scale ultrasound contrast agents have a strong penetrating power and can pass through the vascular wall space of tumor tissues. Its development and application are also conducive to further development of targeting. A new type of ultrasound contrast agent that is highly efficient, miniaturized, and has an adjuvant therapeutic effect. Nano-scale ultrasound contrast agent is an important direction for the development of ultrasound molecular imaging, and it is also a new direction for tumor targeting, visualization and precision treatment.

Shell membrane material is a key element in determining the performance of ultrasound contrast agents. At present, a variety of materials are used for the preparation of ultrasound contrast agents, including: lipids, polymeric polymers, surfactants, etc., but all have certain disadvantages. Lipid ultrasound contrast agents have good stability and good imaging results, but they are expensive and potentially toxic to the kidneys and liver. There are safety problems. Polymers such as lactic acid/glycolic acid copolymer (PLGA) can be prepared. A more stable ultrasound contrast agent prolongs the diagnosis time, but because the contrast agent shell is hard, the elasticity is poor, the imaging is poor, and the high ultrasonic output frequency is required, which may cause non-therapeutic loss to normal tissues; surfactant Ultrasound contrast imaging is good, but its stability is poor and it is not easy to be modified. The chitosan material is non-toxic, non-irritating, not only has good biocompatibility and biodegradability, but also has unique physiological and pharmacological activities. Most importantly, it is rich in active groups such as free amino groups and hydroxyl groups. With good modification, it is an ideal ultrasound contrast agent shell membrane material. Chinese patent document CN106139174A provides a preparation method of a nanometer-based ultrasonic contrast agent based on chitosan derivative encapsulating liquid fluorocarbon, which is modified by acylation reaction to synthesize carboxymethyl chitosan, and has amphiphilic synthesis. The n-hexyl carboxymethyl chitosan was added with liquid fluorocarbon, and a nano-scale ultrasonic contrast agent composed of a liquid fluorocarbon core and a chitosan derivative shell was obtained by ultrasonic emulsification. The polymer material used in the method is safe and non-toxic, and the preparation process of the contrast agent is simple and the operating conditions are mild. However, the nano-scale ultrasound contrast agent prepared by the method is in the state of nano-droplet. In practical application, a certain external stimulus is required to cause liquid-gas phase change, and no further research and evaluation are made on its targeting and drug-loading characteristics.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a chitosan nano-grade ultrasonic contrast agent carrying doxorubicin and a preparation method thereof, and a nano-scale ultrasonic contrast agent prepared by using chitosan as a shell material, the contrast agent It has strong enhanced imaging ability, high drug encapsulation rate and drug loading, and high biosafety.

The invention also provides the application of the above-mentioned doxorubicin-loaded chitosan nanometer ultrasound contrast agent in treating tumors and preparing antitumor drugs.

Explanation of terms:

Doxorubicin (DOX), an anti-tumor antibiotic that inhibits the synthesis of RNA and DNA, has the strongest inhibitory effect on RNA, has a broad anti-tumor spectrum, and has a role in a variety of tumors. The drug has a killing effect on tumor cells of various growth cycles.

Room temperature: 25 ± 2 ° C.

The technical solution of the present invention is:

A chitosan nano-level ultrasound contrast agent carrying doxorubicin, the nano-scale ultrasound contrast agent adopts chitosan as a shell membrane, and the chitosan shell membrane is internally coated with doxorubicin and gaseous fluorocarbon, the nanometer The particle size of the ultrasonic contrast agent is 300-900 nm, wherein the mass ratio of chitosan to doxorubicin is 1:1 to 4.

The preparation method of the above-mentioned doxorubicin-loaded chitosan nano-level ultrasound contrast agent is as follows:

(1) Dispersing palmitic acid and lecithin in a phosphate buffer solution to prepare a suspension, adding chitosan and doxorubicin to the suspension, and incubating at 20 ° C for 20 to 40 mim to obtain an incubation product;

(2) The incubation product prepared in the step (1) is filled with gaseous fluorocarbon, and ultrasonically shaken at room temperature for 1 to 5 minutes to obtain an emulsified product;

(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C, the standing time is ≥ 2 h, and the lower layer liquid is taken after stratification, and the mixture is diluted and centrifuged at a low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent.

