CN102399301B - Preparation method for amphiphilic konjac glucomannan cholesterol grafted polymer and application - Google Patents

Abstract

The invention belongs to the technical field of functional polymer chemistry, and in particular relates to a preparation method of amphiphilic carboxymethyl konjac glucomannan cholesterol graft and its application in drug carrier materials. Firstly, the amphiphilic carboxymethyl konjac glucomannan cholesterol graft was prepared by reacting EDCI-activated carboxymethyl konjac glucomannan with cholesterylamine in DMF. The graft is self-assembled in a selective solvent to form stable nano micelles, and the micelles are used for the encapsulation and sustained release of drugs. The amphiphilic carboxymethyl konjac glucomannan cholesterol graft prepared by the invention has good biocompatibility and biodegradability, and has good application prospects in the field of biomedicine.

Description

Preparation method and application of amphiphilic konjac glucomannan cholesterol graft

technical field

The invention belongs to the technical field of functional polymer chemistry, and in particular relates to a preparation method of amphiphilic carboxymethyl konjac glucomannan cholesterol graft and its application in drug carrier materials.

Background technique

As a natural polymer material, polysaccharide has good biocompatibility and degradability. In addition, polysaccharides also have the characteristics of abundant resources and low processing costs. More importantly, most natural polysaccharide molecules contain hydrophilic groups such as carboxyl, hydroxyl and amino groups, which can have strong non-covalent bonding with functional macromolecules in organisms such as proteins , thereby affecting life activities. All these advantages endow natural polysaccharides with bright prospects as biomaterials (Macromolecules, 1993, 26: 3062-3068). Amphiphilic polymers prepared from aliphatic segments and natural polysaccharides have biodegradability, good biocompatibility, and self-assembly properties to form molecularly ordered bodies, and have broad application prospects (Current nanoscience, 2010, 6: 298-306). In recent years, the study of polymer materials that can be used for drug controlled release has become a research hotspot in medicinal chemistry and material chemistry. Taking advantage of the biocompatibility, degradability, and non-toxicity of polysaccharides, the grafted polysaccharides It is undoubtedly the first choice of researchers to form micelles with lipophilic chain segments that can be used as drug-controlled release carriers in selective solvents. This type of self-assembly in selective solvents forms nano-core-shell micelles that can be used for drug delivery. , can enhance the stability of drugs, reduce side effects, enhance the water solubility of fat-soluble drugs, and control the release of drugs (European polymer journal, 2008, 44:555-565). In addition, they can also be used as high-molecular surfactants with excellent performance. So far, the amphiphilic polysaccharide grafts based on lipophilic segment modification have limited polysaccharide types, limited to chitosan, sodium alginate, dextran, pullulan and heparin. However, the selection of these materials as biomedical materials is far from enough, and the critical concentration of amphiphilic grafts formed by modifying these materials with lipophilic segments is generally high in solution to form micelles, which limits their Further applications in the field of biomedicine. Therefore, it is a very meaningful research topic to discover more non-toxic, biocompatible and degradable materials, and to study their application in the field of biomedicine.

Konjac glucomannan is a high-molecular-weight water-soluble polysaccharide extracted from the tuber of the natural plant Konjac, which is composed of β-D-glucose and β-D-mannose in a molar ratio of 2:3-1:1.6 Linked by β-(1→4) glycosidic bonds. Konjac glucomannan has the effects of reducing blood cholesterol and blood sugar levels, reducing body weight, promoting gastrointestinal motility and reducing the risk of diabetes and heart disease (Agricultural biology and chemistry, 1975, 39: 301-312). At present, some chemical modification technologies have been applied to the development of biofunctional materials with the same properties as konjac glucomannan. Carboxymethyl konjac glucomannan is an anionic polymer obtained by carboxymethylating konjac glucomannan. When it is mixed with oppositely charged polymers, it can exhibit good water solubility, bioactivity, biocompatibility, and gelling ability, which endow carboxymethyl konjac glucomannan as a drug sustained-release biomaterial bright future (Macromolecule rapid communication, 2004, 25: 954-958; International journal of pharmaceuticals, 2008, 364: 102-107). However, the agglomeration phenomenon of the aggregates obtained by the electrostatic attraction of opposite charges is relatively serious, which seriously affects its efficiency as a drug sustained release carrier, and this type of aggregates is not suitable for the currently widely used small molecule and poorly water-soluble drugs. Can not play the role of sustained release.

