CN1128167C - Nanometer microball of chitosan-polyacrylic acid composite and its producing method and use - Google Patents

Abstract

The present invention relates to a nanometer microsphere of chitosan-polyacrylic acid compounds, the average particle diameter of which is from 200 to 300 nm, wherein the molecular weight of chitosan is from 10, 000 to 500, 000; the deacetylation degree is from 50 to 100%; the molecular weight of polyacrylic acid is from 20, 000 to 200, 000; the content of polyacrylic acid is from 20 to 50%. The nanometer microsphere can be used as a carrier of medicine, particularly a carrier of a magnetic resonance imaging and developing reinforcing agent. The present invention discloses a preparation method of the nanometer microsphere.

Description

A kind of nano microsphere of chitosan-polyacrylic acid composite and its preparation method and application

The invention relates to a biodegradable macromolecular nano microsphere, which can be used as a carrier of medicines and proteins, and can also be used as a carrier of magnetic resonance imaging enhancers.

The use of biocompatible polymer materials to prepare drugs and gene carriers for their controlled release has received more and more attention. Degradable polymer nanospheres are a new type of drug carrier developed in the late 1970s with slow-controlled release and in vivo targeting. According to the specificity of particle distribution in the body, the drug can be delivered to the disease site and released slowly, so that the concentration of the drug in the lesion site can be significantly increased, the action time can be prolonged, and the therapeutic effect of the drug can be significantly improved; Toxic and side effects, so that the drug can achieve the purpose of slow release and targeted drug delivery at the lesion site in the body, which has great application value for clinical application (J. Exp. Med. 1988. (67). 440-451).

The materials used for nano-microspheres as drug carriers generally adopt biodegradable polymers, mainly including albumin, gelatin, polysaccharides and polyester compounds. Polyester compounds are the earliest synthetic materials recognized as safe for use in the human body. Such as polylactide, polyglycolide, polycaprolactone and their copolymers (Contraception, 1976, (13), 275-384). Recently, a new type of multi-block polymer embedded with hydrophilic segment PEG has received attention. (Colloids and surfaces B: Biointerfaces, 2000, 18: 371-379) These materials have some shortcomings, such as: the drug encapsulation rate is too low, the controlled release of the drug is difficult to control, peptides and other biologically active substances are easily inactivated during the encapsulation process, etc. . And the chitosan adopted in the present invention becomes a kind of excellent material as drug carrier because of its good biocompatibility, biodegradability, low toxicity and excellent hydrophilicity.

Magnetic resonance imaging (MRI: Magnetic Resonance Imaging) technology is one of the latest achievements in medical imaging since the 1980s. It uses the different magnetic resonance signals generated by different tissues in the body under the influence of an external magnetic field to image. The strength of the magnetic resonance signal depends on the relaxation time of the protons in the molecules in the tissue. The local magnetic field generated by the unpaired electron spins of paramagnetic metal ions (such as Gd ³⁺ , Mn ²⁺ , Fe ³⁺ ) can shorten the relaxation time of protons in adjacent water molecules, thereby increasing the magnetic resonance signal intensity in the adjacent area to increase the contrast of the image.

In order to improve the signal intensity of the diagnosed site, the enhancement agent must reach a certain concentration in the site, so as to improve the sensitivity and accuracy of diagnosis. However, the traditional magnetic resonance imaging enhancer (such as: gadolinium diethylenetriaminepentaacetate DTPA-Gd) is an interstitial enhancer, which is distributed extracellularly and has no specificity in biological distribution, so it is impossible to enrich in specific parts. , it is necessary to increase the amount of enhancer in order to obtain a better image. Because DTPA-Gd has certain side effects, increasing the dose will definitely affect its practical safety, thereby limiting the further application of magnetic resonance imaging technology in medicine.

The use of nano-microspheres or nanoparticles to load magnetic resonance imaging enhancers is a relatively new field, which has been greatly developed in recent years. How to deliver the contrast enhancing agent to the site of interest through the targeting characteristics of the nano-polymer carrier, and improve the signal of the low-concentration receptor is a hot spot in the research of the contrast enhancing agent for magnetic resonance imaging. Through the polymer nano-controlled release system with good biocompatibility, the DTPA-Gd contrast agent can be loaded to locally enrich the contrast agent with a lower concentration and improve the contrast of the magnetic resonance image. At the same time, the nanoparticle contrast agent also has the function of long circulation time and targeting in the body, (collods and surfaces B: Biointerfaces 1999, (16): 305-319) has great potential in the fields of medical blood pool angiography, liver and lung tumor diagnosis, etc. Good application prospects.

