CN107412151B - Phytohemagglutinin-polysaccharide hydrogel capable of intelligently regulating and controlling insulin release as well as preparation and application thereof - Google Patents

Phytohemagglutinin-polysaccharide hydrogel capable of intelligently regulating and controlling insulin release as well as preparation and application thereof Download PDF

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CN107412151B
CN107412151B CN201710612832.2A CN201710612832A CN107412151B CN 107412151 B CN107412151 B CN 107412151B CN 201710612832 A CN201710612832 A CN 201710612832A CN 107412151 B CN107412151 B CN 107412151B
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hydrogel
polysaccharide
phytohemagglutinin
insulin
solution
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CN107412151A (en
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林坤华
易菊珍
杨立群
吴昊
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

Abstract

The invention discloses a phytohemagglutinin-polysaccharide hydrogel capable of intelligently regulating and controlling insulin release, and a preparation method and application thereof. The phytohemagglutinin-polysaccharide hydrogel with the porous structure is prepared by taking natural polysaccharide with better biocompatibility and degradability as a raw material through chemical crosslinking with phytohemagglutinin and under the action of a pore-forming agent. The phytohemagglutinin-polysaccharide hydrogel can be used for loading insulin, so that the polysaccharide hydrogel capable of intelligently regulating and controlling the release of the insulin is prepared. The preparation method has mild preparation conditions and safe used raw materials, keeps the specific binding activity of the phytohemagglutinin on glucose molecules and the structural stability and the biological activity of the insulin, and has simple process, and cheap and easily obtained used equipment and raw materials. The polysaccharide hydrogel capable of intelligently regulating and controlling the release of the insulin is expected to be used as an insulin delivery carrier for intelligently regulating and controlling the release of the insulin, and has a wide application prospect in the aspect of treating diabetes.

Description

Phytohemagglutinin-polysaccharide hydrogel capable of intelligently regulating and controlling insulin release as well as preparation and application thereof
Technical Field
The invention belongs to the technical field of biological medicine materials. More particularly, relates to a phytohemagglutinin-polysaccharide hydrogel for intelligently regulating and controlling insulin release, and a preparation method and application thereof.
Background
Diabetes is a disease with high morbidity, and the current treatment of diabetes mainly relies on subcutaneous insulin injection for many times a day, but the method cannot intelligently regulate and control the release of insulin according to the blood glucose level of a patient, so that the insulin needs to be injected for many times, and excessive insulin can cause acute complications of hypoglycemia, and even syncope and shock in severe cases; meanwhile, long-term injection of insulin can cause skin infection, subcutaneous blood stasis and other problems.
In recent years, intelligent materials are widely concerned, namely environment response materials, for example, glucose intelligent materials are materials which can respond to the change of blood sugar concentration in vivo to a certain extent, namely, the materials can release insulin in time according to the change of blood sugar, so that the blood sugar is maintained at a normal level (4-6 mmol/L). The glucose response self-regulation system is a novel idea for treating diabetes, and the key points of the glucose response self-regulation system are an insulin-loaded carrier and a switch for generating response according to blood glucose change.
At present, there are several relatively mature glucose response self-regulation systems, such as glucose oxidase system and phenylboronic acid group system. However, these systems have limitations that result in failure to achieve the desired effect. For example, in a phenylboronic acid group system, the pKa value of the phenylboronic acid group is about 8-9, so that the application of the phenylboronic acid group in a human physiological state is limited; the glucose oxidase system has a problem of delayed insulin release response because it cannot directly receive a change in glucose concentration as a signal. In addition, patent No. 2007101786.6 discloses a glucose-responsive polyphosphazene hydrogel and a method for preparing the same, which can obtain hydrogel systems having different degrees of crosslinking and different water contents by means of increasing the ratio of glucose side groups and selecting suitable second and third substituents, etc., by using the interaction between polyphosphazene containing glucose side groups and phytohemagglutinin Concanavalin a (Con a). Polyphosphazene is used as a novel degradable medical high polymer material and is widely used in recent years, but degradation products of polyphosphazene are not all nontoxic, so that certain potential safety hazards exist; and the preparation process is complicated, the raw materials are complex, and a plurality of violent reaction conditions can influence the activity of the phytohemagglutinin on the glucose, thereby influencing the final use effect.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the phytohemagglutinin-polysaccharide hydrogel for intelligently regulating and controlling the release of insulin. The hydrogel is mild in preparation conditions, safe in use of natural polysaccharide raw materials, simple in process, cheap and easily available in used equipment and raw materials, and keeps the specific binding activity of the phytohemagglutinin on glucose molecules and the structural stability and biological activity of insulin; meanwhile, the hydrogel can be further used for preparing a medicament for intelligently regulating and controlling the release of insulin, and has a certain application prospect in the aspect of treating diabetes.
The invention aims to provide a phytohemagglutinin-polysaccharide hydrogel for intelligently regulating and controlling insulin release.
Another object of the present invention is to provide a method for preparing the lectin-polysaccharide hydrogel.
It is still another object of the present invention to provide use of the lectin-polysaccharide hydrogel.
The above object of the present invention is achieved by the following technical solutions:
a method for preparing lectin-polysaccharide hydrogel capable of intelligently regulating insulin release comprises reacting polysaccharide with acid anhydride to obtain carboxylated polysaccharide derivative; and dripping the carboxylated polysaccharide derivative into the phytohemagglutinin to perform a crosslinking reaction to obtain the phytohemagglutinin-polysaccharide hydrogel.