As an essential element of the human body, cholesterol is a necessary substance for the synthesis of cell membranes and hormones; the human liver can synthesize about 1 gram of cholesterol per day to meet the needs of various physiological activities. High cholesterol is unhealthy, but low cholesterol is equally deadly. Although medical experts recommend that the cholesterol level be kept below 200 mg/dl, if the value is lower than 160 mg/dl, various health problems will come to your door, including cancer. Studies have shown that women with too low total cholesterol levels are prone to premature birth, and too low cholesterol can also lead to anxiety and depression (Science and Culture, 2010, 4, 45).

Contents of the invention

In order to provide more materials that can be applied in the field of biomedicine, the primary purpose of the present invention is to provide a non-toxic, biocompatible, degradable amphiphilic polysaccharide graft that contains cholesterol segment carboxymethyl konjac glucomannan graft (CHCKGM). The present invention utilizes 1-ethyl-(3-dimethylaminopropyl) carbodiimide and hydroxysuccinimide ester to activate the carboxyl group on carboxymethyl konjac glucomannan to obtain hydrophobic cholesterol-modified Carboxymethyl konjac glucomannan amphiphilic graft. After being modified with hydrophobic cholesterol fragments, carboxymethyl konjac glucomannan has amphiphilic characteristics, so that it can self-assemble in a selective solvent to form a hydrophilic carboxymethyl konjac glucomannan with hydrophobic cholesterol as the core. Monodisperse and stable core-shell structure micelles with sugar as the shell. This well-dispersed core-shell structure micelles can solubilize small molecules and poorly hydrophilic drugs into the micelles to form nano-micelles The drug-loading system can improve the stability and water solubility of the drug, and then realize the controlled release of the drug.

Another object of the present invention is to provide a method for preparing the above-mentioned degradable amphiphilic polysaccharide graft, that is, carboxymethyl konjac glucomannan graft (CHCKGM) containing a cholesterol segment. The preparation method has the following steps: Simple, mild reaction conditions, and easy implementation.

Another object of the present invention is to provide the application of the above-mentioned amphiphilic grafts in the construction of nano drug carrier materials (ie drug-loaded micelles).

The object of the present invention is achieved through the following technical solutions, and the preparation method of the amphiphilic carboxymethyl konjac glucomannan cholesterol graft comprises the steps:

(1) Synthesis of N-tert-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine-CH): cholesterol, N-tert-butoxycarbonyl-glycine and 4-dimethylaminopyridine (cholesterol: N-tert-butoxycarbonyl - Glycine molar ratio is 1:1-1:2, cholesterol: 4-dimethylaminopyridine molar ratio is 1:0.2-1:0.5), dissolved in dichloromethane, followed by adding dicyclohexylcarbodiimide ( Cholesterol: dicyclohexylcarbodiimide molar ratio is 1:1-1:2), stirring, controlling the temperature at -5-30°C, reacting for 10-30h, filtering to remove the white precipitate dicyclohexylurea, and evaporating the solvent to obtain Solid N-t-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine-CH).

(2) Synthesis of glycine cholesteryl ester (Glycine-CH): Dissolve the product in step (1) in dichloromethane, add excess trifluoroacetic acid under ice bath stirring, react for 1-5 hours under ice bath stirring, and the reaction is over Afterwards, the sample was evaporated to dryness, and the product was dissolved in 15% sodium chloride solution, and the pH was adjusted to be 5, filtered, and the filtrate was extracted three times with chloroform, and the organic phase was dried overnight with anhydrous sodium sulfate, and evaporated to dryness to obtain cholesteryl glycine ( Glycine-CH) solid.