Synthesis of drug-loaded chitosan microspheres in the prior art mainly includes precipitation method, reverse phase method (W/O) and spray method etc., the preparation technology of these several methods is more complicated, and the purification steps of gained chitosan microspheres are compared Many and more cumbersome. The preparation method (referring to Polymer Bullitin, 1999, (43): 67-73) of the chitosan nano microsphere close to the present invention is that polyaminomethylpropanesulfonic acid is added dropwise in the dilute solution of chitosan, Polyelectrolyte complex particles are formed under stirring conditions. One aspect of this method is that the particle size of the synthesized particles is relatively large, greater than 1 micron. On the other hand, since such particles are formed in an extremely dilute solution, the yield of finished products is relatively low, and the particle sizes of the formed particles are not uniform.

And prior art preparation is loaded with the approach of magnetic resonance imaging enhancement agent microsphere mainly to contain: (1) embedding development enhancement agent DTPA-Gd with liposome; (collods and surfaces B: Biointerfaces 2000, (18): 293- 299), (2) modify the liposome surface, connect some multifunctional ligands, and then make Gd ³⁺ coordinate with this modified liposome to form a high molecular complex of gadolinium, etc. ( collods and surfaces B: Biointerfaces 1999, (16): 305-319). There are some disadvantages to the above method. In the first method, the small-molecule development enhancer will permeate out from the liposome, and the permeated development enhancer will destroy the liposome membrane. For the second method, it is necessary to ensure that most of the metal atoms and the functional groups on the surface of the liposome are coordinated to ensure the rapid exchange of metal atoms and water molecules. In addition, liposomes used as DTPA-Gd carriers are hydrophobic, which limits the exchange of gadolinium atoms with water, thus the enhancement effect is not very ideal. The chemical process of liposome surface modification is complicated, and the embedding process is also complicated.

The purpose of the present invention is:

1. Provide a kind of nano-microsphere of biodegradable hydrophilic chitosan-polyacrylic acid composite;

2. Provide a kind of preparation method of above-mentioned nano microsphere;

3. Provide a chitosan-polyacrylic acid composite nanosphere loaded with drugs, especially a chitosan-polyacrylic acid composite nanosphere loaded with a magnetic resonance imaging development enhancer.

Technical scheme of the present invention is as follows:

A chitosan-polyacrylic acid composite nanosphere, wherein the molecular weight of chitosan is in the range of 10,000-500,000, the degree of deacetylation is 50-100%, the molecular weight of polyacrylic acid is in the range of 20,000-200,000, the nanosphere The average particle diameter is 200-300nm, and the content of polyacrylic acid is 20-50%.

The above-mentioned nano-microspheres can be nano-microspheres of chitosan-polyacrylic acid composite cross-linked by glutaraldehyde cross-linking agent. The cross-linked nano-microspheres are more stable and have better alkali resistance.

A kind of method for preparing above-mentioned chitosan-polyacrylic acid composite nano microsphere, it is that chitosan is dissolved in acidic (for example can add acetic acid to make acidic) distilled water, add polyacrylic acid aqueous solution under stirring, form microemulsion and filter to obtain the nanometer microspheres of the chitosan-polyacrylic acid composite.

A method for preparing the above-mentioned chitosan-polyacrylic acid composite nano-microspheres, which is to add chitosan and acrylic acid in distilled water under stirring at 40-60 ° C, after the dissolution is complete, add an initiator to react, the initiator Potassium persulfate may be used, and microemulsion is formed after the reaction is completed, and nanometer microspheres of chitosan-polyacrylic acid composite are obtained by filtering.

A method for preparing the above-mentioned cross-linked chitosan-polyacrylic acid composite nanospheres, which is to dissolve chitosan in acidic distilled water (for example, acetic acid can be added to make acidic), add polyacrylic acid aqueous solution under stirring, Form a microemulsion, add a glutaraldehyde aqueous solution with a concentration of 1% (m%), heat up to 40° C., complete the reaction, and filter to obtain cross-linked chitosan-polyacrylic acid composite nanospheres.