Specifically, reacting a polysaccharide with an acid anhydride to produce a carboxylated polysaccharide derivative; the carboxylated polysaccharide derivative is then added dropwise to the lectin in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) andNand (3) carrying out a crosslinking reaction under the catalytic action of-hydroxysuccinimide (NHS) to obtain the phytohemagglutinin-polysaccharide hydrogel.
The polysaccharide with better biocompatibility and degradability is selected as a raw material for preparing the hydrogel, the polysaccharide hydrogel intelligently regulating and controlling the release of insulin is prepared by carrying out chemical crosslinking with phytohemagglutinin under the action of a pore-forming agent, and a hydrogel drug delivery system for efficiently delivering drugs is adopted; meanwhile, the phytohemagglutinin with specific binding property to glucose and the polysaccharide are selected to prepare the chemically crosslinked hydrogel through chemical reaction, the hydrogel has good sensitivity to glucose concentration change and good biocompatibility, and degradation products are nontoxic.
Meanwhile, in order to improve the loading efficiency of the hydrogel, the porous phytohemagglutinin-polysaccharide hydrogel containing a porous structure is further prepared; specifically, a pore-forming agent and a carboxylated polysaccharide derivative are simultaneously dripped into the phytohemagglutinin to prepare the porous phytohemagglutinin-polysaccharide hydrogel containing a porous structure.
Preferably, the polysaccharide is natural polysaccharide such as pullulan, amylopectin, guar gum, lentinan, amylose, glycogen or konjac polysaccharide, and the like, and has good biocompatibility and nontoxic degradation products.
Preferably, the lectin is a legume lectin.
More preferably, the legume lectin is black bean lectin, mung bean lectin, sword bean lectin, peanut lectin, chickpea lectin, kidney bean lectin or winged bean lectin.
Preferably, the anhydride is malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride and pimelic anhydride.
Preferably, the pore-forming agent is polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, water-soluble lignin, calcium lactate, and poly-ferric chloride, which are preferably water-soluble, so that they can be easily washed away with water after forming pores in the hydrogel.
Specifically, the preparation method of the phytohemagglutinin-polysaccharide hydrogel comprises the following steps:
s1, according to polysaccharide: organic solvent = 0.1-1 g: adding polysaccharide into an organic solvent according to the proportion of 10-100 mL, and stirring and dissolving at room temperature;
s2, according to the proportion that an anhydride organic solvent = 1-10 mg: adding 1-10 mL of acid anhydride into the organic solvent, and stirring and dissolving at room temperature;
s3, adding the solution obtained in the step S2 into the mixed solution obtained in the step S1, and mixing the mixed solution: organic amine = 11-110 mL: 1-20 mg, dropping organic amine into the mixed solution, and stirring and reacting for 1-24 hours at room temperature; after the reaction is finished, dialyzing the reaction solution, and freeze-drying to obtain a carboxylated polysaccharide derivative;
s4, according to the carboxylated polysaccharide derivative: buffer solution = 0.1-1 g: 1-10 mL, adding the carboxylated polysaccharide derivative into the buffer solution, and stirring at room temperature to dissolve;
s5, according to the plant agglutinin: buffer = 0.1-10 mg: sequentially adding phytohemagglutinin into the buffer solution according to the proportion of 0.1-2 mL, stirring and dissolving, and standing for 1-10 hours at 4 ℃;
s6, according to a pore-foaming agent: buffer = 1-10 mg: adding a pore-foaming agent into the buffer solution in a proportion of 1-10 mL, and stirring and dissolving at room temperature;
s7, dropwise adding the solution obtained in the step S5 and the solution obtained in the step S6 to the solution obtained in the step S4 to obtain a mixed solution, and stirring and reacting at room temperature for 1-10 hours;
s8, according to the formula, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl):N-hydroxysuccinimide (NHS): buffer = 5-50 mg: 1-10 mg: adding EDC & HCl and NHS into the buffer solution according to the proportion of 0.1-1 mL, dropwise adding the solution into the mixed solution obtained in S7, and stirring and reacting for 1-2 hours at room temperature; after obtaining the hydrogel, soaking the hydrogel for a plurality of times by using a buffer solution, then taking out the hydrogel and freeze-drying the hydrogel to obtain the phytohemagglutinin-polysaccharide hydrogel;
the pH of the buffer solution in the steps S4, S5, S6 or S8 is 4-7.
Preferably, the organic solvent is dimethyl sulfoxide,N,N-dimethylformamide, formamide, acetonitrile.
Preferably, the acid anhydride in step S2 is selected from the group consisting of malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride and pimelic anhydride.
Preferably, the organic amines in step S3 are ethylamine, diethylamine and triethylamine.
Preferably, step S5 is performed according to the metal ion: buffer solution: phytohemagglutinin = 0.03-3 mg: 0.1-2 mL: 0.1-10 mg, adding metal ions and phytohemagglutinin into the buffer solution.
The purpose of adding various metal ions in step S5 is to activate the plant lectin and thereby improve the ability of the plant lectin to bind to the glucose-producing property.
Still preferably, the metal ion is one or more of magnesium chloride, calcium chloride or manganese chloride.