(3) Preparation of cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft (CHCKGM): Dissolve carboxymethyl konjac glucomannan in formamide (concentration: 0.5-8 %), add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide ester, stir at room temperature for 15min, then add 0.15-0.5mmol dissolved in 10ml dimethylformamide Glycine cholesteryl ester (concentration is 0.5-8%, the molar ratio of carboxymethyl konjac glucomannan and glycine cholesteryl ester is 1:0.2-1:2), stirred for 12-24h under the protection of nitrogen, and the reaction temperature is 0- 30°C. After the reaction, precipitate with a large amount of acetone, and wash the precipitate with water and tetrahydrofuran respectively. The obtained precipitate is dispersed with water, dialyzed for more than 72 hours, and the dialysate is freeze-dried to obtain cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft.

The carboxymethyl konjac glucomannan described in step (3) is prepared by reacting konjac glucomannan and chloroacetic acid, and the degree of carboxymethylation is 20-80%.

The molar ratio of carboxyl groups on the 1-ethyl-(3-dimethylaminopropyl) carbodiimide and hydroxysuccinimide ester and konjac glucomannan described in step (3) is 0.5-1 :0.5:2.

The whole reaction equation of the CHCKGM graft prepared by the present invention can be expressed as:

The amphiphilic konjac glucomannan cholesterol graft, that is, the carboxymethyl konjac glucomannan graft containing a cholesterol segment, is prepared by the above preparation method.

The micellization method of the konjac glucomannan cholesterol graft obtained in step (3) in a selective solvent is as follows: disperse carboxymethyl konjac glucomannan containing cholesterol in deionized water (concentration is 0.1-1 %), add the drug solution dissolved in solvent (concentration: 0.5-5%), stir and disperse at room temperature for 12-24h, then ultrasonically in the ultrasonic instrument for 2-5min, rotate and centrifuge after the ultrasonic, the lower layer is the interface for loading the drug branched micelles.

use:

Compared with existing materials, the present invention has the following advantages and beneficial effects:

The preparation method of the biocompatible and fully biodegradable amphiphilic polysaccharide grafts provided by the present invention, that is, the carboxymethyl konjac glucomannan grafts containing cholesterol segments has the advantages of simple steps, mild reaction conditions and easy implementation. features. The base material konjac glucomannan of the present invention has many biological activities, and the introduced hydrophobic segment cholesterol itself is non-toxic. Cholesterol is an essential nutrient element for the human body. Although cholesterol has been criticized by people, the fact is that cholesterol plays a vital role in building cell membranes and synthesizing hormones. In conclusion, carboxymethyl konjac glucomannan grafts containing cholesterol segments have good biocompatibility. Therefore, the carboxymethyl konjac glucomannan graft containing cholesterol chain segment of the present invention has the advantages of non-toxicity, good biocompatibility, degradability and the like. This material not only has the ability to self-assemble into micelles in selective solvents, but also has a very low critical aggregation concentration, and also has modifiability (carboxyl, hydroxyl). The biocompatible and fully biodegradable amphiphilic polysaccharide grafts, that is, the carboxymethyl konjac glucomannan grafts containing cholesterol segments, will have potential application value in the field of biomedicine.

Description of drawings

Figure 1 is the infrared spectrum of glycine cholesteryl ester.

Figure 2 is the infrared spectrum of cholesterol and carboxymethyl konjac glucomannan graft.

Figure 3 is the nuclear magnetic spectrum of cholesterol and carboxymethyl konjac glucomannan graft.

Figure 4 is the particle size distribution of CHCKGM grafts.

Figure 5 shows the critical aggregation concentration of CHCKGM grafts (circles: CHCKGM ₁ , triangles: CHCKGM ₂ , squares: CHCKGM ₃ ).

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and drawings, but the implementation of the invention is not limited thereto.

The preparation of embodiment 1 CHCKGM graft

(1) Synthesis of N-tert-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine-CH): cholesterol 1mmol (0.386g), N-tert-butoxycarbonyl-glycine 1.1mmol (0.1925g), 4-dimethylamino Pyridine 0.25mmol (0.031g), dissolved in 5ml of dichloromethane, then added 1.1mmol of dicyclohexylcarbodiimide (0.226g), stirred in an ice bath, reacted for 24h, after the reaction, filtered to remove the white precipitate Dicyclohexylurea, remove the solvent at room temperature, dissolve the product with a small amount of acetone, overnight in the refrigerator, filter and remove the precipitated DCU, evaporate the solvent to obtain solid N-tert-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine- CH).

