CN112694546B - Amphiphilic chitosan derivative and preparation method thereof - Google Patents
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Abstract
The invention discloses an amphiphilic chitosan derivative and a preparation method thereof, and relates to the technical field of biology. The invention grafts 18 beta glycyrrhetinic acid and sialic acid on a chitosan molecular chain in the presence of HOBt and EDAC & HCl, improves the water solubility of chitosan, and simultaneously, the modified amphiphilic chitosan derivative has no toxicity to cells, has good blood compatibility and has good application prospect in the aspects of anti-inflammation, antibiosis, antivirus and drug carrier. The preparation method is simple, short in reaction time, free of complex instruments and suitable for industrial mass production.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an amphiphilic chitosan derivative and a preparation method thereof.
Background
With the vigorous development and major breakthrough of biotechnology, biomedical materials have become hot spots for research and development. At present, amphiphilic biomaterials with good biocompatibility have the characteristic of spontaneously forming stable micelles in aqueous solution due to the hydrophilic and hydrophobic groups, and are widely concerned in the field of medicine. On one hand, the solubilization and transportation of the hydrophobic drug can be realized by utilizing the action of the hydrophobic core of the micelle and the drug. On the other hand, the specific recognition of the micelle on the focus part can be realized through targeted modification of the micelle, the drug effect is greatly improved, the toxic and side effects are reduced, or the high-resolution imaging of the pathological tissue is realized, and the disease detection sensitivity is improved.
The 18 beta-glycyrrhetinic acid is an active ingredient in liquorice, is an important raw material for medicines and high-grade cosmetics, and has the effects of resisting inflammation and allergy, inhibiting bacterial reproduction and the like. It can be used in cosmetics for regulating skin immunity, enhancing skin immunity, eliminating inflammation, and preventing allergy. Can relieve the adverse side effects of related components and other external factors in cosmetic. It also has effects in inhibiting tyrosinase activity, preventing melanin generation, and whitening skin. Can be used for oral products for preventing and eliminating gingival inflammation and oral ulcer.
Sialic acid is a natural carbohydrate, widely present in the human body and plays an important role in the metabolism of cells. Sialic acid protects macromolecules and cells from enzymatic and immunological attack at cell surface sites and promotes innate immunity. Sialic acid at the terminal of a sugar chain can serve as a recognition site or mask the recognition site between molecules and cells, between cells and cells, and between cells and the outside.
Chitosan (Chitin) is a product obtained by deacetylating Chitin (Chitin) under a strong alkali condition, is the only alkaline polysaccharide in natural polysaccharides, has no irritation, good biocompatibility and biodegradability, and is nontoxic and can be completely absorbed by organisms, so that the chitosan (Chitin) is an ideal nano microcapsule wall material. Compared with chitin, the deacetylated chitosan has improved solubility to a great extent, but can only be dissolved in an acidic solution with pH less than 6.0. Because the chitosan has poor solubility and high viscosity, and the application range of the chitosan is limited, the improvement of the solubility of the chitosan is one of the most important directions in the research of chitosan modification.
The chitosan is chemically modified, after a proper amount of hydrophilic groups or hydrophobic groups are introduced into chitosan molecules, the crystal structure of the chitosan can be damaged, and the solubility of the chitosan is greatly improved, namely, by utilizing the reactivity of amino groups and hydroxyl groups on a chitosan main chain, the chitosan main chain is simultaneously connected with side groups or side chains with hydrophobic side chains and hydrophilic side chains through a chemical modification method, so that the chitosan has both hydrophilicity and hydrophobicity, namely amphiphilicity, and the chitosan is respectively modified by the hydrophilic groups and the hydrophobic groups by adjusting the proportion of the hydrophilic side chains to the hydrophobic side chains, so that the amphiphilicity of the chitosan is controlled in a reasonable range, the amphiphilic chitosan derivative can be obtained, and the application range of the chitosan is favorably expanded. Meanwhile, the medicine can be embedded with fat-soluble medicines, so that the targeting delivery to the focus part is realized, the bioavailability of the medicine is improved to the maximum extent, and the toxic and side effects of the medicine are reduced.
