CN107537044B - Chitosan nano microbubble and preparation method and application thereof - Google Patents
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Abstract
The invention provides a chitosan nano microbubble and a preparation method and application thereof, wherein the method comprises the following steps: (1) preparing an acrylic acid aqueous solution, adding chitosan, dissolving, heating the solution to 60-80 ℃ in the presence of protective gas, adding an initiator for reaction, then reducing the temperature of the solution, carrying out reaction, filtering and dialyzing to remove impurities, adding a cross-linking agent after the treatment is finished, heating, and reacting to obtain a nanoparticle suspension; (2) and adding a freeze-drying protective agent into the nanoparticle suspension, and freeze-drying to obtain the chitosan nanoparticle microbubble. According to the invention, chitosan is dissolved in an acrylic acid solution according to a certain proportion, an initiator is added to enable acrylic acid and chitosan to carry out polymerization reaction to obtain hollow nanoparticles containing water inside, the water in the hollow structure inside is removed, and the prepared nano microbubbles have synergistic effects of ultrasonic development and high-intensity focused ultrasound tissue ablation after being dissolved again.
Description
Technical Field
The invention relates to the field of contrast agents, in particular to chitosan nano microbubble and a preparation method and application thereof.
Background
At present, the mature ultrasonic contrast agents are micron-sized contrast agents, the diameter range of the contrast agents is about 2-8 mu m, the contrast agents can smoothly pass through pulmonary capillaries without causing embolism, but cannot pass through the outside of blood vessels, the developing capability of the contrast agents on extravascular diseases is limited, blood vessels at inflammation and embolism positions are narrow, and the micron-sized ultrasonic contrast agents sometimes cannot smoothly pass through the contrast agents. With the development of molecular biology and nanotechnology, nanopharmaceutics have grown up rapidly. The nano-scale contrast agent has the particle size of 700nm, can effectively realize extravascular imaging, has more advantages than micro-scale microbubbles in clinical diagnosis and treatment, and becomes a direction for microbubble research.
At present, the nano-scale ultrasonic contrast agents comprise nano particles coated with solid particles, nano emulsions coated with liquid fluorocarbon and nano microbubbles coated with gas, and the prepared contrast agents have larger particle size difference and different imaging effects because of different preparation methods, film forming materials and coated contents. At present, the common polymer material is polylactic-co-glycolic acid (PLGA), and the domestic scholars adopt the PLGA as the shell material to prepare polymer microvesicles, but the minimum particle size which can be prepared can only reach about 600 nm. Foreign scholars prepare PLGA nano microbubbles with the average particle size of 150-200nm, but the echo of the agent after intravenous injection is weak, ultrasonic examination cannot image, and the related technology of the PLGA nano contrast agent for developing is not mature.
At present, researches are carried out to prepare chitosan nano-microbubbles by using nano-silica particles as templates, and ultrasonic imaging can be realized after perfluoro-n-pentane is filled into the chitosan hollow nanoparticles, but the preparation process is complex and the cost is high.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a chitosan nanobubble, a preparation method thereof, and a use thereof, for solving the problems of complicated preparation process, high cost, poor imaging effect, and the like of the nanobubble in the prior art.
In order to achieve the above objects and other related objects, the present invention provides a method for preparing chitosan nanobubbles, comprising the steps of:
(1) preparing an acrylic acid aqueous solution, adding chitosan, dissolving, heating the solution to 60-80 ℃ in the presence of protective gas, adding an initiator to react for 10-60 min, then reducing the temperature of the solution to 50-65 ℃, reacting for 60-120min, filtering and dialyzing to remove impurities after the reaction is finished, adding a cross-linking agent after the treatment is finished, heating, and reacting to obtain a nanoparticle suspension;
(2) and adding a freeze-drying protective agent into the nanoparticle suspension, and freeze-drying to obtain the chitosan nanoparticle microbubble.
Further, in the step (1), the mass concentration of the acrylic acid aqueous solution is 0.1-1.6%.
Preferably, in the step (1), the mass concentration of the acrylic acid aqueous solution is 0.22-0.44%.
Further, in the step (1), the mass (in g) of chitosan added is 0.5-3.5% of the volume (in mL) of the acrylic acid aqueous solution.
Preferably, in step (1), the mass (in g) of chitosan added is 0.5-1% of the volume (in mL) of the acrylic acid aqueous solution.