More preferably, in the metal ions of S5, the ratio of magnesium chloride: calcium chloride: manganese chloride = 0.01-1 mg: 0.01-1 mg: 0.01-1 mg.
In order to fully dissolve the polysaccharide and improve the degree of polysaccharide crosslinking reaction, the polysaccharide needs to be dissolved in the solution for a certain time to fully extend polysaccharide chain segments; preferably, the room temperature in the steps S1-S8 is 15-40 ℃; the stirring condition is 300-1000 rpm/min for 1-24 hours.
Preferably, in order to maintain the reactivity of the acid anhydride with the hydroxyl groups of the polysaccharide, the acid anhydride is anhydrous acid anhydride in step S2; the organic amine used in step S3 is intended to remove water, a by-product generated during the reaction of the acid anhydride with the hydroxyl groups of the polysaccharide, and to increase the degree of reaction.
Preferably, the temperature of the freeze-drying in step S3 or S8 is 0 to-60 ℃ for 1-24 hours, so as to complete the drying of the polysaccharide derivative at a low temperature, ensure that the activity of the polysaccharide derivative is not changed, and maintain the spatial structure of the hydrogel to the maximum extent.
Step S3 is carried out by dialysis with pure water for removing solvent and unreacted raw materials; preferably, the dialysis in step S3 is performed by using a dialysis bag with a cut-off molecular weight of 500-50000, so as to ensure that the polysaccharide derivative is not dialyzed while removing small molecular impurities, and the dialysis time is 1-5 days.
In the steps S4, S5, S6 or S8, a buffer solution (pH = 4-7) is used as a reaction solvent, so that the conformational change of the protein in the subsequent reaction in which the protein participates is avoided. A buffer solution (pH = 4-7) commonly used in the art can be selected; preferably, the buffer is an acetic acid-sodium acetate buffer (ph 4.0), a citric acid-sodium citrate buffer (ph 4.4), a citric acid-sodium hydroxide-hydrochloric acid buffer (ph 5.3), a disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (ph 7.4), a disodium hydrogenphosphate-potassium dihydrogenphosphate buffer (ph 6.2), a potassium dihydrogenphosphate-sodium hydroxide buffer (ph 6.8), or the like.
In order to obtain the chemically crosslinked hydrogel of the present invention, the chemical reaction of step S8 is to carboxylated-COOH groups of polysaccharide derivative and-NH groups of lectin2Obtaining a chemically cross-linked lectin-polysaccharide hydrogel through amidation reaction under the catalytic action of EDC and NHS; the purpose of soaking the hydrogel for multiple times by using the buffer solution after obtaining the hydrogel is to wash and remove the water-soluble pore-forming agent remained in the hydrogel, and leave holes at the original positions of the pore-forming agent, so that the phytohemagglutinin-polysaccharide hydrogel with a porous structure is obtained.
Meanwhile, the phytohemagglutinin-polysaccharide hydrogel prepared by the preparation method is also within the protection scope of the invention.
The phytohemagglutinin-polysaccharide hydrogel is used as a hydrogel drug delivery system for efficiently delivering drugs, and the phytohemagglutinin has the performance of specifically binding with glucose and has better sensitivity to the change of the glucose concentration, so that the phytohemagglutinin-polysaccharide hydrogel can be further used for preparing drugs for intelligently regulating and controlling insulin. Therefore, the application of the phytohemagglutinin-polysaccharide hydrogel in the preparation of the medicine for treating diabetes is also within the protection scope of the invention.
In addition, the invention also provides a medicament for intelligently regulating and controlling the release of insulin, which is prepared by loading insulin on the lectin-polysaccharide hydrogel.
Specifically, the lectin-polysaccharide hydrogel is soaked in an insulin solution at room temperature (15-40 ℃) to prepare the lectin-polysaccharide hydrogel loaded with insulin.
Preferably, the soaking time is 1-10 hours, and the concentration of the insulin solution is 1-20 mmol/L.
The mechanism for intelligently regulating and controlling the release of insulin by loading the insulin on the polysaccharide hydrogel is as follows: an insulin-containing polysaccharide hydrogel which in a glucose solution causes the hydrogel to expand in volume to release insulin due to the fact that phytohemagglutinin has a higher ability to bind to free glucose molecules than to glucose units on the polysaccharide chain.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a phytohemagglutinin-polysaccharide hydrogel which is mild in preparation conditions, safe in use of raw materials, simple in process, cheap and easily available in used equipment and raw materials, and keeps the specific binding activity of the phytohemagglutinin on glucose molecules and the structural stability and biological activity of insulin.
(2) The insulin-loaded polysaccharide hydrogel prepared by the invention has sensitivity to glucose concentration change, namely, the insulin is intelligently controlled to be released according to the change of the glucose concentration, and the insulin-loaded polysaccharide hydrogel can be expected to be used as an insulin delivery carrier for intelligently controlling and releasing the insulin, and has a larger application prospect in the aspect of diabetes treatment.