(2) Synthesis of glycine cholesteryl ester (Glycine-CH): Dissolve the product in step (1) in 5 ml of dichloromethane, add 5 ml of trifluoroacetic acid with stirring in ice bath, and react for 3 h under ice bath stirring, After the reaction, evaporate the sample to dryness, dissolve the product in 15% sodium chloride solution, adjust the pH to 5, filter, extract the filtrate three times with chloroform, dry the organic phase with anhydrous sodium sulfate overnight, and evaporate to dryness to obtain glycine cholesterol Ester (Glycine-CH) solid. As shown in Figure 1 infrared spectrum: 3434cm ^-1 is the stretching vibration peak of hydroxyl group, 2938cm ^-1 is the stretching vibration peak of CH bond, 1750cm ^-1 is the stretching vibration peak of carbonyl group, 1676cm ^-1 is the bending vibration peak of amino group, 1264cm ^-1 is the stretching vibration peak of CN bond, and the stretching vibration peak of 1179cm ^-1 COC.

(3) Preparation of cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft (CHCKGM): 0.5 mmol of carboxymethyl konjac glucomannan was dissolved in 10 ml of formamide in a water bath at 80°C, Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide ester, stir at room temperature for 15min, then add 0.15mmol glycine cholesteryl ester dissolved in 10ml dimethylformamide , stirred at room temperature for 18 h under the protection of nitrogen, after the reaction was completed, precipitated with a large amount of acetone, and the precipitate was washed with water and tetrahydrofuran respectively. The obtained precipitate was dispersed with water, and then dialyzed at 25° C. for 72 h with a dialysis bag with a molecular weight cutoff of 8000-14000, and the dialysate was freeze-dried to obtain cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft (CHCKGM). As shown in Figure 2 infrared spectrum: 3411cm ^-1 is the stretching vibration peak of OH, 2936cm ^-1 is the stretching vibration peak of CH bond, 1746cm ^-1 is the stretching vibration peak of carbonyl group, and 1674cm ^-1 has obvious stretching vibration of amide bond Peak. As shown in the triple nuclear magnetic spectrum (DMSO, ppm): 0.67 (3H, s,) is the 18th methyl group on cholesterol, 0.8–2.4 (28H) is 1-H ₂ , 2-H ₂ on cholesterol , 4-H ₂ , 7-H ₂ , 8-H ₁ , 9-H ₁ , 11-H ₂ , 12-H ₂ , 14-H ₁ , 15-H 2 , 16-H _{2 ,} 17-H ₁ , 20-H ₁ , 22-H ₂ , 23-H ₂ , 24-H ₂ and 25-H ₁ peaks, 0.88 (6H, d J = 6.4Hz) are 26-H ₃ and 27-H ₃ on cholesterol 0.93 (3H, d, J = 6.24Hz) is the peak of 21-H ₃ on cholesterol, 1.01 (3H, s) is the peak of cholesterol 19-H ₃ , 3.0-4.5 (m) is the peak of glucose and The peak of 2,3,4,5,6-H ₁ of mannose unit, 4.0(2H,m) is the peak of methylene on OCOCH ₂ -NH ₂ , 4.73(1H,m) is the peak of 3-H on cholesterol ₁ , 5.37(1H,m) is the peak of 6-H ₁ on cholesterol.

The preparation of embodiment 2 CHCKGM grafts

(1) Synthesis of N-t-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine-CH): cholesterol 1mmol (0.386g), N-tert-butoxycarbonyl-glycine 1.5mmol (0.2625g) 4-dimethylaminopyridine 0.25mmol (0.031g), dissolved in 10ml of dichloromethane, then added 1.1mmol of dicyclohexylcarbodiimide (0.226g), stirred in an ice bath, reacted for 24h, after the reaction, filtered to remove the white precipitate di Cyclohexylurea, remove the solvent at room temperature, dissolve the product in a small amount of acetone, overnight in the refrigerator, filter and remove the precipitated DCU, evaporate the solvent to obtain solid N-tert-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine-CH ).

