阅读说明：本技术 一种牛血清白蛋白-疏水改性壳聚糖纳米微囊及其制备方法 (Bovine serum albumin-hydrophobic modified chitosan nano microcapsule and preparation method thereof ) 是由 秦大伟 王利振 于 2021-10-18 设计创作，主要内容包括：本发明涉及多糖技术领域,尤其涉及一种牛血清白蛋白-疏水改性壳聚糖纳米微囊及其制备方法,以正十六烷酸改性壳聚糖,并进一步制备负载抗肿瘤药物的牛血清白蛋白-疏水改性壳聚糖纳米微囊。本发明所制备的纳米微囊为两层结构,内层为疏水改性壳聚糖、9-O-正十八烷基小檗碱和紫杉醇,外层为牛血清白蛋白。该纳米药物能够显著增强紫杉醇对肿瘤细胞的抑制活性。(The invention relates to the technical field of polysaccharide, in particular to a bovine serum albumin-hydrophobic modified chitosan nano microcapsule and a preparation method thereof. The nano microcapsule prepared by the invention has a two-layer structure, the inner layer is hydrophobic modified chitosan, 9-O-n-octadecyl berberine and paclitaxel, and the outer layer is bovine serum albumin. The nanometer medicine can remarkably enhance the inhibiting activity of paclitaxel on tumor cells.)

1. A preparation method of bovine serum albumin-hydrophobic modified chitosan nano-microcapsule is characterized by comprising the following steps: the method comprises the following steps:

preparation of hydrophobic modified chitosan: weighing N-hexadecanoic acid and chitosan, adding the N-hexadecanoic acid and chitosan into a first solvent, stirring for 10-20 minutes, controlling the temperature to be 15-25 ℃, adding a condensing agent and N-hydroxysuccinimide, stirring for 0.5-1.5 hours, heating to 60-90 ℃, stirring for 12-24 hours, cooling to 20-40 ℃ to obtain a mixed solution, pouring the mixed solution into a second solvent, stirring for 20-40 minutes, filtering, washing a filter cake with a detergent, and performing vacuum drying to obtain the hydrophobically modified chitosan;

the first solvent is dimethyl sulfoxide or ethanol;

the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride or N, N-diisopropylcarbodiimide;

the second solvent is isopropanol or ethanol;

the detergent is an ethanol-water mixture, wherein the mass ratio of ethanol to water is (2-4): 1;

preparing a bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with an antitumor drug: adding the hydrophobic modified chitosan obtained in the step into dimethyl sulfoxide, adding 9-O-n-octadecyl berberine and paclitaxel, stirring for 5-10 minutes, dropwise adding a bovine serum albumin aqueous solution at the dropping speed of 2-3 drops/second, performing ultrasound for 10-30 minutes after the dropwise adding is finished, dialyzing for 24-72 hours by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain a bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with an anti-tumor drug;

the mass concentration of the bovine serum albumin in the bovine serum albumin aqueous solution is 0.5-1.5%;

wherein the mass ratio of the N-hexadecanoic acid, the chitosan, the solvent I, the condensing agent, the N-hydroxysuccinimide, the solvent II, the detergent, the dimethyl sulfoxide, the 9-O-N-octadecyl berberine, the paclitaxel and the bovine serum albumin aqueous solution is 0.2-0.5: 0.8-1.4: 100-200: 0.3-0.5: 0.25-0.4: 100-300: 5-15: 20-40: 0.02-0.18: 0.06-0.26: 200 to 400.

2. The method for preparing bovine serum albumin-hydrophobically modified chitosan nanocapsule according to claim 1, wherein the method comprises the following steps: the average molecular weight of the chitosan is 20 ten thousand, and the degree of deacetylation is more than 80%.

3. The method for preparing bovine serum albumin-hydrophobically modified chitosan nanocapsule according to claim 1, wherein the method comprises the following steps: and the first solvent is dimethyl sulfoxide.

4. The method for preparing bovine serum albumin-hydrophobically modified chitosan nanocapsule according to claim 1, wherein the method comprises the following steps: the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

5. The method for preparing bovine serum albumin-hydrophobically modified chitosan nanocapsule according to claim 1, wherein the method comprises the following steps: and the second solvent is isopropanol.