Disclosure of Invention
The invention aims to provide an amphiphilic chitosan derivative and a preparation method thereof, which are used for solving the problems in the prior art, enriching the variety of the chitosan derivative, expanding the application range of chitosan in biomedicine and simultaneously simplifying the preparation method.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides an amphiphilic chitosan derivative, which has a structure shown in a formula I:
the invention also provides a preparation method of the amphiphilic chitosan derivative, which comprises the following steps:
(1)18 beta glycyrrhetinic acid grafted chitosan:
a. mixing chitosan, water and HOBt, stirring, and dissolving chitosan;
b. adding 18 beta glycyrrhetinic acid into the system in the step a, uniformly stirring, adding EDAC & HCl, uniformly stirring, adjusting the pH value of the system to 4.8-5.2, and stirring at room temperature for reaction for 1-12 h;
c. after the reaction is finished, dialyzing the system obtained in the step b in water by using a dialysis bag, and carrying out vacuum drying, washing and vacuum drying on the dialyzed product again to obtain 18 beta glycyrrhetinic acid-chitosan;
(2) sialic acid grafted 18 β glycyrrhetinic acid-chitosan:
d. mixing and stirring the 18 beta glycyrrhetinic acid-chitosan prepared in the step (1) with water and HOBt, and dissolving the 18 beta glycyrrhetinic acid-chitosan for later use;
e. adding sialic acid into the system obtained in the step d, uniformly stirring, adding EDAC & HCl, uniformly stirring, adjusting the pH value of the system to 4.8-5.2, and then stirring at room temperature for reaction for 1-12 hours;
f. and e, after the reaction is finished, dialyzing the system obtained in the step e by using a dialysis bag in water, and drying the dialyzed product in vacuum to obtain a sialic acid-chitosan 18 beta glycyrrhetinic acid grafted product, namely the amphiphilic chitosan derivative.
Further, the concentration of the chitosan in the step a is 5-20mg/ml, and the molar ratio of the chitosan amino group to the HOBt is 2: 1-1: 2.
Further, the molar ratio of chitosan amino groups to HOBt in step a is 1: 1.
Further, the molar ratio of the EDAC & HCl and the 18 beta glycyrrhetinic acid in the step b to the HOBt in the step a is (1-6): 1-3): 1; the reaction time of the step b is 3 h; and (c) adjusting the pH value of the system in the step b by using dilute hydrochloric acid or acidic PBS.
Further, the molar ratio of EDAC & HCl, 18 beta glycyrrhetinic acid in the step b to HOBt in the step a is 4: 2: 1.
Further, the solvent used for washing in step c is one of ethanol, methanol or dichloromethane; the cut-off molecular weight of the dialysis bag in the step c and the step f is 2000-3500Da, and the dialysis time is 48-72 h.
Further, the washing solvent in step c is ethanol; the molecular weight cut-off of the dialysis bag in the steps c and f is 3500Da, and the dialysis time is 72 h.
Further, in the step d, the concentration of the 18 beta glycyrrhetinic acid-chitosan is 5-20mg/mL, and the molar ratio of the 18 beta glycyrrhetinic acid-chitosan amino to the HOBt is 2: 1-1: 2;
the molar ratio of the EDAC & HCl and the sialic acid in the step e to the HOBt in the step d is (1-6): 1-3): 1; adjusting the pH value of the system in the step e by using dilute hydrochloric acid or acidic PBS; the reaction time of step e was 3 h.
Further, the molar ratio of the 8 beta glycyrrhetinic acid-chitosan amino to HOBt in the step d is 1: 1; the molar ratio of the EDAC & HCl and the sialic acid in the step e to the HOBt in the step d is 4: 2: 1.
The invention discloses the following technical effects:
the chitosan which is one of the raw materials used by the SA-CS-GA prepared by the invention is the only natural cationic polysaccharide in the nature, is derived from shrimp and crab shells and the like, and has abundant resources; the SA-CS-GA prepared by the invention has good water solubility, no toxic or side effect on cells and good blood compatibility, and the amphiphilic chitosan derivative has an amphiphilic group, can be used as a carrier of a hydrophobic drug, has good application prospects in the aspects of anti-inflammation, antibacterial and antiviral properties and drug carriers, and has potential application values in the field of biological medicine.