According to the present invention, the mass ratio of palmitic acid to lecithin in step (1) is 1:2, the concentration of palmitic acid in the suspension is 0.4-0.5 g/L; wherein the purity of palmitic acid is ≥99%. , purchased from sigma, product number 200-312-9; lecithin purity ≥ 99%, purchased from sigma, product number 232-715-0.

According to a preferred embodiment of the invention, the phosphate buffer component of step (1) has a composition of: Na ₂ HPO ₄ 8 mM, KH ₂ PO ₄ 2 mM, pH 7.2-7.4.

According to the preferred embodiment of the present invention, the mass ratio of chitosan to doxorubicin in step (1) is 1:1 to 4, and the concentration of chitosan in the suspension is 0.4 to 0.6 g/L; The molecular weight of chitosan was 160 kDa, purchased from sigma, product number MFCD00161512; the purity of doxorubicin was ≥98%, purchased from sigma, product number 246-818-3.

Further preferably, the mass ratio of the chitosan to the doxorubicin is 1:2,

According to a preferred embodiment of the invention, the gaseous fluorocarbon in step (2) is a perfluoropropane gas.

According to a preferred embodiment of the invention, the dilution in step (3) is 5 to 10 times.

According to a preferred embodiment of the invention, the low speed centrifugation in step (3) is centrifuged at 500 rpm for 5 min.

The above-mentioned doxorubicin-loaded chitosan nano-sound ultrasound contrast agent combined with ultrasonic targeted destruction of microbubbles in the treatment of tumors.

The above-mentioned doxorubicin-loaded chitosan nano-sound ultrasound contrast agent combined with ultrasonic targeted destruction of microbubbles for the preparation of antitumor drugs.

The technical features of the invention: palmitic acid and lecithin contribute to the dissolution of chitosan, and the preparation of the incubation product is facilitated by the dissolution of chitosan, and the incubation product of chitosan and doxorubicin is obtained by phacoemulsification. An emulsion product with chitosan as the outer shell and doxorubicin and gaseous fluorocarbon as the core. After standing, the ultrasonic contrast agent with larger particle size is distributed in the upper layer, and the nano-sized ultrasound contrast agent with relatively small particle size is distributed. In the lower layer, the lower layer liquid was subjected to dilution and low-speed centrifugation to obtain a nano-sized ultrasonic contrast agent having a small particle size and uniformity.

Beneficial effects:

1. The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared by the invention is prepared by chitosan as a shell membrane, and doxorubicin (DOX) and perfluoropropane gas are encapsulated in the chitosan shell membrane. The particle size is 300-900 nm, and the average particle size is 641 nm (polymer dispersion index PDI: 0.256). In the nanometer range, it can pass through the blood vessel wall gap of the tumor tissue, and is targeted and highly effective for the treatment of tumors.

2. The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared by the invention has strong enhanced imaging ability, and can maintain development for a long time, and has a high drug encapsulation rate and The drug loading, biosafety, irradiation intensity is 0.5W/cm ² , irradiation time is 30sec or 60sec, cell survival rate is >80%, suitable for ultrasound imaging and adjuvant treatment of tumor diseases.

3. The invention utilizes the method of ultrasonic irradiation to enhance the development of chitosan nano-sound ultrasound contrast agent carrying doxorubicin on the one hand, and promote the doxorubicin by the sonic hole effect generated by ultrasonic irradiation on the other hand. Into the tumor cells, to achieve integration of diagnosis and treatment.

4. The DOX-loaded chitosan nano-scale ultrasound contrast agent prepared by the invention combined with ultrasonic targeted destruction microbubbles (UTMD) can significantly improve the extraction rate of DOX and reduce the survival rate of tumor cells, and is beneficial to the treatment of tumor diseases.