6. The method for preparing bovine serum albumin-hydrophobically modified chitosan nanocapsule according to claim 1, wherein the method comprises the following steps: the mass ratio of ethanol to water in the detergent is 3: 1.

7. the method for preparing bovine serum albumin-hydrophobically modified chitosan nanocapsule according to claim 1, wherein the method comprises the following steps: the mass concentration of the bovine serum albumin in the bovine serum albumin aqueous solution is 1.0%.

8. A bovine serum albumin-hydrophobic modified chitosan nano microcapsule is characterized in that: the preparation method according to any one of claims 1 to 7, wherein the bovine serum albumin-hydrophobically modified chitosan nanocapsule has a two-layer spherical structure, the inner layer is hydrophobically modified chitosan, 9-O-n-octadecyl berberine and paclitaxel, and the outer layer is bovine serum albumin.

Technical Field

The invention relates to the technical field of polysaccharide, in particular to a bovine serum albumin-hydrophobic modified chitosan nano microcapsule and a preparation method thereof.

Background

Paclitaxel is one of the world famous anti-tumor drugs, and has unique action mechanism and wide anti-tumor activity. However, paclitaxel is slightly soluble in water and is clinically used in combination with ethanol and polyoxyethylated castor oil in order to improve its solubility, but the use of ethanol and polyoxyethylated castor oil in combination increases hypersensitivity during chemotherapy.

In the prior art, paclitaxel is usually made into liposome preparation, for example, the method for preparing paclitaxel liposome preparation disclosed in chinese patent CN101011357 adopts a film dispersion method or a spray drying method to prepare long-circulating paclitaxel liposome, takes cholesterol, distearyl phosphatidylcholine and tetradecanoic acid as stabilizers, sucrose as a freeze-drying protective agent, and chloroform and methanol as organic solvents; the liposome membrane is modified by amphiphilic polyethylene glycol derivative, and the liposome has particle size not more than 100nm and entrapment rate not less than 85% by extrusion or high-pressure homogenization. The paclitaxel liposome preparation prepared by the method has the advantages of low toxicity and high stability, improves the solubility of paclitaxel, but the liposome is decomposed and metabolized in vivo quickly, and the paclitaxel liposome still has serious side effects, is easy to generate symptoms such as anaphylaxis, nausea and vomiting, and limits the use.

The nano-carrier has high drug loading rate and can prolong the circulation time of the paclitaxel in vivo. The chitosan is a naturally-occurring polysaccharide, has the advantages of no toxic or side effect, good biocompatibility, degradability, good cell adhesion and the like, and is an ideal material for preparing the nano-drug carrier. Bovine serum albumin is a globulin from serum, plays a role in maintaining blood osmotic pressure, pH buffer and carrier, and has excellent biocompatibility, so that bovine serum albumin can be used for preparing nano-carriers, has a good physical protection effect on anti-tumor drugs, and prolongs the action time of the drugs in vivo. However, chitosan and bovine serum albumin are both composed of hydrophilic monomers, and after the chitosan and bovine serum albumin are prepared into nano-carriers, the drug loading rate of the hydrophobic paclitaxel is not high, and the paclitaxel is easy to leak in vivo.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a preparation method of a bovine serum albumin-hydrophobic modified chitosan nano microcapsule, which introduces hydrophobic alkyl into a chitosan structure, can efficiently load hydrophobic paclitaxel, prolong the transportation time of a medicament in a body and prevent the medicament from leaking.

The technical scheme of the invention is as follows:

a preparation method of bovine serum albumin-hydrophobic modified chitosan nano-microcapsule comprises the following steps:

preparation of hydrophobic modified chitosan: weighing N-hexadecanoic acid and chitosan, adding the N-hexadecanoic acid and chitosan into a first solvent, stirring for 10-20 minutes, controlling the temperature to be 15-25 ℃, adding a condensing agent and N-hydroxysuccinimide, stirring for 0.5-1.5 hours, heating to 60-90 ℃, stirring for 12-24 hours, cooling to 20-40 ℃ to obtain a mixed solution, pouring the mixed solution into a second solvent, stirring for 20-40 minutes, filtering, washing a filter cake with a detergent, and performing vacuum drying to obtain the hydrophobically modified chitosan;