The method has mild reaction conditions, can graft 18 beta glycyrrhetinic acid and sialic acid onto a chitosan molecular chain only in the presence of HOBt and EDAC & HCl, has simple preparation method, short reaction time, mild conditions and high reaction efficiency, is convenient to implement, does not need complex equipment, and is convenient for industrial batch production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a 1H-NMR spectrum of SA-CS-GA synthesized in example 1 of the present invention;
FIG. 2 is an infrared spectrum of SA-CS-GA synthesized in example 1 of the present invention;
FIG. 3 is a graph showing the results of the cytotoxicity test of SA-CS-GA synthesized in example 1 of the present invention;
FIG. 4 is a graph showing the results of the blood compatibility test of SA-CS-GA synthesized in example 1 of the present invention, in which (A) is the hemolysis ratio and (B) is a visual chart.
FIG. 5 is a graph showing the results of the SA-CS-GA anti-inflammatory test synthesized in example 1 of the present invention, wherein (A) is the difference in ear thickness among the different treatment groups and (B) is the difference in ear weight among the different treatment groups.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The amphiphilic chitosan derivative has a structure shown in a formula I:
example 1
A preparation method of an amphiphilic chitosan derivative shown as a formula I comprises the following steps:
(1) synthesis of 18 beta glycyrrhetinic acid grafted chitosan:
a. mixing chitosan (concentration of 20mg/ml), deionized water and 1-hydroxybenzotriazole (HOBt), stirring for 10min to dissolve chitosan; wherein the molar ratio of the chitosan amino group to the HOBt is 1: 1.
b. Slowly adding 18 beta glycyrrhetinic acid into the system in the step a, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 4.8 by using dilute hydrochloric acid, and then stirring and reacting at room temperature for 3 hours;
the molar ratio of the EDAC & HCl and 18 beta glycyrrhetinic acid to the HOBt is 4: 2: 1.
c. After the reaction is finished, dialyzing the product in water by using a dialysis bag (the molecular weight cut-off of the dialysis bag is 3500Da), wherein the dialysis time is 72 h; vacuum drying, washing with ethanol, and vacuum drying to obtain 18 beta glycyrrhetinic acid-chitosan.
(2) Sialic acid grafted 18 beta glycyrrhetinic acid-chitosan
d. Mixing the 18 beta glycyrrhetinic acid-chitosan (the concentration is 5mg/mL) prepared in the step 1 with water and HOBt, stirring for 10min, and dissolving for later use;
the molar ratio of the 18 beta glycyrrhetinic acid-chitosan amino to the HOBt is 1: 1.
e. Slowly adding sialic acid into the system in the step d, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 4.8 by using acidic PBS (phosphate buffer solution), and then stirring and reacting at room temperature for 3 hours;
the ratio of the number of moles of EDAC & HCl and sialic acid added to the number of moles of HOBt in step d was 4: 2: 1.
f. After the reaction is finished, dialyzing the product (the molecular weight cutoff is 3.5kDa) in water by using a dialysis bag, dialyzing for 72 hours, and drying in vacuum to obtain the sialic acid-chitosan 18 beta glycyrrhetinic acid grafted product (SA-CS-GA).
FIG. 1 is a 1H-NMR spectrum of SA-CS-GA synthesized in example 1 of the present invention; FIG. 2 is an infrared spectrum of SA-CS-GA synthesized in example 1 of the present invention.