DRAWINGS

Figure 1 is a photomicrograph of a chitosan nano-scale ultrasound contrast agent carrying DOX;

2 is a particle size diagram of a DOX-loaded chitosan nano-scale ultrasound contrast agent;

Figure 3 is an in vitro self-made ultrasound enhanced imaging device;

Figure 4 is an in vitro development of a chitosan nanoscale ultrasound contrast agent carrying DOX;

Figure 5 is a graph showing the trend of in vitro development of a chitosan nano-sized ultrasound contrast agent carrying DOX over time;

Figure 6 is a biosafety evaluation of chitosan nanoscale ultrasound contrast agent carrying DOX under different ultrasound parameters;

Figure 7 is a flow cytometry diagram of DOX uptake in a chitosan nanoscale ultrasound contrast agent carrying DOX;

Figure 8 is a graph showing the effect of DOX-loaded chitosan nano-grade ultrasound contrast agent on the survival rate of breast cancer MCF-7 cells.

Detailed ways

The present invention will be further described below in conjunction with the embodiments, but the scope of protection of the present invention is not limited thereto.

Palmitic acid was purchased from sigma, product number 200-312-9; lecithin was purchased from sigma, product number 232-715-0; chitosan was purchased from sigma, product number MFCD00161512; doxorubicin was purchased from sigma, product number 246- 818-3.

The present embodiment relates to drugs and reagents which are all commercially available products unless otherwise specified.

Example 1: Preparation of DOX-loaded chitosan nano-scale ultrasound contrast agent

A preparation method of chitosan nanometer ultrasonic contrast agent carrying doxorubicin, the steps are as follows:

(1) Weigh palmitic acid and lecithin dispersed in phosphate buffer (Na ₂ HPO ₄ 8 mM, KH ₂ PO ₄ 2 mM, pH 7.2), and mix to prepare a suspension for use in the suspension. , the concentration of palmitic acid is 0.45g / L, the concentration of lecithin is 0.9g / L; chitosan (molecular weight 160kDa) and doxorubicin, chitosan and doxorubicin are mixed into the above suspension. The concentration in the suspension was 0.5 g/L and 1 g/L, respectively, and the mixture was incubated at 25 ° C for 30 mim to obtain an incubation product;

(2) The incubation product prepared in the step (1) is filled with perfluoropropane gas, and ultrasonically shaken at room temperature for 2 minutes to obtain an emulsified product;

(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C for 2 h, and the lower layer liquid is taken after stratification, diluted 5 times, and then centrifuged at 500 rpm for 5 min at low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent (DOX-NB).

The doxorubicin-loaded chitosan nano-sized ultrasound contrast agent prepared above was diluted and added to a glass slide, and the apparent morphology of the contrast agent was observed by an optical microscope. The result is shown in Fig. 1, 1000× optical. Under the microscope, the contrast agents were all spherical, the particle size was uniform, and the dispersion was uniform without aggregation.

The doxorubicin-loaded chitosan nano-scale ultrasound contrast agent prepared above is diluted, and the particle size of the ultrasonic contrast agent is detected by using a nano-laser particle size and a zeta potential analyzer. The result is shown in FIG. The diameter was 300 to 900 nm, the average particle diameter of the contrast agent was 641 nm (polymer dispersion index PDI: 0.256), and the surface potential was 67.12 ± 2.1 mW.

Example 2

A preparation method of chitosan nanometer ultrasonic contrast agent carrying doxorubicin, the steps are as follows:

(1) Weigh palmitic acid and lecithin dispersed in phosphate buffer (Na ₂ HPO ₄ 8 mM, KH ₂ PO ₄ 2 mM, pH 7.3), and mix to prepare a suspension for use in the suspension. , the concentration of palmitic acid is 0.4g / L, the concentration of lecithin is 0.8g / L; chitosan (molecular weight 160kDa) and doxorubicin, chitosan and doxorubicin are mixed into the above suspension. The concentration in the suspension was 0.4 g/L and 1.6 g/L, respectively, and 25 μm was incubated at 25 ° C to obtain an incubation product;

(2) The incubation product prepared in the step (1) is filled with perfluoropropane gas, and ultrasonically shaken at room temperature for 3 minutes to obtain an emulsified product;

(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C for 4 h, and the lower layer liquid is taken after stratification, diluted 6 times, and then centrifuged at 500 rpm for 5 min at low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent (DOX-NB).