Further, in the step (1), the molecular weight of the chitosan is 100kDa-800kDa, and the deacetylation degree is 60% -100%.
Further, in the step (1), the initiator is selected from potassium persulfate.
Further, in the step (1), the amount of the initiator added is 30mg to 70mg, and the volume of the acrylic acid aqueous solution at this time is 50 mL.
Further, in the step (1), the protective gas is selected from nitrogen or other inert gases.
Further, in step (1), the solution is heated to 70-75 ℃ in the presence of a protective gas.
Further, in the step (1), the cross-linking agent is at least one selected from glutaraldehyde and sodium tripolyphosphate.
Further, in the step (1), the dialysis bag containing the reaction solution is placed in a buffer solution, and impurities are removed by dialysis.
Further, in the step (1), the buffer is selected from an acetate-acetate buffer, and the pH is 4.5.
Further, in the step (1), the processing time of the nanoparticle suspension in the buffer solution is 24-48 h.
Further, in the step (1), the mass (unit g) of the crosslinking agent added is 0.05 to 2% of the volume (unit mL) of the reaction solution.
Further, in the step (1), after the cross-linking agent is added, the solution is heated to 40-50 ℃ for reaction.
Further, in the step (1), after the cross-linking agent is added, the reaction time is 1-2 h.
Further, in the step (2), the lyoprotectant is at least one selected from mannitol, glucose, sucrose and lactose.
Further, in the step (2), the mass (unit g) of the added freeze-drying protective agent is 1% -10% of the volume (unit mL) of the nanoparticle suspension.
Further, in the step (2), the mass (unit g) of the freeze-drying protective agent is added to be 1-5% of the volume (unit mL) of the nanoparticle suspension.
Further, in the step (2), the freeze-drying method comprises the following steps: prefreezing at-80 deg.C for 4-5 hr, and freeze drying at-45 deg.C for 48 hr.
Further, in the step (2), after freeze-drying, perfluor compound is filled into the nano-micro-bubbles to prepare the chitosan nano-micro-bubbles.
Further, in the step (2), the perfluoro-containing compound is selected from at least one of perfluoropropane, perfluoroethane, sulfur hexafluoride and perfluoro-n-pentane.
Further, in the step (2), after freeze drying, the nano-microbubbles are firstly vacuumized, and then perfluorinated compounds are filled into the nano-microbubbles.
Further, in the step (2), when liquid perfluoro-n-pentane is filled, vaporization treatment is carried out to prepare the chitosan nano-microbubble coated with perfluoro-n-pentane.
Further, when filling liquid perfluoro-n-pentane, the vaporization temperature is 40-60 ℃.
Further, in the step (1), the anti-tumor drug is added into the reaction solution, and after incubation, the cross-linking agent is added. That is, the drug-loaded nanobubble can be prepared for targeted therapy, and specific drugs, gene targeted therapy drugs and the like can be prepared.
Further, the anti-tumor drug is selected from doxorubicin.
The invention provides chitosan nano-microbubble, which comprises a hollow part and an outer shell containing chitosan and polyacrylic acid, wherein the hollow part is filled with air or perfluorinated compounds. The chitosan nanobubbles may be prepared by the aforementioned method.
Further, the shell comprises a polyacrylic acid layer and a chitosan layer from inside to outside in sequence, and the shell further contains an anti-tumor drug, so that the microvesicle has a drug loading function.
The third aspect of the invention provides the application of the chitosan nano-microbubble in ultrasonic development or high-intensity focused ultrasound synergy.
As described above, the chitosan nanobubble of the present invention, the preparation method and the use thereof, have the following advantageous effects: according to the invention, chitosan is dissolved in an acrylic acid solution according to a certain proportion, an initiator is added to enable acrylic acid and chitosan to have polymerization reaction to obtain hollow nanoparticles containing water inside, the water in the hollow structure inside is removed (or perfluorinated compounds are filled), and the prepared nano microbubbles have the effects of ultrasonic development, high-intensity focused ultrasound tissue ablation synergy and the like after being dissolved again.
Drawings
Fig. 1 shows a transmission electron microscope image of nanoparticles prepared in example 1.
FIG. 2(a) is the ultrasonic development of chitosan nano-microbubble in water sac in example 4 of the present invention.