(2) Synthesis of glycine cholesteryl ester (Glycine-CH): Dissolve the product in step (1) in 5 ml of dichloromethane, add 5 ml of trifluoroacetic acid with stirring in ice bath, and react for 3 h under ice bath stirring, After the reaction, evaporate the sample to dryness, dissolve the product in 15% sodium chloride solution, adjust the pH to 5, filter, extract the filtrate three times with chloroform, dry the organic phase with anhydrous sodium sulfate overnight, and evaporate to dryness to obtain glycine cholesterol Ester (Glycine-CH) solid. As shown in Figure 1 infrared spectrum: 3434cm ^-1 is the stretching vibration peak of hydroxyl group, 2938cm ^-1 is the stretching vibration peak of CH bond, 1750cm ^-1 is the stretching vibration peak of carbonyl group, 1676cm ^-1 is the bending vibration peak of amino group, 1264cm ^-1 is the stretching vibration peak of CN bond, and the stretching vibration peak of 1179cm ^-1 COC.

(3) Preparation of cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft (CHCKGM): 0.5 mmol of carboxymethyl konjac glucomannan was dissolved in 10 ml of formamide in a water bath at 80 degrees Celsius, Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide ester, stir at room temperature for 15min, then add 0.25mmol glycine cholesteryl ester dissolved in 10ml dimethylformamide , stirred at room temperature for 24 h under the protection of nitrogen, after the reaction was completed, precipitated with a large amount of acetone, and the precipitate was washed with water and tetrahydrofuran respectively. The obtained precipitate was dispersed with water, and then dialyzed at 25° C. for 72 h with a 8000-14000 molecular weight cut-off dialysis bag, and the dialysate was freeze-dried to obtain cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft (CHCKGM). As shown in Figure 2 infrared spectrum: 3411cm ^-1 is the stretching vibration peak of OH, 2936cm ^-1 is the stretching vibration peak of CH bond, 1746cm ^-1 is the stretching vibration peak of carbonyl group, and 1674cm ^-1 has obvious stretching vibration of amide bond Peak. As shown in the triple nuclear magnetic spectrum (DMSO, ppm): 0.67 (3H, s,) is the 18th methyl group on cholesterol, 0.8–2.4 (28H) is 1-H ₂ , 2-H ₂ on cholesterol , 4-H ₂ , 7-H ₂ , 8-H ₁ , 9-H ₁ , 11-H ₂ , 12-H ₂ , 14-H ₁ , 15-H 2 , 16-H _{2 ,} 17-H ₁ , 20-H ₁ , 22-H ₂ , 23-H ₂ , 24-H ₂ and 25-H ₁ peaks, 0.88 (6H, d J = 6.4Hz) are 26-H ₃ and 27-H ₃ on cholesterol 0.93 (3H, d, J = 6.24Hz) is the peak of 21-H ₃ on cholesterol, 1.01 (3H, s) is the peak of cholesterol 19-H ₃ , 3.0-4.5 (m) is the peak of glucose and The peak of 2,3,4,5,6-H ₁ of mannose unit, 4.0(2H,m) is the peak of methylene on OCOCH ₂ -NH ₂ , 4.73(1H,m) is the peak of 3-H on cholesterol ₁ , 5.37(1H,m) is the peak of 6-H ₁ on cholesterol.

The preparation of embodiment 3 CHCKGM grafts

(1) Synthesis of N-t-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine-CH): cholesterol 1mmol (0.386g), N-tert-butoxycarbonyl-glycine 1.5mmol (0.2625g) 4-dimethylaminopyridine 0.5mmol (0.062g), dissolved in 5-10ml of dichloromethane, then added 1.1mmol of dicyclohexylcarbodiimide (0.226g), stirred in an ice bath, reacted for 18h, after the reaction, filtered to remove the white Precipitate dicyclohexylurea, remove the solvent at room temperature, dissolve the product with a small amount of acetone, overnight in the refrigerator, filter and remove the precipitated DCU, evaporate the solvent to obtain solid N-tert-butoxycarbonyl-glycine cholesteryl ester (N-t-glycine -CH).