Drawings
FIG. 1 is a process flow diagram of the preparation method of the present invention;
FIG. 2 is a reaction mechanism diagram of the production process of the present invention;
FIG. 3 shows NMR spectra of pullulan raw material (a) and carboxylated pullulan derivative (b) in example 1 of the present invention1H NMR）；
FIG. 4 is an infrared (FTIR) spectrum of pullulan raw material, (b) carboxylated pullulan derivative and (c) concanavalin-pullulan hydrogel according to example 1 of the present invention;
FIG. 5 is a graph of the swelling ratio of the non-activated and (b) activated concanavalin-pullulan hydrogels of example 1 of the present invention (solvent PBS (pH =7.4, swelling ratio is defined as the ratio of the weight (mg) added to the hydrogel at swelling equilibrium to the weight (mg) of the gel after lyophilization) in release media of different concentrations of glucose;
FIG. 6 is a graph showing the cumulative release rate of insulin from insulin-loaded activated concanavalin-pullulan hydrogel in glucose release media of different concentrations in example 1 of the present invention (the cumulative release rate is defined as the ratio of the cumulative released insulin weight (mg) of the hydrogel to the loaded insulin weight (mg) of the hydrogel).
Fig. 7 is a graph showing the effect of a pore-forming agent polyethylene glycol (PEG) on the performance of an activated concanavalin-pullulan hydrogel in adsorbing insulin in example 1 of the present invention: (a) an activated concanavalin-pullulan hydrogel to which no PEG was added, (b) an activated concanavalin-pullulan hydrogel to which PEG was added (insulin loading rate is defined as the ratio of the weight of insulin loaded in the hydrogel (μ g) to the weight of the dried hydrogel (mg)).
Detailed Description
The present invention will be further explained with reference to specific examples, which are not intended to limit the present invention in any way. Unless otherwise indicated, the reagents and methods referred to in the examples are those commonly used in the art. Unless otherwise indicated, all reagents referred to in the examples are commercially available.
The invention provides polysaccharide hydrogel for intelligently regulating and controlling insulin release and a preparation method thereof, the process route is shown as figure 1, and the reaction formulas of the synthesis of carboxylated polysaccharide derivatives and the chemical reaction of the carboxylated polysaccharide derivatives and phytohemagglutinin are respectively shown as figure 2 and figure 3. The following examples are specifically illustrative.
Example 1
1. Preparation of insulin-loaded Canavalia gladiata lectin-Pullulan hydrogel
(1) Weighing 0.2 g of pullulan polysaccharide, named PUL, placing in 20 ml of dimethyl sulfoxide, and dissolving and stirring for 2 hours at 15 ℃;
(2) weighing 3mg succinic anhydride, placing in 1ml dimethyl sulfoxide, and dissolving for 4 hours at 15 ℃;
(3) and (3) adding the solution obtained in the step (2) into the mixed solution obtained in the step (1), then dropwise adding 16 mg of triethylamine into the mixed solution, and stirring and reacting for 1 hour at 15 ℃. After the reaction is finished, dialyzing the reaction solution by using distilled water of a dialysis bag with the interception molecular weight of 1,000, and freeze-drying at the temperature of minus 20 ℃ to obtain a carboxylated polysaccharide derivative, which is named as CPUL;
(4) weighing 0.3 g of CPUL obtained in the step (3), dissolving the CPUL in 2ml of acetic acid-sodium acetate buffer solution (pH4.0), and stirring and dissolving the CPUL at 15 ℃ for 12 hours;
(5) weighing 0.01 mg of magnesium chloride, 0.01 mg of calcium chloride and 0.01 mg of manganese chloride in 0.1ml of acetic acid-sodium acetate buffer solution (pH 4.0), weighing 0.1 mg of concanavalin, adding into the solution, stirring for dissolving, and placing in a refrigerator at 4 ℃ for 8 hours;
(6) weighing 2 mg of polyethylene glycol, dissolving in 2ml of acetic acid-sodium acetate buffer solution (pH4.0), and stirring at 15 deg.C for dissolving for 10 hr;
(7) and (3) dropwise adding the solution obtained in the step (5) and the solution obtained in the step (6) into the solution obtained in the step (4) to obtain a mixed solution, fully contacting the solution and the mixed solution, and stirring and reacting for 10 hours at 15 ℃.
(8) 10mg of EDC. HCl and 10mg of NHS were weighed, dissolved in 0.1mL of acetic acid-sodium acetate buffer (pH 4.0), added dropwise to the mixture obtained in (7), stirred, and reacted at 15 ℃ for 1 hour. After obtaining the hydrogel, soaking the hydrogel for 12 hours in acetic acid-sodium acetate buffer solution (pH 4.0) for many times, then taking out the hydrogel, and freeze-drying the hydrogel at the temperature of-20 ℃ to obtain the porous sword bean agglutinin-pullulan hydrogel.
(9) Soaking the porous sword bean agglutinin-polysaccharide hydrogel in the step (8) in an insulin solution at 15 ℃ to prepare the insulin-loaded sword bean agglutinin-pullulan hydrogel.
2. Results
(1) FIG. 3 shows the pullulan raw material and the carboxylated pullulan derivative1H NMR spectrum. Comparison of pullulan feedstock1H NMR spectrum (FIG. 3 a), of carboxylated polysaccharide derivatives1In the H NMR spectrum (fig. 3 b), the signal peak (peak a) of H1 on the pullulan chain shifts in the high field direction, indicating that the chemical environment of the carboxylated pullulan derivative H1 changes. And two-CHs appear in FIG. 3b2Signal peaks of the groups, namely the b peak (2.62 ppm) and the c peak (2.44 ppm), which are respectively assigned to the two-CH groups to which the carboxylated pullulan derivative is attached to the-COOH group2-a group. Therefore, the temperature of the molten metal is controlled,1the H NMR method further proves that the carboxylated pullulan derivative is synthesized. Further through1H NMR integration method for H1 peak (peak a) and two-CH groups of carboxylated polysaccharide derivative2The integration of the-group peaks (b and c peaks) was carried out, and the degree of substitution of-COOH groups in the carboxylated polysaccharide derivative was calculated to be 0.2 according to formula (1). Definition of degree of substitution: the number of-COOH groups per glucose unit on average in the carboxylated polysaccharide derivative.