FIG. 2(b) shows an ultrasonic development of the buffer control in the water sac according to example 4 of the present invention.
FIG. 3(a) is a diagram showing the ultrasonic development of nanoparticles in water capsules in example 5 of the present invention.
FIG. 3(b) shows an ultrasonic development of the buffer control in the water sac according to example 5 of the present invention.
FIG. 4(a) is the ultrasonic development of chitosan nanobubbles in water sac in example 6 of the present invention.
FIG. 4(b) shows an ultrasonic development of the buffer control in the water sac according to example 6 of the present invention.
FIG. 5 is a schematic view showing the structure of chitosan nanobubbles prepared in examples 1-3 of the present invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In the following examples, the molecular weight of chitosan is 100kDa-800kDa, and the degree of deacetylation is 60% -100%.
Example 1
A preparation method of chitosan nano-micro bubbles comprises the following steps:
(1) preparing 50mL of acrylic acid aqueous solution with the mass concentration of 0.44%, adding chitosan, wherein the mass (unit g) of the added chitosan is 1% of the volume (unit mL) of the acrylic acid aqueous solution, stirring until the chitosan is completely dissolved, heating to 75 ℃ under the protection of nitrogen, adding 70mg of potassium persulfate, reacting for about 1h, setting the temperature to 60 ℃, continuing to react for 80min, then filtering the product to remove impurities, pouring the product into a dialysis bag, placing the dialysis bag in acetic acid-sodium acetate buffer solution with the pH value of 4.5, magnetically stirring the solution for 24 hours, taking the solution out after removing the impurities through dialysis, adding 0.078g of glutaraldehyde, heating the solution to 40 ℃ for reaction, after 2 hours, obtaining nanoparticle suspension with the particle size of about 100nm after the reaction is finished, the nano particle has a hollow structure, the shape of the nano particle is observed by a transmission electron microscope, the transmission electron microscope figure is shown in figure 1, the nano particle is round and round, and the particle size is about 100 nm.
(2) Adding mannitol into the nanoparticle suspension prepared in the step (1), wherein the mass (unit g) of mannitol is 5% of the volume (unit mL) of the nanoparticle suspension, pre-freezing at-80 ℃ for 4h, taking out the mannitol from a pre-freezing device at-80 ℃, putting the mannitol into a freeze dryer, freeze-drying at-45 ℃ for 48h, taking out the mannitol, putting the mannitol into a glass tubule, pumping negative pressure by using a vacuum pump, adding a proper amount of liquid perfluoro-n-pentane, and vaporizing the mannitol for 30min at 40 ℃.
Example 2
A preparation method of chitosan nano-micro bubbles comprises the following steps:
(1) preparing 50mL of acrylic acid solution with the mass concentration of 0.22%, adding chitosan, wherein the mass (unit g) of the added chitosan is 0.5% of the volume (unit mL) of the acrylic acid aqueous solution, stirring until the chitosan is completely dissolved, heating to 75 ℃ under the protection of nitrogen, adding 45mg of potassium persulfate, reacting for about 1h, setting the temperature to 60 ℃, filtering the product to remove impurities after 100min, pouring the product into a dialysis bag, placing the dialysis bag into acetic acid-sodium acetate buffer solution with the pH value of 4.5, magnetically stirring for 24h, taking out, adding 0.035g of glutaraldehyde, heating to 40 ℃ for reaction, and after 2h, finishing the reaction to obtain suspension nanoparticles, wherein the particle size of the nanoparticles is about 100nm and has a hollow structure, and the structure form of the nanoparticles is similar to that of the suspension shown in the figure 1.
(2) And (2) adding lactose into the nanoparticle suspension prepared in the step (1), wherein the mass (unit g) of the lactose is 5% of the volume (unit mL) of the nanoparticle suspension, pre-freezing at-80 ℃ for 5h, taking out the lactose from a pre-freezing device at-80 ℃, putting the lactose into a freeze dryer, freeze-drying at-45 ℃ for 48h, taking out the lactose, putting the lactose into a glass tubule, pumping negative pressure by using a vacuum pump, filling perfluoropropane gas, and replacing for three times to prepare the chitosan nanobubbles wrapped with the perfluoropropane gas.