(2) Synthesis of glycine cholesteryl ester (Glycine-CH): Dissolve the product in step (1) in 5 ml of dichloromethane, add 5 ml of trifluoroacetic acid with stirring in ice bath, and react for 3 h under ice bath stirring, After the reaction, evaporate the sample to dryness, dissolve the product in 15% sodium chloride solution, adjust the pH to 5, filter, extract the filtrate three times with chloroform, dry the organic phase with anhydrous sodium sulfate overnight, and evaporate to dryness to obtain glycine cholesterol Ester (Glycine-CH) solid. As shown in Figure 1 infrared spectrum: 3434cm ^-1 is the stretching vibration peak of hydroxyl group, 2938cm ^-1 is the stretching vibration peak of CH bond, 1750cm ^-1 is the stretching vibration peak of carbonyl group, 1676cm ^-1 is the bending vibration peak of amino group, 1264cm ^-1 is the stretching vibration peak of CN bond, and the stretching vibration peak of 1179cm ^-1 COC.

(3) Preparation of cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft (CHCKGM): 0.5 mmol of carboxymethyl konjac glucomannan was dissolved in 10 ml of formamide in a water bath at 80°C, Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinimide ester, stir at room temperature for 15min, then add 0.5mmol glycine cholesteryl ester dissolved in 10ml dimethylformamide , stirred at room temperature for 24 h under the protection of nitrogen, after the reaction was completed, precipitated with a large amount of acetone, and the precipitate was washed with water and tetrahydrofuran respectively. The obtained precipitate was dispersed with water, and then dialyzed at 25° C. for 72 h with a dialysis bag with a molecular weight cutoff of 8000-14000, and the dialysate was freeze-dried to obtain cholesterol-containing carboxymethyl konjac glucomannan amphiphilic graft (CHCKGM). As shown in Figure 2 infrared spectrum: 3411cm ^-1 is the stretching vibration peak of OH, 2936cm ^-1 is the stretching vibration peak of CH bond, 1746cm ^-1 is the stretching vibration peak of carbonyl group, and 1674cm ^-1 has obvious stretching vibration of amide bond Peak. As shown in the triple nuclear magnetic spectrum (DMSO, ppm): 0.67 (3H, s,) is the 18th methyl group on cholesterol, 0.8–2.4 (28H) is 1-H ₂ , 2-H ₂ on cholesterol , 4-H ₂ , 7-H ₂ , 8-H ₁ , 9-H ₁ , 11-H ₂ , 12-H ₂ , 14-H ₁ , 15-H 2 , 16-H _{2 ,} 17-H ₁ , 20-H ₁ , 22-H ₂ , 23-H ₂ , 24-H ₂ and 25-H ₁ peaks, 0.88 (6H, d J = 6.4Hz) are 26-H ₃ and 27-H ₃ on cholesterol 0.93 (3H, d, J = 6.24Hz) is the peak of 21-H ₃ on cholesterol, 1.01 (3H, s) is the peak of cholesterol 19-H ₃ , 3.0-4.5 (m) is the peak of glucose and The peak of 2,3,4,5,6-H ₁ of mannose unit, 4.0(2H,m) is the peak of methylene on OCOCH ₂ -NH ₂ , 4.73(1H,m) is the peak of 3-H on cholesterol ₁ , 5.37(1H,m) is the peak of 6-H ₁ on cholesterol.

The micellization of embodiment 4 CHCKGM grafts in selective solvents

4 mg of CHCKGM ₁ was dispersed in 4 ml of deionized water, stirred and dispersed at room temperature for 24 hours, and then sonicated in an ultrasonic instrument for 2 minutes. After the sonication, the solution was centrifuged at 7000 rpm to remove impurities. The particle size data obtained by testing the obtained micellar solution with a laser dynamic light scattering instrument are shown in Figure 4. The effective particle size of the micelles is 984 nm, and the polydispersity coefficient is 0.279. As shown in Figure 5: the critical aggregation concentration (CAC) obtained by the fluorescent probe method is 5.89×10 ^-3 (mg/ml).