DS=N -COOH /N Glc=I b,c/(4I a)（1）
Wherein DS is the degree of substitution of the-COOH group in the carboxylated polysaccharide derivative, N-COOHIs the number of-COOH groups,N Glcis the number of glucose units that are present,I b,candI aare respectively b andthe sum of the integrated areas of the c peaks and the integrated area of the a peak.
(2) Fig. 4 is an FTIR spectrum of pullulan raw material, carboxylated polysaccharide derivative and concanavalin-pullulan hydrogel. Comparison of FTIR spectra of pullulan starting material (FIG. 4 a) and carboxylated polysaccharide derivative (FIG. 4 b) at 1723cm-1The characteristic absorption peak of-COOH group appears, which indicates that the carboxylated polysaccharide derivative is synthesized. The absorption peak disappeared in the FTIR spectrum of the concanavalin-pullulan hydrogel (FIG. 4 c), indicating that the-COOH group of the carboxylated polysaccharide derivative and the-NH group of the concanavalin2A reaction takes place.
(3) Fig. 5 is a graph showing the swelling ratio of unactivated and activated concanavalin-pullulan hydrogel in release media of glucose with different concentrations, and the hydrogel prepared by concanavalin after being activated by calcium ions and manganese ions shows higher swelling ratio, which shows that the activated concanavalin-pullulan hydrogel has the property of specific binding to glucose.
(4) FIG. 6 is a graph of cumulative release rate of insulin from insulin-loaded activated concanavalin-pullulan hydrogel in release media of varying concentrations of glucose. It can be seen that the cumulative release rate of insulin in the glucose solution medium is higher than that in the PBS solution, and the cumulative release rate of insulin increases with the increase of the glucose concentration in the release medium. These results indicate that the hydrogel exhibits better properties for intelligently regulating insulin release according to changes in glucose concentration.
(6) Fig. 7 is a graph of the performance of the activated concanavalin-pullulan hydrogel without or with the addition of the pore-forming agent PEG in absorbing insulin, and the hydrogel with the addition of PEG shows a higher insulin loading rate (fig. 7 b) compared with the hydrogel without the addition of PEG (fig. 7 a), indicating that the addition of PEG contributes to the improvement of the performance of the hydrogel in loading insulin.
Example 2
1. Preparation of insulin-loaded peanut lectin-amylopectin hydrogel
(1) 0.5 g of amylopectin, named AMYP, is weighed out and placed at 50 mmLifting of wineN,N-dissolving in dimethylformamide, stirring at 30 ℃ for 24 hours;
(2) 5 mg of malonic anhydride are weighed and placed in 5mlN,N-dissolution in dimethylformamide at 30 ℃ for 24 hours;
(3) and (3) adding the solution obtained in the step (2) into the mixed solution obtained in the step (1), dropwise adding 10mg of diethylamine into the mixed solution, and stirring and reacting at 30 ℃ for 24 hours. After the reaction is finished, dialyzing the reaction solution by using dialysis bag distilled water with the cutoff molecular weight of 50,000, and freeze-drying at the temperature of minus 30 ℃ to obtain a carboxylated polysaccharide derivative, which is named as CAMYP;
(4) weighing 0.5 g of CAMYP obtained in the step (3), dissolving in 5ml of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.4), and dissolving at 30 ℃ for 24 hours;
(5) weighing 0.04 mg of magnesium chloride, 0.04 mg of calcium chloride and 0.05 mg of manganese chloride in 1ml of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.4), weighing 2 mg of peanut agglutinin, adding into the above solution, stirring for dissolving, and placing in a refrigerator at 4 deg.C for 4 hr;
(6) weighing 5 mg of polyvinyl alcohol, dissolving in 5ml of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.4), and stirring and dissolving at 15 ℃ for 24 hours;
(7) and (4) dropwise adding the solution obtained in the step (5) and the solution obtained in the step (6) into the solution obtained in the step (4) to obtain a mixed solution, fully contacting the solution and the mixed solution, and reacting for 5 hours at 30 ℃.
(8) 20mg of EDC. HCl and 5 mg of NHS were weighed, dissolved in 0.5mL of disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH 7.4), added dropwise to the mixture obtained in (7), stirred, and reacted at 30 ℃ for 1.5 hours. After obtaining the hydrogel, soaking the peanut skin in a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH 7.4) for 24 hours for many times, and then taking out the hydrogel to be frozen and dried at the temperature of-30 ℃ to obtain the porous peanut agglutinin-amylopectin hydrogel.
(9) And (3) soaking the porous peanut agglutinin-polysaccharide hydrogel obtained in the step (8) in an insulin solution at the temperature of 30 ℃ to prepare the peanut agglutinin-amylopectin hydrogel loaded with insulin.