the first solvent is dimethyl sulfoxide or ethanol;

the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride or N, N-diisopropylcarbodiimide;

the second solvent is isopropanol or ethanol;

the detergent is an ethanol-water mixture, wherein the mass ratio of ethanol to water is (2-4): 1;

preparing a bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with an antitumor drug: adding the hydrophobic modified chitosan obtained in the step into dimethyl sulfoxide, adding 9-O-n-octadecyl berberine and paclitaxel, stirring for 5-10 minutes, dropwise adding a bovine serum albumin aqueous solution at the dropping speed of 2-3 drops/second, performing ultrasound for 10-30 minutes after the dropwise adding is finished, dialyzing for 24-72 hours by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain a bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with an anti-tumor drug;

the mass concentration of the bovine serum albumin aqueous solution is 0.5-1.5%;

wherein the mass ratio of the N-hexadecanoic acid, the chitosan, the solvent I, the condensing agent, the N-hydroxysuccinimide, the solvent II, the detergent, the dimethyl sulfoxide, the 9-O-N-octadecyl berberine, the paclitaxel and the bovine serum albumin aqueous solution is 0.2-0.5: 0.8-1.4: 100-200: 0.3-0.5: 0.25-0.4: 100-300: 5-15: 20-40: 0.02-0.18: 0.06-0.26: 200 to 400.

Preferably, the chitosan has an average molecular weight of 20 ten thousand and a degree of deacetylation of more than 80%.

Preferably, the first solvent is dimethyl sulfoxide.

Preferably, the condensing agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

Preferably, the second solvent is isopropanol.

Preferably, in the detergent, the mass ratio of ethanol to water is 3: 1.

preferably, the mass concentration of the bovine serum albumin in the bovine serum albumin aqueous solution is 1.0%.

A bovine serum albumin-hydrophobic modified chitosan nanometer microcapsule has two-layer spherical structure, wherein the inner layer is hydrophobic modified chitosan, 9-O-n-octadecyl berberine and paclitaxel, and the outer layer is bovine serum albumin.

The invention has the beneficial effects that: the product obtained by the invention is of a core-shell structure, the inner core layer is hydrophobic modified chitosan, paclitaxel and 9-O-n-octadecyl berberine, and the outer shell layer is bovine serum albumin. Firstly, n-hexadecanoic acid reacts with amino on chitosan, hydrophobic alkyl is introduced on chitosan molecules to obtain an amphiphilic polymer material, the amphiphilic polymer can be self-assembled in water to form a microcapsule structure, the hydrophobic end is wrapped in the core, and the hydrophilic end is out; according to a similar compatibility principle, long-chain alkyl of the hydrophobically modified chitosan is combined with hydrophobic paclitaxel, is wrapped inside, and is protected by carbohydrate outside, so that the defect of poor water solubility of the paclitaxel can be effectively improved, and the chitosan has a long circulation time in vivo so as to slowly release the medicine;

secondly, the 9-O-n-octadecyl berberine introduces hydrophobic long-chain alkyl at the 9-O position of the berberine, and is more easily combined with hydrophobic modified chitosan to form a micro-capsule structure according to the similar compatibility principle, and the berberine has a certain anti-tumor effect and can realize a better anti-tumor effect under the synergistic action with paclitaxel; the rest part of naked amino in the hydrophobically modified chitosan can be protonated in an acidic microenvironment of tumor cells, and the rapid release of the drug is realized;

in addition, when the nano-carrier is prepared, bovine serum albumin is added finally, contains a large amount of carboxyl and can be combined with the rest amino in the hydrophobic modified chitosan through static electricity to form a protective layer outside the microcapsule, so that the drug is further prevented from being released too fast, the biocompatibility of the carrier is increased, and the in-vivo circulation time of the carrier is prolonged.

Drawings

FIG. 1 shows the results of cytotoxicity tests;

FIG. 2 is the appearance of the nano-material prepared in example 3 measured by transmission electron microscopy;

FIG. 3 is the appearance of the nanomaterial prepared in example 3 measured by atomic force microscope.