The micellization of embodiment 5 CHCKGM grafts in selective solvent

4 mg of CHCKGM ₂ was dispersed in 10 ml of deionized water, stirred and dispersed at room temperature for 36 hours, and then ultrasonicated in an ultrasonic instrument for 5 minutes. After ultrasonication, the solution was centrifuged at 7000 rpm to remove impurities. The particle size data obtained by testing the obtained micellar solution with a laser dynamic light scattering instrument are shown in Figure 4: the effective particle size of the micelles is 648nm, and the polydispersity coefficient is 0.169. As shown in Figure 5: the critical aggregation concentration (CAC) obtained by the fluorescent probe method is 3.89×10 ^-3 (mg/ml).

The micellization of embodiment 6 CHCKGM grafts in selective solvents

4 mg of CHCKGM ₃ was dispersed in 10 ml of deionized water, stirred and dispersed at room temperature for 24 hours, and then sonicated in an ultrasonic instrument for two minutes. After the ultrasonic was completed, the solution was centrifuged at 10,000 rpm to remove impurities. Put the solution into a dialysis bag and dialyze at 25°C for 24 hours. The particle size data obtained by testing the obtained micellar solution with a laser dynamic light scattering instrument are shown in Figure 5: the effective particle size of the micelles is 439nm, and the polydispersity coefficient is 0.109 . As shown in Figure 5: the critical aggregation concentration (CAC) obtained by the fluorescent probe method is 2.59×10 ^-3 (mg/ml).

Example 7 CHCKGM graft nano drug-loaded micelles and drug release in vitro

Disperse 4 mg of CHCKGM ₃ in 3.8 ml of deionized water, add 1 mg of indomethacine dissolved in 0.2 ml of methanol, stir and disperse at room temperature for 24 hours, and then ultrasonicate for two minutes in an ultrasonic instrument. Centrifuge for 15 minutes to separate, the lower layer is the grafted micelles loaded with drugs, and the supernatant liquid is detected with a spectrophotometer at 294nm for the content of indomethacine, and the total drug dosage minus the drug content in the upper layer is the encapsulation rate. The encapsulation rate obtained during the experiment was 17.5%. After lyophilization, the drug-loaded graft micelles in the lower layer were dispersed into 4mL of PBS buffer, and then put into a dialysis bag with a molecular weight cut-off of 8000-14000 and dialyzed in a beaker at 25°C, and the solution in the beaker was taken out within a certain period of time Use a spectrophotometer to measure the content of indomethacin, and the time is maintained within 24 hours.

Example 8 CHCKGM graft nano drug-loaded micelles and drug release in vitro

Disperse 4 mg of CHCKGM ₃ in 3.8 ml of deionized water, add 1 mg of indomethacine dissolved in 0.2 ml of ethanol, stir and disperse at room temperature for 24 hours, and then ultrasonicate for two minutes in an ultrasonic instrument. The ultrasonic mode is 2 seconds on and 2 seconds off. After the ultrasound is over, put the solution into a dialysis bag, dialyze at 30°C for 9-12 hours, freeze-dry the dialysate to obtain the drug-loaded graft micelles, and extract the indomethacin in the drug-loaded micelles with methanol , using a spectrophotometer to detect the content of indomethacin, and the encapsulation rate was 20%. Disperse 4 mg of lyophilized drug-loaded micelles into 4 mL of PBS buffer, then put them into a dialysis bag with a molecular weight cut-off of 8,000-14,000, and dialyze in a beaker at 25°C, take out the solution in the beaker within a certain period of time and use a spectrometer Photometer measures the content of indomethacin, and the time is maintained within 24h.