2. The result shows that the obtained peanut agglutinin-amylopectin hydrogel has specific binding performance to glucose, and the hydrogel shows better performance of intelligently regulating and controlling insulin release according to the change of glucose concentration.
Example 3
1. Preparation of insulin-loaded chickpea lectin-guar gum gel
(1) Weighing 0.8 g of guar gum, named as GUA, placing the guar gum in 40 ml of formamide, and stirring and dissolving for 15 hours at 35 ℃;
(2) weighing 10mg succinic anhydride, placing in 10ml formamide, and dissolving for 12 hours at 35 ℃;
(3) and (3) adding the solution obtained in the step (2) into the mixed solution obtained in the step (1), dropwise adding 15 mg of triethylamine into the mixed solution, and reacting at 35 ℃ for 12 hours. After the reaction is finished, dialyzing the reaction solution by using distilled water of a dialysis bag with the cutoff molecular weight of 10,000, and freeze-drying at the temperature of minus 60 ℃ to obtain a carboxylated polysaccharide derivative, which is named as CGUA;
(4) weighing 0.1 g of CGUA obtained in the step (3), dissolving in 1ml of citric acid-sodium hydroxide-hydrochloric acid buffer solution (pH5.3), and stirring at 35 ℃ for dissolving for 10 hours;
(5) weighing 0.05 mg of magnesium chloride, 0.05 mg of calcium chloride and 0.05 mg of manganese chloride in 0.2ml of citric acid-sodium hydroxide-hydrochloric acid buffer solution (pH 5.3), weighing 10mg of chickpea agglutinin, adding the solution, stirring to dissolve, and placing in a refrigerator at 4 ℃ for 6 hours;
(6) weighing 10mg of polyvinylpyrrolidone, dissolving in 10ml of citric acid-sodium hydroxide-hydrochloric acid buffer solution (pH 5.3), and stirring at 35 deg.C for dissolving for 12 hr;
(7) and (3) dropwise adding the solution obtained in the step (5) and the solution obtained in the step (6) into the solution obtained in the step (4) to obtain a mixed solution, fully contacting the solution and the mixed solution, and stirring and reacting at 35 ℃ for 8 hours.
(8) 5 mg of EDC. HCl and 6 mg of NHS were weighed, dissolved in 0.4mL of citric acid-sodium hydroxide-hydrochloric acid buffer (pH 5.3), added dropwise to the mixture obtained in (7), stirred, and reacted at 35 ℃ for 1.2 hours. After obtaining the hydrogel, soaking the hydrogel for multiple times by using citric acid-sodium hydroxide-hydrochloric acid buffer solution (pH 5.3), wherein the soaking time is 10 hours each time, and then taking out the hydrogel to be frozen and dried at 60 ℃ to prepare the porous chickpea agglutinin-guar gum gel.
(9) Soaking the porous chick pea agglutinin-polysaccharide hydrogel in the step (8) in an insulin solution at 35 ℃ to prepare the chick pea agglutinin-guar gum water gel loaded with insulin.
2. The result shows that the obtained chickpea agglutinin-guar gum water gel has the specific binding performance to glucose, and the hydrogel shows better performance of intelligently regulating and controlling the release of insulin according to the change of the concentration of the glucose.
Example 4
1. Preparation of insulin-loaded black bean lectin-lentinan hydrogel
(1) Weighing 0.1 g of lentinan, named as LEN, placing in 10ml of acetonitrile, and stirring at 20 ℃ for dissolving for 10 hours;
(2) weighing 8 mg of glutaric anhydride, placing the glutaric anhydride in 2ml of acetonitrile, and dissolving the glutaric anhydride for 10 hours at 20 ℃;
(3) and (3) adding the solution obtained in the step (2) into the mixed solution obtained in the step (1), dropwise adding 20mg of diethylamine into the mixed solution, and stirring and reacting for 10 hours at 20 ℃. After the reaction is finished, dialyzing the reaction solution by using distilled water of a dialysis bag with the cut-off molecular weight of 20,000, and freeze-drying at the temperature of minus 50 ℃ to obtain a carboxylated polysaccharide derivative, which is named as CLEN;
(4) weighing 0.4 g of CLEN obtained in the step (3), dissolving in 5ml of citric acid-sodium citrate buffer solution (pH4.4), and stirring at 20 ℃ for dissolving for 15 hours;
(5) weighing 1mg of magnesium chloride, 1mg of calcium chloride and 0.04 mg of manganese chloride in 2ml of citric acid-sodium citrate buffer solution (pH 4.4), weighing 5 mg of black bean agglutinin, adding into the solution, stirring for dissolving, and placing in a refrigerator at 4 ℃ for 5 hours;
(6) weighing 8 mg of water-soluble lignin, dissolving in 7 ml of citric acid-sodium citrate buffer solution (pH4.4), and dissolving at 20 deg.C for 15 hr;
(7) and (3) dropwise adding the solution obtained in the step (5) and the solution obtained in the step (6) into the solution obtained in the step (4) to obtain a mixed solution, fully contacting the solution and the mixed solution, and stirring and reacting for 1 hour at 20 ℃.