Example 3
A preparation method of chitosan nano-micro bubbles comprises the following steps:
(1) preparing 50mL of acrylic acid solution with the mass concentration of 0.44%, adding chitosan, wherein the mass (unit g) of the added chitosan is 1% of the volume (unit mL) of the acrylic acid aqueous solution, heating to 70 ℃ under the protection of nitrogen, adding 60mg of potassium persulfate, reacting for about 1h, setting the temperature to 60 ℃, filtering the product to remove impurities after 80min, pouring the product into a dialysis bag, placing the dialysis bag into acetic acid-sodium acetate buffer solution with the pH value of 4.5, magnetically stirring for 24h, taking out, adding 0.078g of glutaraldehyde, heating to 40 ℃ for reaction, and reacting for 2h to obtain a nanoparticle suspension, wherein the particle size of the nanoparticles is about 100nm, the nanoparticles have a hollow structure, and the structural form of the nanoparticles is similar to that of the graph 1.
(2) Adding mannitol into the nanoparticle suspension prepared in the step (1), wherein the mass (unit g) of mannitol is 5% of the volume (unit mL) of the nanoparticle suspension, pre-freezing at-80 ℃ for 5h, taking out the mannitol from a pre-freezing device at-80 ℃, putting the mannitol into a freeze dryer, freeze-drying at-45 ℃ for 48h, taking out the mannitol, and not charging fluorocarbon gas, removing the water in the hollow area inside the nanoparticles obtained after freeze-drying, filling the inside of the nanoparticles with air, wherein the nanobubbles wrapped with air have a certain ultrasonic development function.
Example 4
Evaluation of chitosan nanobubbles obtained in example 1 by development
The ultrasonic diagnostic apparatus MYLAB90 is used, the ultrasonic probe is a superficial probe LA523, and the ultrasonic frequency is 5.0-10 MHz. The nanobubbles obtained by the steps (1) and (2) in the above example 1 were filled into a water bag, and the water bag was placed into a container filled with degassed water for development test, and the degassed water bag filled with acetic acid-sodium acetate buffer solution with ph4.5 was used as a control for comparative observation of the development of the two water bags.
Fig. 2(a) shows the ultrasonic development of the nanobubbles in the water sac, and fig. 2(b) shows the ultrasonic development of the buffer solution control in the water sac, as can be seen from the results, the nanobubbles loaded with perfluoro-n-pentane prepared in example 1 has stronger development effect, while the control group of the water sac of acetic acid-sodium acetate buffer solution with pH4.5 can only see the wall curve of the water sac under the ultrasonic development, which indicates that the nanobubbles prepared in example 1 have development function.
The nano-microbubbles obtained in example 2 also have a developing function similar to that of the nano-microbubbles obtained in example 1.
Example 5
Evaluation of hollow nanoparticles obtained in step (1) of example 3 by ultrasonic development
DP3300 ultrasonic diagnostic apparatus was used, ultrasonic frequency 6.5 MHz. The nanoparticles prepared in step (1) of example 3 were loaded into a water sac, and the water sac was placed in a container filled with degassed water for development test, and the degassed water sac filled with an acetic acid-sodium acetate buffer solution having a pH of 4.5 was used as a control for comparative observation of the development of the two water sacs.
Fig. 3(a) shows an ultrasonic development image of the nanoparticles in the water sac in this example, and fig. 3(b) shows an ultrasonic development image of the buffer solution control in the water sac in this example, and as a result, the nanoparticles prepared in step (1) and the buffer solution water sac control can only see the wall curve of the water sac under the ultrasonic development, which indicates that the nanoparticles prepared in step (1) do not have the development function.
Example 6
Ultrasonic development evaluation of the chitosan nanobubbles obtained in step (2) of example 3
DP3300 ultrasonic diagnostic apparatus was used, ultrasonic frequency 6.5 MHz. Weighing a proper amount of the nanobubbles prepared in the step (2) in the example 3, dissolving the nanobubbles in a buffer solution with pH4.5 again, putting the dissolved nanobubbles into a water sac, putting the water sac into a container with degassed water for a development test, taking the degassed water sac with the buffer solution with pH4.5 as a reference, and comparing and observing the development conditions of the two water sacs.
Fig. 4(a) shows the ultrasonic development of the chitosan nanobubble in the water sac of the present embodiment, and fig. 4(b) shows the ultrasonic development of the buffer solution contrast in the water sac of the present embodiment, as a result, the chitosan nanobubble water sac has a stronger development effect, while the buffer solution water sac contrast only shows the wall curve of the water sac under the ultrasonic development, which indicates that the microbubble prepared by filling air in step (2) of example 3 has a development function.