Example 2
A preparation method of an amphiphilic chitosan derivative shown as a formula I comprises the following steps:
(1) synthesis of 18 beta glycyrrhetinic acid grafted chitosan:
a. mixing chitosan (concentration of 5mg/ml), deionized water and 1-hydroxybenzotriazole (HOBt), stirring for 10min, and dissolving chitosan;
the molar ratio of the chitosan amino group to the HOBt is 1.2: 2.
b. Slowly adding 18 beta glycyrrhetinic acid into the system in the step a, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 5.0 by using dilute hydrochloric acid, and then stirring and reacting at room temperature for 12 hours;
the molar ratio of the molar number of EDAC & HCl and 18 beta glycyrrhetinic acid to the molar number of HOBt is 1: 1.
c. After the reaction is finished, dialyzing the product in water by using a dialysis bag (the cut-off molecular weight of the dialysis bag is 3000Da) for 48 h; vacuum drying, washing with methanol, and vacuum drying to obtain 18 beta glycyrrhetinic acid-chitosan;
(2) sialic acid grafted 18 beta glycyrrhetinic acid-chitosan
d. Mixing the 18 beta glycyrrhetinic acid-chitosan (the concentration is 20mg/mL) prepared in the step 1 with water and HOBt, stirring for 10min, and dissolving for later use;
the molar ratio of the 18 beta glycyrrhetinic acid-chitosan amino to the HOBt is 1.5: 1.
e. Slowly adding sialic acid into the system in the step d, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 5.0 by using acidic PBS (phosphate buffer solution), and then stirring and reacting at room temperature for 12 hours;
the molar ratio of the added EDAC & HCl and sialic acid to the HOBt in the step d is 3: 2: 1.
f. After the reaction is finished, dialyzing the product (the molecular weight cutoff is 3.0kDa) in water by using a dialysis bag, wherein the dialysis time is 48 hours, and drying in vacuum to obtain the sialic acid-chitosan 18 beta glycyrrhetinic acid grafted product.
Example 3
A preparation method of an amphiphilic chitosan derivative shown as a formula I comprises the following steps:
(1) synthesis of 18 beta glycyrrhetinic acid grafted chitosan:
a. mixing chitosan (concentration of 15mg/ml), deionized water and 1-hydroxybenzotriazole (HOBt), stirring for 11min, and dissolving chitosan;
the molar ratio of the chitosan amino group to the HOBt is 2: 1.5;
b. slowly adding 18 beta glycyrrhetinic acid into the system in the step a, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 5.2 by using dilute hydrochloric acid, and then stirring and reacting for 1h at room temperature;
the molar ratio of the molar number of EDAC & HCl and 18 beta glycyrrhetinic acid to the molar number of HOBt is 5: 3: 1.
c. After the reaction is finished, dialyzing the product in water by using a dialysis bag (the cut-off molecular weight of the dialysis bag is 2500Da), wherein the dialysis time is 50 h; vacuum drying, washing with dichloromethane, and vacuum drying to obtain 18 beta glycyrrhetinic acid-chitosan.
(2) Sialic acid grafted 18 beta glycyrrhetinic acid-chitosan
d. Mixing the 18 beta glycyrrhetinic acid-chitosan (the concentration is 15mg/mL) prepared in the step 1 with water and HOBt, stirring for 12min, and dissolving for later use;
the mol ratio of the 18 beta glycyrrhetinic acid-chitosan amino to the HOBt is 1: 2;
e. slowly adding sialic acid into the system in the step d, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 5.2 by using dilute hydrochloric acid, and then stirring and reacting at room temperature for 10 hours;
the molar ratio of the added EDAC & HCl and sialic acid to the HOBt in step d is 5: 3: 1.
f. After the reaction is finished, dialyzing the product (the cut-off molecular weight is 2000Da) in water by using a dialysis bag, wherein the dialysis time is 55h, and drying in vacuum to obtain the sialic acid-chitosan 18 beta glycyrrhetinic acid grafted product.
Example 4
A preparation method of an amphiphilic chitosan derivative shown as a formula I comprises the following steps:
(1) synthesis of 18 beta glycyrrhetinic acid grafted chitosan:
a. mixing chitosan (concentration of 10mg/ml), deionized water and 1-hydroxybenzotriazole (HOBt), stirring for 10min, and dissolving chitosan; the molar ratio of the chitosan amino group to the HOBt is 1.5: 1.
b. Slowly adding 18 beta glycyrrhetinic acid into the system in the step a, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 5.2 by using dilute hydrochloric acid, and then stirring and reacting at room temperature for 6 hours;
the molar ratio of the EDAC & HCl and 18 beta glycyrrhetinic acid to the HOBt is 4: 1.
c. After the reaction is finished, dialyzing the product in water by using a dialysis bag (the molecular weight cut-off of the dialysis bag is 2700Da), wherein the dialysis time is 60 h; vacuum drying, washing with ethanol, and vacuum drying to obtain 18 beta glycyrrhetinic acid-chitosan.