The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared in this embodiment has a spherical shape under a 1000× optical microscope, uniform particle size, uniform dispersion and no aggregation, and the particle size of the contrast agent is 300-900 nm.

Example 3

A preparation method of chitosan nanometer ultrasonic contrast agent carrying doxorubicin, the steps are as follows:

(1) Weigh palmitic acid and lecithin dispersed in phosphate buffer (Na ₂ HPO ₄ 8 mM, KH ₂ PO ₄ 2 mM, pH 7.4), and mix to prepare a suspension for use in the suspension. The concentration of palmitic acid is 0.5g/L, and the concentration of lecithin is 1.0g/L; chitosan (molecular weight 160kDa) and doxorubicin are added to the above suspension, and chitosan and doxorubicin are mixed. The concentrations in the suspension were 0.6 g/L and 1.8 g/L, respectively, and the mixture was incubated at 25 ° C for 35 mim to obtain the incubation product;

(2) The incubation product prepared in the step (1) is filled with perfluoropropane gas, and ultrasonically shaken at room temperature for 4 minutes to obtain an emulsified product;

(3) The emulsified product obtained in the step (2) is allowed to stand at 4 ° C for 6 h, and the lower layer liquid is taken after stratification, diluted 8 times, and then centrifuged at 500 rpm for 5 min at low speed to take the lower layer liquid, which is a shell carrying doxorubicin. A nanometer-scale ultrasound contrast agent (DOX-NB).

The doxorubicin-loaded chitosan nano-sound ultrasound contrast agent prepared in this embodiment has a spherical shape under a 1000× optical microscope, uniform particle size, uniform dispersion and no aggregation, and the particle size of the contrast agent is 300-900 nm.

Example 4: Drug Encapsulation Rate and Drug Loading of DOX-NB

Taking the lower liquid after standing in the preparation of DOX-NB in Examples 1 to 3, the absorbance of the bottom liquid in the lower liquid at 480 nm was detected by a microplate reader, and the free DOX mass in the bottom liquid was calculated according to the standard curve, and then according to the following The formula calculates the drug encapsulation efficiency (EE%) and drug loading (LD%) of DOX-NB:

EE% = (loaded DOX quality / initial DOX quality added) x 100%.

LD%=(quality of DOX loaded/mass of added chitosan nano-sound ultrasound contrast agent)×100%

among them,

DOX quality carried = initial DOX quality added - free DOX mass,

The mass of chitosan nano-scale ultrasound contrast agent added is the total mass of the starting materials added, including chitosan, doxorubicin, palmitic acid and lecithin.

The calculation results are shown in Table 1. The DOX-NB prepared by the present invention has a drug encapsulation efficiency of 50% or more and a drug loading amount of ≥59.38 mg of DOX/g contrast agent.

Table 1 Encapsulation efficiency and drug loading of different chitosan ultrasonic nano-scale contrast agents carrying DOX

Example 5: In vitro ultrasonic development capability detection of DOX-NB

The 4% agar gel model was prepared, and the enhanced imaging ability of DOX-NB prepared in Example 1 was observed. The operation device was as shown in Fig. 3. DOX-NB at a concentration of 1.0 × 10 ⁶ bubbles/mL was added to the agar gel model. In the circular hole, the ultrasonic imaging capability was measured by a clinical diagnostic ultrasound system (LOGIQ E9; GE, USA). The main parameters were: frequency 9.0 MHz; mechanical index: 0.12; focal length: 3.0 cm; dynamic range: 60 dB, two-dimensional and ultrasound The contrast mode is observed synchronously and the parameter settings remain unchanged.