From the experimental results of example 5 and example 6, it was found that the nanoparticle suspension had to be freeze-dried before the nanoparticle suspension had the ultrasonic imaging function.
Experiments show that the nano-microbubbles obtained by freeze-drying without adding the freeze-drying protective agent are difficult to redissolve, and after the nano-microbubbles are added with the buffer solution, flocculent precipitates and suspended matters appear and cannot be used for injection, so that the freeze-drying protective agent is very important for preparing the nano-microbubbles meeting the requirements.
Example 7
Evaluation of high-intensity focused ultrasound synergistic function of chitosan nanobubbles
Cutting fresh isolated ox liver into cube blocks of 10cm x 6cm, placing in degassing barrel, degassing for 40min, placing in plastic container with sound-transmitting film at bottom, and soaking the container bottom in degassed water. Observing a small cavity area in a bovine liver tissue by ultrasonic, dividing the bovine liver into 3-4 layers, wherein each layer has 2-3 points, the interval between each layer and each point is more than 1cm, sucking 0.5mL of nano microbubble solution with the concentration of 0.068g/mL (the microbubble solution is prepared by dissolving the nano microbubbles prepared in the embodiment 3 in acetic acid-sodium acetate buffer solution) into an injector, avoiding the position of an inner cavity of the bovine liver, vertically penetrating a needle into the middle part of the bovine liver with the depth of about 2cm, positioning the injection position by ultrasonic radiography, injecting nano microbubbles, rapidly pulling the needle out, immediately starting HIFU (high intensity focused ultrasound) to irradiate, wherein the irradiation power P is 120W, the irradiation time is 5s, taking out the bovine liver after the irradiation is finished, searching a treatment area by slicing, taking a picture, calculating the size of the damaged area of the irradiation area, repeating the test for 3 times, and counting the experimental result; in addition, the bovine liver tissue without injected microbubbles is directly treated by HIFU ablation as a contrast test. The experimental result shows that the average damage area of the bovine liver tissue after the chitosan nano-microbubble is injected is 36.9mm2The average damage area of the bovine liver tissue without chitosan nano-microbubble injection is 8.5mm2The chitosan nano microbubble prepared by the invention can obviously increase the damage area of HIFU treatment.
Example 8
The preparation method of the drug-loaded chitosan nano microbubble comprises the following steps:
(1) preparing 50mL of acrylic acid solution with the mass concentration of 0.44%, adding chitosan, wherein the mass (unit g) of the added chitosan is 1% of the volume (unit mL) of the acrylic acid aqueous solution, heating to 75 ℃ under the protection of nitrogen, adding 30mg of potassium persulfate, reacting for about 1h, setting the temperature to 65 ℃, filtering the product to remove impurities after 60min, pouring the product into a dialysis bag, placing the dialysis bag into acetic acid-sodium acetate buffer solution with the pH value of 4.5, and taking out the product after magnetically stirring for 24 h.
(2) Adding 0.5g of doxorubicin into the reaction solution, incubating at 40 ℃ for 48h, adding 0.078g of glutaraldehyde, heating to 40 ℃ for reaction, and after 2h, completing the reaction to obtain a nanoparticle suspension, wherein the particle size of the nanoparticles is about 100nm, the nanoparticles have a hollow structure, and the structural form of the nanoparticle suspension is similar to that of the nanoparticle in figure 1.
(3) And (3) adding mannitol into the nanoparticle suspension prepared in the step (2), wherein the mass (unit g) of the mannitol is 5% of the volume (unit mL) of the nanoparticle suspension, pre-freezing at-80 ℃ for 5h, taking the mannitol out of a pre-freezing device at-80 ℃, putting the mannitol into a freeze dryer, freeze-drying at-45 ℃ for 48h, taking out, pumping negative pressure by using a vacuum pump, adding a proper amount of liquid perfluoron-pentane, and evaporating at 40 ℃ for 30min to obtain the nano-microbubble with the double functions of developing and drug loading.