Detailed Description

In order to make the technical means, technical features, objects and technical effects of the present invention easily understandable, the present invention is further described below with reference to the specific drawings.

Example 1:

a preparation method of bovine serum albumin-hydrophobic modified chitosan nano-microcapsule comprises the following steps:

preparation of hydrophobic modified chitosan: weighing 2g of n-hexadecanoic acid and 8g of chitosan, adding the n-hexadecanoic acid and 8g of chitosan into 1000g of dimethyl sulfoxide, stirring for 10 minutes, controlling the temperature to be 15 ℃, adding 3g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2.5g N-hydroxysuccinimide, stirring for 0.5 hour, heating to 60 ℃, stirring for 12 hours, cooling to 20 ℃ to obtain a mixed solution, pouring the obtained mixed solution into 1000g of isopropanol, stirring for 20 minutes, filtering, washing a filter cake with 50g of an ethanol-water mixture (the mass ratio of ethanol to water is 2: 1), and performing vacuum drying to obtain hydrophobically modified chitosan;

preparing a bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with an antitumor drug: adding the hydrophobic modified chitosan obtained in the step into 200g of dimethyl sulfoxide, adding 9-O-n-octadecyl berberine and 0.6g of paclitaxel, stirring for 5 minutes, dropwise adding 2000g of bovine serum albumin aqueous solution (the mass concentration is 0.5%), stirring at the speed of 2 drops/second, performing ultrasonic treatment for 10 minutes, dialyzing for 24 hours by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain the bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with the antitumor drug.

Example 2:

a preparation method of bovine serum albumin-hydrophobic modified chitosan nano-microcapsule comprises the following steps:

preparation of hydrophobic modified chitosan: weighing 5g of N-hexadecanoic acid and 14g of chitosan, adding the N-hexadecanoic acid and 14g of chitosan into 2000g of ethanol, stirring for 20 minutes, controlling the temperature at 25 ℃, adding 5g of N, N-diisopropylcarbodiimide and 4g of N-hydroxysuccinimide, stirring for 1.5 hours, heating to 90 ℃, stirring for 24 hours, cooling to 40 ℃ to obtain a mixed solution, pouring the obtained mixed solution into 3000g of ethanol, stirring for 40 minutes, filtering, washing a filter cake with 150g of ethanol-water mixture (the mass ratio of ethanol to water is 4: 1), and drying in vacuum to obtain hydrophobically modified chitosan;

preparing a bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with an antitumor drug: adding the hydrophobic modified chitosan obtained in the step into 400g of dimethyl sulfoxide, adding 1.8g of 9-O-n-octadecyl berberine and 2.6g of paclitaxel, stirring for 10 minutes, dropwise adding 4000g of bovine serum albumin aqueous solution (the mass concentration is 1.5%), dropwise adding at the speed of 3 drops/second, performing ultrasonic treatment for 30 minutes, dialyzing for 72 hours by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain the bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with the antitumor drug.

Example 3:

a preparation method of bovine serum albumin-hydrophobic modified chitosan nano-microcapsule comprises the following steps:

preparation of hydrophobic modified chitosan: weighing 4g of n-hexadecanoic acid and 10g of chitosan, adding the n-hexadecanoic acid and 10g of chitosan into 1500g of dimethyl sulfoxide, stirring for 15 minutes, controlling the temperature at 20 ℃, adding 4g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 3.5g N-hydroxysuccinimide, stirring for 1 hour, heating to 80 ℃, stirring for 18 hours, cooling to 30 ℃ to obtain a mixed solution, pouring the obtained mixed solution into 1500g of isopropanol, stirring for 30 minutes, filtering, washing a filter cake by using a mixture of 100g of ethanol and water (the mass ratio of the ethanol to the water is 3: 1), and drying in vacuum to obtain hydrophobically modified chitosan;

preparing a bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with an antitumor drug: adding the hydrophobic modified chitosan obtained in the step into 300g of dimethyl sulfoxide, adding 9-O-n-octadecyl berberine 1.2g and paclitaxel 2.0g, stirring for 8 minutes, dropwise adding 3000g of bovine serum albumin aqueous solution (the mass concentration is 1.0%), dropwise adding at the speed of 3 drops/second, performing ultrasonic treatment for 20 minutes, dialyzing for 48 hours by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain the bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with the antitumor drug.