Cross-linked chitosan-polyacrylic acid composite nano-microspheres can also be prepared by the following method. Add chitosan and acrylic acid in distilled water under stirring at 40-60 ° C, and add initiator potassium persulfate after dissolution is complete. After the reaction is completed, a microemulsion is formed, and then a cross-linking agent glutaraldehyde aqueous solution is added to continue the reaction, and the cross-linked chitosan-polyacrylic acid composite nanometer microspheres are obtained after the reaction is completed.

The cross-linking agent glutaraldehyde aqueous solution can also be added in advance and the initiator potassium persulfate at the same time, which has no effect on the cross-linking reaction.

The chitosan-polyacrylic acid composite nano microsphere of the invention can be used as a carrier of medicine, especially as a carrier of a magnetic resonance imaging enhancer.

As a drug carrier, it can add the drug or magnetic resonance imaging enhancer to be loaded into the chitosan-polyacrylic acid of the present invention, and after static adsorption and dialysis, the drug or magnetic resonance imaging enhancer can be obtained. Nanospheres of Chitosan-Polyacrylic Acid Composite.

As a drug carrier, it can also be added to the loading agent before the polymerization of acrylic acid (that is, when the initiator is added), such as the magnetic resonance enhancement agent gadolinium diethylenetriaminepentaacetate (DTPA-Gd) or 1,4,7 , 10-tetraazacyclododecane-N, N, N, N-tetraacetate gadolinium (DOTA-Gd), then add initiator, cross-linking agent, after the reaction is complete, filter out the nano-microspheres, use secondary Water dialysis is used to remove small inorganic salt molecules and unreacted monomers in the system, and the nano-microspheres of chitosan-polyacrylic acid composites wrapped with magnetic resonance imaging agents can be obtained.

The invention provides a chitosan-polyacrylic acid composite nano-microsphere with an average particle diameter of 200-300nm, which is hydrophilic, has good stability in a pH3-8 buffer solution, and is biodegradable , good biocompatibility. The preparation method of the invention adopts the method of water phase polymerization to prepare the chitosan nano microspheres. No organic reagents and surfactants are used in the preparation process. The problem of separation and purification of chitosan microspheres is well solved. The surface of the microsphere is regular and has a certain strength. DTPA-Gd or DOTA-Gd has a high loading rate and stable chemical properties. The above characteristics indicate that the microsphere, as a carrier, meets the application requirements in the fields of biomedicine and biochemical engineering in terms of performance.

Description of drawings:

Fig. 1 is the model figure of the nano microsphere of the present invention loaded with DTPA-Gd: 1 is polymer network; 2 is DTPA-Gd.

Fig. 2 is an electron micrograph (magnification 50,000 times) of the cross-linked nano-microspheres of the present invention.

Fig. 3 is the transmission electron micrograph of the loaded DTPA-Gd nano microspheres prepared by the method of the present invention, the particle diameter is less than 300 nanometers (magnification 50000 times).

Fig. 4 is a statistical diagram of the particle size distribution of the nanospheres obtained in each embodiment, wherein Figs. 4-1 to 4-5 are the nanospheres obtained in Examples 1 to 5, respectively.

Fig. 5 is the in vitro drug release curve of nano-microspheres loaded with doxorubicin.

The technical solution of the present invention will be further described below through examples.

Example 1:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 80,000, the degree of deacetylation is 3 grams of chitosan of 90%, add 1 gram of acrylic acid again, under stirring condition, be heated up to 60 ℃. After the chitosan is completely dissolved, 40 mg of initiator—potassium persulfate is added thereto, and reacted at 60° C. for 2 hours to obtain a nano-microemulsion. Stop the reaction, filter and pour the microemulsion into a dialysis bag for dialysis for 48 hours to remove small inorganic salt molecules and unreacted monomers in the system to obtain composite nanospheres.

The content of nanoparticles in the above system is about 4 grams, and the molecular weight of polyacrylic acid is 40,000. The nanoparticles of chitosan observed by transmission electron microscope are relatively regular spherical structures with an average particle diameter of about 200nm. Stable storage.