(2) Sialic acid grafted 18 beta glycyrrhetinic acid-chitosan
d. Mixing the 18 beta glycyrrhetinic acid-chitosan (the concentration is 10mg/mL) prepared in the step 1 with water and HOBt, stirring for 10min, and dissolving for later use;
the mol ratio of the 18 beta glycyrrhetinic acid-chitosan amino to the HOBt is 2: 1;
e. slowly adding sialic acid into the system in the step d, uniformly stirring, slowly adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDAC & HCl), uniformly stirring, adjusting the pH value of the system to 5.2 by using acidic PBS (phosphate buffer solution), and then stirring and reacting at room temperature for 6 hours;
the molar ratio of the added EDAC & HCl and sialic acid to the HOBt in the step d is 4: 1.
f. After the reaction is finished, dialyzing the product (the molecular weight cutoff is 2800Da) in water by using a dialysis bag, wherein the dialysis time is 65 hours; vacuum drying to obtain the sialic acid-chitosan 18 beta glycyrrhetinic acid grafted product.
Effect verification:
firstly, cytotoxicity test:
cytotoxicity test was carried out for SA-CS-GA prepared in example 1, according to the following procedure:
after harvesting HaCaT cells, the cell concentration was adjusted at 2X 104The cells were plated at a density of one well per well (100. mu.l per well) in 96-well culture plates, each set of 6 wells, and MTT assay was performed on the cells after completion of the culture. The specific operation steps of MTT detection are as follows: after the cell treatment, 10. mu.l of MTT solution (5mg/mL) was added to each well, incubated at 37 ℃ for 4h, the liquid in each well was carefully removed, 150. mu.L of DMSO was added to each well, and the wells were shaken in the dark for 10-15min, and the absorbance (OD) of each well at a wavelength of 570nm was measured using a full-automatic enzyme calibration apparatus. Cell viability was calculated according to the following formula:
cell survival (%). test (OD)/control (OD) × 100%
The cytotoxicity test results are shown in fig. 3, and it can be seen from fig. 3 that there is no significant difference in the survival rate of the amphiphilic chitosan derivative compared to normal cells, indicating that the synthesized chitosan derivative is not cytotoxic.
II, blood compatibility test:
a blood compatibility test was performed on the SA-CS-GA prepared in example 1, according to the following procedure:
1. centrifuging fresh anticoagulated mouse blood at 2000rpm/min for 15min, discarding supernatant to obtain erythrocyte pellet, washing erythrocyte pellet with 1 XPBS buffer solution (0.1M, pH 7.2-7.4) for several times until supernatant is clear, and diluting erythrocyte pellet with PBS to volume concentration of 2% (v/v);
2. 500 mul of erythrocyte suspension and 500 mul of samples with different concentrations (dissolved by PBS, filtered, sterilized and then sterilized by ultraviolet for 30min) are mixed evenly in a 1.5mL centrifuge tube, the tube is sealed and shaken evenly and then placed in a water bath with 37 ℃ for incubation for 1h, and simultaneously 500 mul of deionized water and 500 mul of PBS are respectively mixed and cultured with 500 mul of erythrocyte suspension to be used as positive and blank controls for 3 times of parallel experiments.
FIG. 4 shows the results of the blood compatibility test, in which (A) is the hemolysis rate and (B) is a visual chart, and it can be seen from FIG. 4 that the SA-CS-GA treated sample showed no significant erythrocyte disruption compared to the water-treated sample, and that the hemolysis rate was < 5% in the concentration range, similar to the PBS-treated sample. The result shows that SA-CS-GA has no damage effect on erythrocytes when the concentration is less than 500 mug/mL, and has good blood compatibility.