Example 4:

and (3) testing the encapsulation efficiency of the nano-drug:

the encapsulation efficiency of the nano-carrier is determined by adopting a high performance liquid chromatography, paclitaxel and 9-O-n-octadecyl berberine are respectively weighed to prepare solutions with the concentration of 0, 0.5, 1, 4, 7, 10, 13, 16 and 20 mu mol/L, and a liquid chromatogram map of the solutions is determined on a high performance liquid chromatograph to draw a standard curve.

Weighing a certain amount of the bovine serum albumin-hydrophobic modified chitosan nano microcapsule loaded with the antitumor drugs prepared in the examples 1, 2 and 3, dissolving the nano microcapsule with pure water, fixing the volume with methanol, crushing the nano microcapsule by using ultrasonic waves to completely release paclitaxel and 9-O-n-octadecyl berberine in the nano microcapsule, measuring a liquid chromatogram of the nano microcapsule on a high performance liquid chromatograph, and calculating the encapsulation rate of the paclitaxel and the 9-O-n-octadecyl berberine according to the peak area and a standard curve, wherein the calculation formula is as follows:

entrapment efficiency = drug content/drug input x 100% as determined by high performance liquid chromatography.

The test results are shown in table 1:

TABLE 1 encapsulation efficiency test results of nanocapsules on paclitaxel and 9-O-n-octadecyl berberine

The nano microcapsule prepared by the invention has higher encapsulation efficiency on paclitaxel and 9-O-n-octadecyl berberine.

Cytotoxicity test:

cytotoxicity test of materials HeLa cells were treated with samples (in terms of paclitaxel) at concentrations of 2, 4, 6, 8, 10 μmol/L for 48 hours using the MTT method, and the inhibition rate of the cells was determined, as shown in fig. 1, as a result of the cytotoxicity test. The paclitaxel-loaded nanomaterial has obvious inhibition activity on HeLa cells because paclitaxel is added, particularly the nanomaterial prepared in example 3 has an inhibition rate on cells of about 76% when the concentration of paclitaxel is 4 μmol/L, which is much higher than that of paclitaxel drugs without nanomaterial coating. The nano material prepared by the invention can obviously improve the anti-tumor activity of the paclitaxel medicament.

Evaluation of in vivo antitumor Activity:

BALB/C mice (Liaoning Biotechnology GmbH) were selected to determine the antitumor activity of the nano-drugs in vivo. HeLa cells were injected into BALB/C mice at an injection rate of 200. mu.L and an injection concentration of 10⁵And (4) feeding each cell/mL at normal temperature for 2 weeks to construct a mouse tumor xenograft model. Grouping the tumor-transplanted mice, and setting a drug group, a control group and a model group, wherein the drug group is administered with oral gavage nanometer drugs, the administration dosage is 2mg/kg in terms of paclitaxel content, the control group is administered with pure paclitaxel or 9-O-n-octadecyl berberine, the administration dosage is 2mg/kg, the model group is the tumor-transplanted mice, and the mice are normally bred. Once daily for 20 consecutive days, tumors were surgically removed from the mice, weighed, and the average tumor weight was calculated, as shown in table 2.

TABLE 2 in vivo antitumor Activity of antitumor drug-loaded nanocapsules

Examples 1-3 the antitumor activity of the nano-drug encapsulating paclitaxel and 9-O-n-octadecyl berberine is much better than that of paclitaxel, the average tumor weight is less than 0.47g, and the average tumor weight is 0.71g after the pure paclitaxel is administered for 20 days.

Appearance morphology testing of the materials:

as shown in FIG. 2, the appearance of the nanomaterial prepared in example 3 is determined by transmission electron microscopy, and the nanomaterial prepared by the invention has an average particle size of about 200nm, good dispersibility and uniform particle size. As shown in fig. 3, the appearance of the nanomaterial prepared in example 3 is measured by an atomic force microscope, and the atomic force microscope shows that the nanomaterial has a spherical structure.

In summary, the embodiments of the present invention are merely exemplary and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made according to the content of the claims of the present invention should fall within the technical scope of the present invention.