As shown in fig. 5, the chitosan nanobubble structure obtained in examples 1 to 3 is schematically shown, and includes a hollow part 1 and a shell 2, wherein the hollow part 1 is filled with air or perfluoro compound, the shell 2 obtained in examples 1 to 3 contains chitosan and polyacrylic acid, the shell 2 sequentially comprises a polyacrylic acid layer 21 and a chitosan layer 22 from inside to outside, and the shell 2 obtained in example 8 contains not only chitosan and polyacrylic acid, but also the anti-tumor drug doxorubicin, so that the chitosan nanobubble also has a drug loading function. The outer diameter of the housing 2 is approximately 100 nm.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (8)
1. A preparation method of chitosan nano-microbubble is characterized by comprising the following steps:
(1) preparing an acrylic acid aqueous solution, wherein the mass concentration of the acrylic acid aqueous solution is 0.1-1.6%, adding chitosan, the mass g of the added chitosan is 0.5-3.5% of the volume mL of the acrylic acid aqueous solution, heating the solution to 70-80 ℃ in the presence of protective gas after dissolution, adding an initiator potassium persulfate, the adding amount of the initiator in every 50mL of the acrylic acid aqueous solution is 30-70mg, reacting for 10min-60min, then reducing the temperature of the solution to 50-65 ℃, reacting for 60-120min, filtering and dialyzing to remove impurities after the reaction is finished, adding a cross-linking agent into the reaction solution after the treatment is finished, heating the solution to 40-50 ℃, and reacting for 1-2 h to obtain a nanoparticle suspension, wherein the nanoparticles have a hollow structure; the cross-linking agent is at least one of glutaraldehyde and sodium tripolyphosphate, and the mass g of the added cross-linking agent is 0.05-2% of the volume mL of the reaction solution;
(2) adding a freeze-drying protective agent into the nanoparticle suspension, and directly freezing and drying to obtain chitosan nano-microbubbles which are filled with air and have the ultrasonic development function, or taking out after freezing and drying, placing in a glass tubule, pumping negative pressure by using a vacuum pump, and filling perfluorinated compounds into the nano-microbubbles to obtain the chitosan nano-microbubbles which are wrapped with perfluorinated compounds and have the ultrasonic development function; the freeze-drying protective agent is selected from at least one of mannitol, glucose, sucrose and lactose, and the mass g of the added freeze-drying protective agent is 1-10% of the volume mL of the nanoparticle suspension.
2. The method of claim 1, wherein: in the step (1), the temperature of the solution is reduced to 50-65 ℃, and the reaction is carried out for 80-100 min;
and/or, in the step (1), the mass concentration of the acrylic acid aqueous solution is 0.22-0.44%;
and/or, in the step (1), the mass g of the added chitosan is 0.5-1% of the volume mL of the acrylic acid aqueous solution;
and/or, in the step (1), the molecular weight of the chitosan is 100kDa-800kDa, and the deacetylation degree is 60% -100%.
3. The method of claim 1, wherein: in the step (1), the protective gas is selected from nitrogen or other inert gases;
and/or, in step (1), heating the solution to 70-75 ℃ in the presence of a protective gas.
4. The method of claim 1, wherein: in the step (2), adding a freeze-drying protective agent, wherein the mass g of the freeze-drying protective agent is 1-5% of the volume mL of the nanoparticle suspension;
and/or in the step (2), the freeze drying method comprises the following steps: pre-freezing at-80 deg.C for 4-5 hr, and freeze-drying at-45 deg.C for 24-48 hr;
and/or, in the step (2), the perfluorinated compound is selected from at least one of perfluoropropane, perfluoroethane, sulfur hexafluoride and perfluoro-n-pentane.
5. The method of claim 1, wherein: in the step (1), the antitumor drug is added into the reaction liquid after filtration and impurity removal, and after incubation, the cross-linking agent is added for subsequent operation, so that the nano-microbubble with double functions of ultrasonic development and drug loading can be prepared.
6. The method of claim 5, wherein: the anti-tumor drug is selected from doxorubicin.
7. A chitosan nano microbubble is characterized in that: the method according to any one of claims 1 to 4, comprising a hollow part (1) and a shell (2) comprising chitosan and polyacrylic acid, wherein the hollow part (1) is filled with air or perfluoro compound.
8. Chitosan nanobubble, characterized in that it is prepared according to any one of claims 5 to 6, the shell (2) of which comprises, in order from the inside towards the outside, a polyacrylic layer (21), a chitosan layer (22), said shell (2) also containing an anti-tumor drug.
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