Example 2:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 80,000, the degree of deacetylation is 3 grams of chitosan of 85%, add 1 gram of acrylic acid again, under stirring condition, be heated up to 60 ℃. After the chitosan is completely dissolved, 40 mg of initiator—potassium persulfate is added thereto, and reacted at 60° C. for 2 hours to obtain a nano-microemulsion. Then add 10ml of 1% cross-linking agent glutaraldehyde and react at 40°C for 2 hours to ensure complete reaction of glutaraldehyde. Stop the reaction, filter and pour the microemulsion into a dialysis bag for dialysis for 48 hours to remove small inorganic salt molecules and unreacted monomers in the system to obtain composite nanospheres.

The content of nanoparticles in the above system is about 4 grams, and the molecular weight of polyacrylic acid is 40,000. The nano-particles of chitosan observed by transmission electron microscopy are relatively regular spherical structures with an average particle size of about 200nm, and can be preserved for a long time in the range of pH3-8.

Example 3:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 80,000, the degree of deacetylation is 3 grams of chitosan of 70%, add 1 gram of acrylic acid again, under stirring condition, be heated up to 60 ℃. After the chitosan is completely dissolved, 0.3 gram of DTPA-Gd small molecular compound is added thereto, and 30 mg of potassium persulfate is added as an initiator. React at 60°C for 2 hours to obtain nano-microemulsion. Cool down to 40°C, add 10ml of 1% cross-linking agent glutaraldehyde, and react at 40°C for 2 hours to ensure complete reaction of glutaraldehyde. After stopping the reaction, filter, pour the microemulsion into a dialysis bag and dialyze with secondary water for more than 1 hour to remove small inorganic salt molecules and unreacted monomers in the system. Composite nano microspheres wrapped with the magnetic resonance imaging agent can be obtained.

The content of nanoparticles in the above system is 4 grams, the molecular weight of polyacrylic acid is 40,000, and the coating rate of DTPA-Gd is about 60%. The nano-particles of chitosan observed by transmission electron microscope are relatively regular spherical structures with an average particle diameter of about 210nm, and can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Example 4:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 80,000, the degree of deacetylation is 1 gram of chitosan of 55%, add 1 gram of acrylic acid again, stir. After the chitosan was completely dissolved, 15 mg of potassium persulfate and 5 ml of glutaraldehyde aqueous solution (1%, wt%) were added thereto. React at 50°C for 4 hours to obtain nano-microemulsion. Then react at 40° C. for 2 hours to ensure complete reaction of glutaraldehyde. After stopping the reaction, filter and pour the microemulsion into a dialysis bag for dialysis for more than 1 hour to remove small inorganic salt molecules and unreacted monomers in the system. Add 0.1 g of DTPA-Gd compound therein, let it stand for 48 hours, and dialyze in secondary water for more than 1 hour to obtain composite nanospheres wrapped with magnetic resonance imaging agent.

The content of nanoparticles in the above system is 2 grams, the molecular weight of polyacrylic acid is 60,000, and the coating rate of DTPA-Gd is about 60%. The nano-particles of chitosan observed by transmission electron microscopy are relatively regular spherical structures with an average particle size of about 200nm, and can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Example 5:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 80,000, the degree of deacetylation is 1 gram of chitosan of 60%, add 1 gram of acrylic acid again, stir. After the chitosan was completely dissolved, 15 mg of potassium persulfate, 0.1 g of DTPA-Gd compound and 5 ml of glutaraldehyde aqueous solution (1%, wt%) were added thereto. React at 50°C for 4 hours to obtain nano-microemulsion. Then react at 40° C. for 2 hours to ensure complete reaction of glutaraldehyde. After stopping the reaction, pour the microemulsion into a dialysis bag after filtration, and dialyze in secondary water for more than 1 hour to remove small inorganic salt molecules and unreacted monomers in the system. , the composite nano-microspheres wrapped with the magnetic resonance imaging agent can be obtained.

The content of nanoparticles in the above system is 2 grams, the molecular weight of polyacrylic acid is 40,000, and the coating rate of DTPA-Gd is about 60%. The nano-particles of chitosan observed by transmission electron microscope are relatively regular spherical structures with an average particle diameter of about 230nm, and can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Embodiment 6:

Configuration 2% (wt %) molecular weight is 80,000, and deacetylation degree is the acetic acid solution 100ml of 1% (wt %) of chitosan of 90%, then adds the DTPA-Gd of 0.2 gram wherein, after dissolving completely, to 50 ml of 1% (wt%) polyacrylic acid solution with a molecular weight of 100,000 was added to form a microemulsion. After filtration, 10 ml of 1% (wt%) glutaraldehyde aqueous solution was added to the system, the temperature was raised to 40° C., and the mixture was stirred for 2 hours, filtered, and dialyzed in secondary water for more than 1 hour. Composite nano microspheres loaded with magnetic resonance imaging enhancer can be obtained.