Claims (7)

1. the preparation method of CHCKGM, is characterized in that comprising the steps:

(1) N-tertbutyloxycarbonyl-glycine cholesterol ester synthesis: cholesterol, N-tertbutyloxycarbonyl-glycine and DMAP are dissolved in dichloromethane, consumption is: cholesterol and N-tertbutyloxycarbonyl-glycine mol ratio are 1:1-1:2, and cholesterol and DMAP mol ratio are 1:0.2-1:0.5; Add subsequently dicyclohexylcarbodiimide, consumption is: cholesterol and dicyclohexylcarbodiimide mol ratio are 1:1-1:2, stir, control temperature in-5-30 ℃, reaction 10-30h, remove by filter the white precipitate dicyclohexylurea, drain solvent, obtain the N-tertbutyloxycarbonyl of solid-glycine cholesteryl ester;

(2) glycine cholesterol ester synthesis: the product in step (1) is dissolved in dichloromethane, ice bath adds excessive trifluoroacetic acid under stirring, and ice bath stirs lower reaction 1-5h, after reaction finishes, the evaporate to dryness sample, product is dissolved in 15% sodium chloride solution, regulating pH is 5, filters, filtrate is used chloroform extraction three times, organic facies is spent the night with anhydrous sodium sulfate drying, and evaporate to dryness obtains the solid of glycine cholesteryl ester;

(3) contain the preparation of the Carboxymethyl Konjac Glucomannan amphipathic stem-grafting thing of cholesterol: Carboxymethyl Konjac Glucomannan is dissolved into Methanamide under 80 ℃ of water-baths in, after dissolving, the concentration of Carboxymethyl Konjac Glucomannan is 0.5-8%, add 1-ethyl-(3-dimethyl aminopropyl) carbodiimides and hydroxysuccinimide eater, the 10 milliliters of glycine cholesteryl esters in dimethyl formamide that are dissolved in that add 0.15-0.5mmol after stirring at room 15min, the concentration of glycine cholesteryl ester is 0.5-8%, Carboxymethyl Konjac Glucomannan and glycine cholesteryl ester mol ratio are 1:0.2-1:2, stir 12-24h under nitrogen protection, reaction temperature is 0-30 ℃, after reaction finishes, use a large amount of acetone precipitations, precipitate water and oxolane wash respectively, the precipitation aqueous dispersion that obtains, more than dialysis 72h, the dialysis solution lyophilization obtains containing the Carboxymethyl Konjac Glucomannan amphipathic stem-grafting thing of cholesterol.

2. the preparation method of CHCKGM according to claim 1, is characterized in that: be 20-80% in the Carboxymethyl Konjac Glucomannan carboxy methylation degree described in step (3).

3. the preparation method of CHCKGM according to claim 1, it is characterized in that: the molar ratio of carboxyl is 0.5-1:0.5-2 on the 1-ethyl described in step (3)-(3-dimethyl aminopropyl) carbodiimides and hydroxysuccinimide eater and Carboxymethyl Konjac Glucomannan.

4. the preparation method of CHCKGM according to claim 1, it is characterized in that: the micellization method of the Carboxymethyl Konjac Glucomannan cholesterol grafted polymer that described step (3) obtains in selective solvent is as follows: the Carboxymethyl Konjac Glucomannan that will contain cholesterol is dispersed in deionized water, making its concentration is 0.1-1%, dispersed with stirring 12-24h at room temperature, then ultrasonic 2-5min in Ultrasound Instrument, after ultrasonic end, rotating centrifugal is removed impurity.

5. the application of CHCKGM in the structure drug carrier material of the described method preparation of claim 1.

6. the application of the CHCKGM described according to claim 5 in the structure drug carrier material, it is characterized in that: build pharmaceutical carrier by micellization, the Carboxymethyl Konjac Glucomannan that is about to contain cholesterol is dispersed in deionized water, making its concentration is 0.1-1%, add the drug solution with dissolution with solvents, the concentration of drug solution Chinese medicine is 0.5-5%, dispersed with stirring 12-24h under room temperature, then ultrasonic 2-5min in Ultrasound Instrument, rotating centrifugal after ultrasonic end, lower floor is the graft micelle of carrying medicament.

7. the application in the structure of the CHCKGM described according to claim 6 drug carrier material, it is characterized in that: said solvent is water or methanol or ethanol or oxolane or dioxane or acetone or chloroform or dichloromethane.

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