Thirdly, testing the acute anti-inflammatory effect:
the acute anti-inflammatory effect test was performed on SA-CS-GA prepared in example 1, and the procedure was as follows:
male ICR mice, 6 weeks old, were raised for 1 week with free access to food and water for acclimation to the experiment and subsequent experiments were started. Mice were divided into 4 groups, normal, model, CS, SA-CS-GA, 8 mice per group. Normal group is not treated and fed normally; the right ear of the other group of mice was treated with TPA acetone solution (5. mu.g/20. mu.L) (3 consecutive days), the left ear was treated with TPA-free pure acetone liquid (20. mu.L), and the right ear of the mice was treated with CS and SA-CS-GA treatment after 1 hour as shown in FIG. 5. On day four, the ears of the mice were photographed and the mice were sacrificed for subsequent testing. The thickness of the right and left mouse ears was measured with a thickness gauge, and then the right and left mouse ear tissues were taken with a 8mm mouse ear punch, and the ear weights were weighed with an analytical balance.
FIG. 5 is a graph of ear thickness difference and ear weight difference of TPA-induced ear swelling in mice. FIG. 5(A) is the difference in ear thickness between the different treatment groups, and the results show that the difference in thickness between the right and left ears of the model group is significantly increased after the right ear of the mice is induced with TPA, compared with the normal group. Compared with the model group, the ear thickness difference of the CS group is not obviously different, while the ear thickness difference of the SA-CS-GA group is obviously reduced, which indicates that the SA-CS-GA has an inhibiting effect on the development of inflammation induced by TPA. Fig. 5(B) shows the difference in ear weight for the different treatment groups, which is similar to the difference in ear weight, and the difference in ear weight for the model group is increased and has a high significance compared to the normal group. Compared with the model group, the difference in ear weight of the CS group was not significantly different, whereas the difference in ear weight of the SA-CS-GA group was significantly reduced. Indicating that SA-CS-GA has anti-inflammatory effects.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. An amphiphilic chitosan derivative, characterized by having the structure shown in formula I:
2. a method for preparing the amphiphilic chitosan derivative of claim 1, comprising the steps of:
(1)18 beta-glycyrrhetinic acid grafted chitosan:
a. mixing chitosan, water and HOBt, stirring, and dissolving chitosan;
b. adding 18 beta glycyrrhetinic acid into the system in the step a, uniformly stirring, adding EDAC & HCl, uniformly stirring, adjusting the pH value of the system to 4.8-5.2, and stirring at room temperature for reaction for 1-12 h;
c. after the reaction is finished, dialyzing the system obtained in the step b in water by using a dialysis bag, and carrying out vacuum drying, washing and vacuum drying on the dialyzed product again to obtain 18 beta glycyrrhetinic acid-chitosan;
(2) sialic acid grafted 18 β glycyrrhetinic acid-chitosan:
d. mixing and stirring the 18 beta glycyrrhetinic acid-chitosan prepared in the step (1) with water and HOBt, and dissolving the 18 beta glycyrrhetinic acid-chitosan for later use;
e. adding sialic acid into the system obtained in the step d, uniformly stirring, adding EDAC & HCl, uniformly stirring, adjusting the pH value of the system to 4.8-5.2, and then stirring at room temperature for reaction for 1-12 hours;
f. and e, after the reaction is finished, dialyzing the system obtained in the step e by using a dialysis bag in water, and drying the dialyzed product in vacuum to obtain a sialic acid-chitosan 18 beta glycyrrhetinic acid grafted product, namely the amphiphilic chitosan derivative.
3. The method of claim 2, wherein the concentration of chitosan in step a is 5-20mg/ml, and the molar ratio of chitosan amino groups to HOBt is 2: 1-1: 2.
4. The method of claim 3, wherein the molar ratio of chitosan amino groups to HOBt in step a is 1: 1.
5. The method for preparing amphiphilic chitosan derivatives according to claim 2, wherein the molar ratio of EDAC-HCl, 18 β glycyrrhetinic acid in step b to HOBt in step a is (1-6): (1-3): 1; the reaction time of the step b is 3 h;
and (c) adjusting the pH value of the system in the step b by using dilute hydrochloric acid or acidic PBS.