The content of nanoparticles in the above system is 2 grams, and the coverage rate of DTPA-Gd is about 60%. The nano-particles of chitosan were observed by transmission electron microscope as a relatively regular spherical structure with an average particle diameter of about 290nm and a wide particle size distribution. It can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Table 1 illustrates the relaxation time (T1) of different DTPA-Gd concentrations containing DTPA-Gd nanospheres.

Embodiment 7:

Configuration 2% (wt %) molecular weight is 80,000, and deacetylation degree is 1% (wt %) acetic acid solution 50ml of chitosan of 90%, then adds 0.2 gram of DTPA-Gd wherein, after dissolving completely, will This solution is added to 1% (wt %), the polyacrylic acid solution 100ml that molecular weight is 100,000, forms a microemulsion. After filtration, 10 ml of 1% (wt%) glutaraldehyde aqueous solution was added to the system, the temperature was raised to 40° C., and the mixture was stirred for 2 hours, filtered, and dialyzed in secondary water for more than 1 hour. Composite nano microspheres loaded with magnetic resonance imaging enhancer can be obtained.

The content of nanoparticles in the above system is 2 grams, and the coverage rate of DTPA-Gd is about 60%. The nano-particles of chitosan were observed by transmission electron microscope as a relatively regular spherical structure with an average particle diameter of about 280nm and a wide particle size distribution. It can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Embodiment 8:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 20,000, the degree of deacetylation is 2 grams of chitosan of 90%, then add 1 gram of acrylic acid, under stirring condition, be heated up to 60 ℃. After the chitosan is completely dissolved, 0.2 gram of DTPA-Gd small molecular compound is added thereto, and 20 mg of potassium persulfate is added as an initiator. React at 60°C for 2 hours to obtain nano-microemulsion. Cool down to 40°C, add 10ml of 1% cross-linking agent glutaraldehyde, and react at 40°C for 2 hours to ensure complete reaction of glutaraldehyde. After stopping the reaction, filter, pour the microemulsion into a dialysis bag and dialyze with secondary water for more than 1 hour to remove small inorganic salt molecules and unreacted monomers in the system. Composite nano microspheres wrapped with the magnetic resonance imaging agent can be obtained.

The content of nanoparticles in the above system is 2.5 grams, the molecular weight of polyacrylic acid is 15,000, and the coating rate of DTPA-Gd is about 50%. The nano-particles of chitosan observed by transmission electron microscope are relatively regular spherical structures with an average particle diameter of about 210nm, and can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Embodiment 9:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 200,000, the degree of deacetylation is 2 grams of chitosan of 85%, add 1 gram of acrylic acid again, under stirring condition, be heated up to 60 ℃. After the chitosan is completely dissolved, 0.2 gram of DTPA-Gd small molecular compound is added thereto, and 20 mg of potassium persulfate is added as an initiator. React at 60°C for 2 hours to obtain nano-microemulsion. Cool down to 40°C, add 10ml of 1% cross-linking agent glutaraldehyde, and react at 40°C for 2 hours to ensure complete reaction of glutaraldehyde. After stopping the reaction, filter, pour the microemulsion into a dialysis bag and dialyze with secondary water for more than 1 hour to remove small inorganic salt molecules and unreacted monomers in the system. Composite nano microspheres wrapped with the magnetic resonance imaging agent can be obtained.

The content of nanoparticles in the above system is 2.0 grams, the molecular weight of polyacrylic acid is 80,000, and the coating rate of DTPA-Gd is about 50%. The nano-particles of chitosan observed by transmission electron microscope are relatively regular spherical structures with an average particle diameter of about 240nm, and can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Example 10:

In the 100ml stirred reactor that fills 50ml distilled water, add molecular weight and be 450,000, the degree of deacetylation is 4 grams of chitosan of 85%, add 1.5 grams of acrylic acid again, under stirring condition, be heated up to 60 ℃. After the chitosan is completely dissolved, 0.2 gram of DTPA-Gd small molecular compound is added thereto, and 20 mg of potassium persulfate is added as an initiator. React at 60°C for 2 hours to obtain nano-microemulsion. Cool down to 40°C, add 10ml of 1% cross-linking agent glutaraldehyde, and react at 40°C for 2 hours to ensure complete reaction of glutaraldehyde. After stopping the reaction, filter, pour the microemulsion into a dialysis bag and dialyze with secondary water for more than 1 hour to remove small inorganic salt molecules and unreacted monomers in the system. Composite nano microspheres wrapped with the magnetic resonance imaging agent can be obtained.