The image data is stored by the internal workstation of the ultrasound system. The image is shown in Figure 4. In the gray-scale imaging and enhanced imaging mode, the image can be displayed clearly; the image J software analyzes the gray value of the image, and the result is shown in the figure. As shown in Fig. 5, the above results indicate that the contrast agent has strong ultrasonic development ability and can maintain development for a long time (15 min).

Example 6: Biosafety detection of chitosan nanometer ultrasound contrast agent

The chitosan nano-sound ultrasound contrast agent was prepared according to the preparation method described in Example 1, except that no doxorubicin was added in the step (1). The chitosan nano-scale ultrasound contrast agent prepared above was used to determine the biosafety of the MCF-7 cell line using CCK-8 reagent. The specific operation was as follows: MCF-7 cells were seeded in a 96-well plate to contain 10% FBS DMEM was used as the medium, the density of the cells was 1.0×10 ⁴ cells/well, cultured in a 37° C., 5% CO ₂ incubator for 24 hours, the cells were attached, and the medium was changed to contain the above chitosan nanoscale. Ultrasound contrast agent in fresh 10% FBS DMEM medium, volume fraction of chitosan nanometer ultrasound contrast agent in DMEM medium was 0, 10%, 20%, 30%, respectively, and ultrasonic irradiation, ultrasonic irradiation treatment As shown in the following table:

Table 2 different ultrasonic irradiation treatment methods

After the irradiation, the cells were cultured in a 37 ° C, 5% CO ₂ incubator for 48 h, then the cells were washed with PBS, and fresh 10% FBS DMEM medium containing 10 μL of LCCK-8 reagent was added, and incubation was continued for 1-4 h, using a microplate reader. The light absorption value was measured at a wavelength of 480 nm. The results are shown in Fig. 6. When the volume fraction of chitosan nano-scale ultrasound contrast agent is ≤30% and the irradiation intensity is 0.5W/cm ² , the survival rate of each group is >80%; when the irradiation intensity is increased At 1.0W/cm ² , the volume fraction of chitosan nano-grade ultrasound contrast agent is ≤20%, and the cell survival rate is also above 80%, indicating that the contrast agent has little cytotoxicity within a certain range of ultrasonic intensity. Good security.

Example 7: Effect of DOX-NB combined targeted destruction microbubbles (UTMD) on DOX extraction rate

The DOX-NB prepared in Example 1 was used to detect the DOX uptake rate of MCF-7 cells in DOX-NB. The procedure was as follows: MCF-7 cells were seeded in a 6-well plate at a density of 2.5×10 ⁵ cells per well. Overnight, add fresh 10% FBSDMEM medium, 10% FBS DMEM medium containing free DOX, DOX-NB, free DOX and UTMD, DOX-NB and UTMD, respectively, DOX-NB in the medium The volume fraction was 20%, and the content of DOX in each group remained the same. Incubate for 1 h at 37 ° C, 5% CO ₂ incubator, and give ultrasonic irradiation or no ultrasonic irradiation. The treatment methods are as follows:

Table 3. Different ultrasonic irradiation treatments in DMEM medium containing different components

After the end of the ultrasonic irradiation treatment, MCF-7 cells were collected for flow cytometry. The results are shown in Figure 7. The results showed that the DOX uptake efficiency of DOX-NB combined with UTMD group was significantly higher than that of the control group and free DOX combined with UTMD group. Moreover, the uptake efficiency at the irradiation time of 60 sec was higher than the uptake efficiency at the irradiation time of 30 sec.

Example 8: Effect of DOX-NB combined with targeted disruption of microvesicles (UTMD) on MCF-7 cells

The DOX-NB prepared in Example 1 was used to determine the effect of combined UTMD on MCF-7 cells using CCK-8 reagent, and the application value of DOX-NB in tumor therapy was predicted. The specific operation was as follows: MCF-7 cells were seeded in a 96-well plate, and DMEM containing 10% FBS was used as a medium. The density of the cells was 1.0×10 ⁴ cells/well, and the cells were incubated overnight at 37° C. in a 5% CO ₂ incubator. Culture, cell adherence, replacement of fresh 10% FBS DMEM medium containing free DOX, DOX-NB, free DOX and UTMD, DOX-NB and UTMD, respectively, wherein the volume of DOX-NB in the medium The score was 20%. The content of DOX in each group was consistent. Ultrasonic irradiation or no ultrasonic irradiation was applied. The treatment was as shown in Table 3. In addition, MCF-cultured in 10% FBS DMEM medium containing no other ingredients. 7 cells were blank controls and were not treated with ultrasound.