(8) 4 mg of EDC. HCl and 8 mg of NHS were weighed, dissolved in 0.2mL of citric acid-sodium citrate buffer (pH 4.4), added dropwise to the mixture obtained in (7), stirred, and reacted at 20 ℃ for 0.5 hour. After obtaining the hydrogel, soaking the black bean in citric acid-sodium citrate buffer solution (pH 4.4) for multiple times, wherein the soaking time is 8 hours each time, and then taking out the hydrogel to be frozen and dried at 50 ℃ to obtain the porous black bean agglutinin-lentinan hydrogel.
(9) And (3) soaking the porous black bean agglutinin-polysaccharide hydrogel obtained in the step (8) in an insulin solution at the temperature of 20 ℃ to prepare the black bean agglutinin-lentinan hydrogel loaded with insulin.
2. The result shows that the obtained black bean agglutinin-lentinan hydrogel has specific binding performance to glucose, and the hydrogel shows better performance of intelligently regulating and controlling insulin release according to the change of glucose concentration.
Example 5
1. Preparation of insulin-loaded Tetragonopsis tetragonoloba lectin-amylose hydrogel
(1) Weighing 0.4 g of amylose, named as AMY, placing the amylose in 60 ml of dimethyl sulfoxide, and dissolving the amylose for 16 hours at 25 ℃;
(2) weighing 9 mg of adipic anhydride, placing the adipic anhydride in 8 ml of dimethyl sulfoxide, and dissolving the adipic anhydride for 16 hours at 25 ℃;
(3) and (3) adding the solution obtained in the step (2) into the mixed solution obtained in the step (1), then dropwise adding 12 mg of ethylamine into the mixed solution, and stirring and reacting for 8 hours at 25 ℃. After the reaction is finished, dialyzing the reaction solution by using distilled water of a dialysis bag with the cutoff molecular weight of 40,000, and freeze-drying at the temperature of minus 40 ℃ to obtain a carboxylated polysaccharide derivative, which is named as CAMY;
(4) weighing 0.6 g of CAMY obtained in the step (3), dissolving in 6ml of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.2), and stirring at 25 ℃ for dissolving for 14 hours;
(5) weighing 0.08 mg of magnesium chloride, 0.08 mg of calcium chloride and 0.08 mg of manganese chloride in 0.7 ml of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.2), weighing 6 mg of winged bean agglutinin, adding into the above solution, stirring for dissolving, and placing in a refrigerator at 4 deg.C for 1 hr;
(6) weighing 1mg of calcium lactate, dissolving in 2ml of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.2), and stirring at 25 deg.C for dissolving for 9 hr;
(7) and (3) dropwise adding the solution obtained in the step (5) and the solution obtained in the step (6) into the solution obtained in the step (4) to obtain a mixed solution, fully contacting the solution and the mixed solution, and stirring and reacting at 25 ℃ for 7 hours.
(8) 50mg of EDC. HCl and 1mg of NHS were weighed, dissolved in 1mL of disodium hydrogenphosphate-potassium dihydrogenphosphate buffer (pH 6.2), added dropwise to the mixture obtained in (7), stirred, and reacted at 25 ℃ for 2 hours. After obtaining the hydrogel, soaking the hydrogel for multiple times by using a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 6.2), wherein the soaking time is 15 hours each time, and freeze-drying the hydrogel at the temperature of minus 40 ℃ to obtain the polyporus tetragon agglutinin-amylose hydrogel.
(9) Soaking the polyporus tetragon agglutinin-polysaccharide hydrogel in the step (8) in an insulin solution at 25 ℃ to prepare the insulin-loaded polyporus tetragon agglutinin-amylose hydrogel.
2. The result shows that the obtained tetragon bean agglutinin-amylose hydrogel has the specific binding performance to glucose, and the hydrogel shows better performance of intelligently regulating and controlling the release of insulin according to the change of the concentration of the glucose.
Example 6
1. Preparation of insulin-loaded mung bean agglutinin-konjac polysaccharide hydrogel
(1) Weighing 1g of konjac polysaccharide, named as KJC, placing the konjac polysaccharide in 100ml of dimethyl sulfoxide, and stirring and dissolving the konjac polysaccharide for 1 hour at 40 ℃;
(2) weighing 1mg of pimelic anhydride, placing in 6ml of dimethyl sulfoxide, and dissolving for 1 hour at 40 ℃;
(3) adding the solution obtained in the step (2) into the mixed solution obtained in the step (1), dropwise adding 17 mg of ethylamine into the mixed solution, and reacting at 40 ℃ for 15 hours. After the reaction is finished, dialyzing the reaction solution by using distilled water of a dialysis bag with the cut-off molecular weight of 500, and freeze-drying at 0 ℃ to obtain a carboxylated polysaccharide derivative, namely CKJC;
(4) weighing 1g of CKJC obtained in the step (3), dissolving the CKJC in 10ml of potassium dihydrogen phosphate-sodium hydroxide buffer solution (pH 6.8) and dissolving the CKJC for 1 hour at the temperature of 40 ℃;
(5) weighing 1mg of magnesium chloride, 1mg of calcium chloride and 1mg of manganese chloride in 0.5ml of potassium dihydrogen phosphate-sodium hydroxide buffer solution (pH 6.8), weighing 8 mg of mung bean agglutinin, adding into the solution, stirring for dissolving, and placing in a refrigerator at 4 ℃ for 10 hours;
(6) weighing 9 mg of polyferric chloride, dissolving in 1ml of potassium dihydrogen phosphate-sodium hydroxide buffer solution (pH 6.8), and dissolving at 40 ℃ for 1 hour;
(7) and (4) dropwise adding the solution obtained in the step (5) and the solution obtained in the step (6) into the solution obtained in the step (4) to obtain a mixed solution, fully contacting the solution and the mixed solution, and reacting for 6 hours at 40 ℃.