The content of nanoparticles in the above system is 2.5 grams, the molecular weight of polyacrylic acid is 80,000, and the coating rate of DTPA-Gd is about 50%. The nano-particles of chitosan observed by transmission electron microscope are relatively regular spherical structure, with an average particle diameter of about 260nm, wide distribution, and can be preserved for a long time in the range of pH3-8. The results of nuclear magnetic resonance experiments show that the nanoparticles have good relaxation properties in vitro.

Example 11: Determination of encapsulation rate of magnetic resonance enhancer DTPA-Gd.

Accurately measure 10ml of the prepared nano-microemulsion, place it in an ultracentrifuge (Ultra ProTM 80, Du Pont) at 50,000 rpm for 1 hour, and collect the supernatant. The concentration of Gd ions in the serum was measured by atomic absorption spectrometry (ICP), and the encapsulation rate of DTPA-Gd could be calculated.

Example 12: Determination of in vitro drug release profile of drug-loaded nanoparticles

Take 100 mg of nanoparticles prepared in Example 2, disperse them in normal saline, add 10 mg of the anticancer drug doxorubicin hydrochloride therein, after dissolving, let stand for 24 hours, and after ultracentrifugation, take the precipitate at 37°C Next, the drug release curve was determined in normal saline. Under this condition, the encapsulation rate of the drug is 80%.

Table 1. Relaxation time (T1) of nanospheres with different DTPA-Gd contents Sample number (embodiment six) Concentration (mmol/mL) T1, (ms) 1 4.46 24.0 2 1.59 64.2 3 1.27 82.6 4 0.637 172.8 5 0.204 383.1

Claims (8)

1. the Nano microsphere of a chitosan-polyacrylic acid composite, the median size that it is characterized in that Nano microsphere is 200-300nm, wherein the molecular weight of chitosan is 10000-500000, deacetylation is 50-100%, polyacrylic molecular weight is 20000-200000, and polyacrylic content is 20-50%.

2. Nano microsphere according to claim 1 is characterized in that the Nano microsphere through the crosslinked chitosan-polyacrylic acid composite of glutaraldehyde cross-linking agent.

3. a method for preparing the described Nano microsphere of claim 1 is characterized in that chitosan is dissolved in the acid distilled water, stirs down to add the polyacrylic acid aqueous solution, forms microemulsion, filters the Nano microsphere that promptly gets chitosan-polyacrylic acid composite.

4. a method for preparing the described Nano microsphere of claim 1 is characterized in that under 40-60 ℃ of stirring, adds chitosan and vinylformic acid in distilled water, after the dissolving fully, add initiator and react, form microemulsion, filter the Nano microsphere that promptly gets chitosan-polyacrylic acid composite.

5. method for preparing the described Nano microsphere of claim 2, it is characterized in that chitosan is dissolved in the acid distilled water, stir and add the polyacrylic acid aqueous solution down, form microemulsion, add the linking agent glutaraldehyde water solution, be warming up to 40 ℃, reaction is finished, and filters the Nano microsphere that promptly gets crosslinked chitosan-polyacrylic acid composite.

6. method for preparing the described Nano microsphere of claim 2, it is characterized in that at 40-60 ℃, stir down, in distilled water, add chitosan and vinylformic acid, dissolving back adding initiator is fully reacted, and forms microemulsion, adds the linking agent glutaraldehyde water solution then, continue reaction, promptly get the Nano microsphere of crosslinked chitosan-polyacrylic acid composite after reacting completely.

7. the method for Nano microsphere according to claim 6 is characterized in that the linking agent glutaraldehyde water solution when adding initiator, adds simultaneously.

8. the purposes of Nano microsphere according to claim 1 and 2 is characterized in that the carrier as medicine or nuclear magnetic resonance developing promotor.

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