After the end of the ultrasonic irradiation, culture in a 37 ° C, 5% CO ₂ incubator for 24 hours, then wash the cells with PBS, add fresh 10% FBS DMEM medium containing 10μLCCK-8 reagent, continue to incubate for 1-4h, using enzyme label The instrument absorbs the light absorption value at 480 nm and calculates the cell survival rate. The results are shown in Fig. 8. The results show that DOX-NB combined with UTMD has an ultrasonic intensity of 0.5 W/cm ² and an irradiation time of 60 sec, MCF- The survival rate of the 7 cells was the lowest, 2.2±0.9%, which was significantly lower than that of the DOX-NB group (21.0±2.2%) and the free DOX group (6.4±0.7%). The difference was statistically significant (p<0.01). DOX-NB combined with UTMD can effectively reduce the survival rate of tumor cells under ultrasound irradiation conditions and is applied to tumor treatment.

Claims (10)

1. The chitosan nano-scale ultrasonic contrast agent carrying adriamycin is characterized in that chitosan is used as a shell membrane, adriamycin and gaseous fluorocarbon are wrapped inside the shell membrane, the particle size of the nano-scale ultrasonic contrast agent is 300-900 nm, and the mass ratio of the chitosan to the adriamycin is 1: 1-4.

2. The method for preparing a chitosan nano-scale ultrasonic contrast agent carrying doxorubicin according to claim 1, characterized by comprising the steps of:

(1) dispersing palmitic acid and lecithin in a phosphate buffer solution to prepare a suspension, adding chitosan and adriamycin into the suspension, and incubating for 20-40 mim at 25 ℃ to obtain an incubation product;

(2) filling gaseous fluorocarbon into the incubation product prepared in the step (1), and performing ultrasonic oscillation for 1-5 min at room temperature to obtain an emulsified product;

(3) and (3) standing the emulsified product prepared in the step (2) at 4 ℃, wherein the standing time is more than or equal to 2 hours, taking the lower-layer liquid after layering, diluting, centrifuging at low speed, and taking the lower-layer liquid, namely the chitosan nano-ultrasonic contrast agent carrying the adriamycin.

3. The preparation method according to claim 2, wherein the mass ratio of the palmitic acid to the lecithin in the step (1) is 1:2, and the concentration of the palmitic acid in the suspension is 0.4-0.5 g/L; wherein the purity of the palmitic acid is more than or equal to 99 percent, and the purity of the lecithin is more than or equal to 99 percent.

4. The method according to claim 2, wherein the phosphate buffer in step (1) has the following composition: na (Na)₂HPO₄8mM，KH₂PO₄2mM，pH 7.2-7.4。

5. The preparation method according to claim 2, wherein the mass ratio of the chitosan to the adriamycin in the step (1) is 1: 1-4, and the concentration of the chitosan in the suspension is 0.4-0.6 g/L; wherein the molecular weight of the chitosan is 160kDa, and the purity of the adriamycin is more than or equal to 98 percent.

6. The method of claim 2, wherein the gaseous fluorocarbon in step (2) is perfluoropropane gas.

7. The method according to claim 2, wherein the dilution in the step (3) is 5 to 10 times.

8. The method according to claim 2, wherein the low-speed centrifugation in the step (3) is centrifugation at 500rpm for 5 min.

9. The use of the chitosan nano-sized ultrasound contrast agent carrying doxorubicin according to claim 1 in combination with ultrasound targeted microbubble destruction for the treatment of tumors.

10. The use of the chitosan nano-sized ultrasound contrast agent carrying doxorubicin in combination with ultrasound targeted microbubble destruction for the preparation of an antitumor drug as claimed in claim 1.