6. The method of claim 5, wherein the molar ratio of EDAC-HCl, 18 β glycyrrhetinic acid in step b to HOBt in step a is 4: 2: 1.
7. The method for preparing amphiphilic chitosan derivative according to claim 2, wherein the solvent used for washing in step c is one of ethanol, methanol or dichloromethane;
the cut-off molecular weight of the dialysis bag in the step c and the step f is 2000-3500Da, and the dialysis time is 48-72 h.
8. The method for preparing amphiphilic chitosan derivative according to claim 7, wherein the solvent used for washing in step c is ethanol;
the molecular weight cut-off of the dialysis bag in the steps c and f is 3500Da, and the dialysis time is 72 h.
9. The method for preparing amphiphilic chitosan derivative according to claim 2, wherein the concentration of 18 β glycyrrhetinic acid-chitosan in step d is 5-20mg/mL, and the molar ratio of 18 β glycyrrhetinic acid-chitosan amino group to HOBt is 2: 1-1: 2;
the molar ratio of the EDAC & HCl and the sialic acid in the step e to the HOBt in the step d is (1-6): 1-3): 1;
adjusting the pH value of the system in the step e by using dilute hydrochloric acid or acidic PBS; the reaction time of step e was 3 h.
10. The method for preparing amphiphilic chitosan derivative according to claim 9, wherein the molar ratio of 8 β glycyrrhetinic acid-chitosan amino group to HOBt in step d is 1: 1;
the molar ratio of the EDAC & HCl and the sialic acid in the step e to the HOBt in the step d is 4: 2: 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200637496A (en) * | 2004-12-01 | 2006-11-01 | Univ Hokkaido Nat Univ Corp | Chitosan complex |
| CN102863556A (en) * | 2012-09-27 | 2013-01-09 | 复旦大学附属上海市第五人民医院 | Lactose acidized glycyrrhetinic chitosan material and preparation method and application thereof |
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2021
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| TW200637496A (en) * | 2004-12-01 | 2006-11-01 | Univ Hokkaido Nat Univ Corp | Chitosan complex |
| CN102863556A (en) * | 2012-09-27 | 2013-01-09 | 复旦大学附属上海市第五人民医院 | Lactose acidized glycyrrhetinic chitosan material and preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| "Biodistribution and biocompatibility of glycyrrhetinic acid and galactose-modified chitosan nanoparticles as a novel targeting vehicle for hepatocellular carcinoma";Min Li等;《Nanomedicine》;20191219;第15卷(第2期);第1-17页 * |
| "Grafting of 18β-Glycyrrhetinic Acid and Sialic Acid onto Chitosan to Produce a New Amphipathic Chitosan Derivative：Synthesis, Characterization, and Cytotoxicity";Wei-Yan Quan等;《Molecules》;20210116;第26卷(第452期);第1-12页 * |
| "Preparation and characterization of glycyrrhetinic acid-modified stearic acid-grafted chitosan micelles";Qian Chen等;《Artificial Cells, Nanomedicine, and Biotechnology》;20131004;第43卷(第4期);第217-223页 * |
| "Targeted treatment of CD22-positive non-Hodgkin"s lymphoma with sialic acid – modified chitosan-PLGA hybrid nanoparticles";Haixi Zhang等;《Journal of Nanoparticle Research》;20190606;第21卷(第154期);第1-13页 * |
| "甘草次酸修饰化壳聚糖的制备及表征";赵瑞娟等;《南京中医药大学学报》;20140510;第30卷(第3期);第274-275页 * |
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| CN112694546A (en) | 2021-04-23 |
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Inventor after: Kong Songzhi Inventor after: Quan Weiyan Inventor after: Li Sidong Inventor after: Li Chengpeng Inventor after: Hu Zhang Inventor after: Liu Huazhong Inventor before: Kong Songzhi Inventor before: Quan Fei Inventor before: Li Sidong Inventor before: Li Chengpeng Inventor before: Hu Zhang Inventor before: Liu Huazhong |