(8) 30 mg of EDC. HCl and 9 mg of NHS were weighed, dissolved in 0.6mL of potassium dihydrogen phosphate-sodium hydroxide buffer (pH 6.8), added dropwise to the mixture obtained in (7), stirred, and reacted at 40 ℃ for 0.5 hour. After obtaining the hydrogel, soaking the hydrogel for 1 hour in potassium dihydrogen phosphate-sodium hydroxide buffer solution (pH 6.8) for several times, taking out the hydrogel, and freeze-drying at 0 deg.C to obtain the porous mung bean agglutinin-konjac polysaccharide hydrogel.
(9) Soaking the porous mung bean agglutinin-polysaccharide hydrogel in the step (8) in an insulin solution at 40 ℃ to prepare the mung bean agglutinin-konjac polysaccharide hydrogel loaded with insulin.
2. The result shows that the mung bean agglutinin-konjac polysaccharide hydrogel has specific binding performance on glucose, and the hydrogel shows better performance of intelligently regulating and controlling insulin release according to the change of glucose concentration.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (7)

1. A preparation method of phytohemagglutinin-polysaccharide hydrogel intelligently regulating and controlling insulin release is characterized in that polysaccharide reacts with acid anhydride to generate carboxylated polysaccharide derivatives; simultaneously dripping a pore-foaming agent and a carboxylated polysaccharide derivative into the phytohemagglutinin, and carrying out a crosslinking reaction under the catalytic action of 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to prepare the porous phytohemagglutinin-polysaccharide hydrogel containing a porous structure; the polysaccharide is pullulan, amylopectin, guar gum, lentinan, amylose and konjak; the plant agglutinin is semen Sojae Atricolor agglutinin, semen Phaseoli Radiati agglutinin, semen Canavaliae agglutinin, semen Arachidis Hypogaeae agglutinin, semen Ciceris Arietini agglutinin, and semen Strychni agglutinin.
2. The preparation method according to claim 1, which specifically comprises the steps of:
s1, according to polysaccharide: 0.1-1 g of organic solvent: adding polysaccharide into an organic solvent according to the proportion of 10-100 mL, and stirring and dissolving at room temperature;
s2, according to anhydride: 1-10 mg of organic solvent: adding 1-10 mL of acid anhydride into the organic solvent, and stirring and dissolving at room temperature;
s3, adding the solution obtained in the step S2 into the mixed solution obtained in the step S1, and mixing the mixed solution: organic amine is 11-110 mL: 1-20 mg, dropping organic amine into the mixed solution, and stirring and reacting for 1-24 hours at room temperature; after the reaction is finished, dialyzing the reaction solution, and freeze-drying to obtain a carboxylated polysaccharide derivative;
s4, according to the carboxylated polysaccharide derivative: 0.1-1 g of buffer solution: 1-10 mL, adding the carboxylated polysaccharide derivative into the buffer solution, and stirring at room temperature to dissolve;
s5, according to the plant agglutinin: 0.1-10 mg of buffer solution: sequentially adding phytohemagglutinin into the buffer solution according to the proportion of 0.1-2 mL, stirring and dissolving, and standing for 1-10 hours at 4 ℃;
s6, according to a pore-foaming agent: 1-10 mg of buffer solution: adding a pore-foaming agent into the buffer solution in a proportion of 1-10 mL, and stirring and dissolving at room temperature;
s7, dropwise adding the solution obtained in the step S5 and the solution obtained in the step S6 to the solution obtained in the step S4 to obtain a mixed solution, and stirring and reacting at room temperature for 1-10 hours;
s8, according to EDC & HCl: NHS: 5-50 mg of buffer solution: 1-10 mg: adding EDC & HCl and NHS into the buffer solution according to the proportion of 0.1-1 mL, dropwise adding the solution into the mixed solution obtained in S7, and stirring and reacting for 1-2 hours at room temperature; after obtaining the hydrogel, soaking the hydrogel for a plurality of times by using a buffer solution, then taking out the hydrogel and freeze-drying the hydrogel to obtain the phytohemagglutinin-polysaccharide hydrogel;
the pH of the buffer solution in the steps S4, S5, S6 or S8 is 4-7.
3. The production method according to claim 2, wherein the organic solvent is dimethyl sulfoxide, N-dimethylformamide, formamide or acetonitrile; the acid anhydride in the step S2 is malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride or pimelic anhydride; and the organic amine in the step S3 is ethylamine, diethylamine or triethylamine.
4. The method of claim 2, wherein the step S5 is performed according to the ratio of metal ions: buffer solution: phytohemagglutinin is 0.03-3 mg: 0.1-2 mL: 0.1-10 mg, adding metal ions and phytohemagglutinin into the buffer solution.
5. A lectin-polysaccharide hydrogel produced by the production method according to any one of claims 1 to 4.
6. The use of the lectin-polysaccharide hydrogel of claim 5 in the preparation of a medicament for the treatment of diabetes.
7. A pharmaceutical preparation for intelligently regulating insulin release, which is prepared by loading insulin on the lectin-polysaccharide hydrogel of claim 5.
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