CN111437381B - Hydrogel particles and preparation method thereof - Google Patents
Hydrogel particles and preparation method thereof Download PDFInfo
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- CN111437381B CN111437381B CN202010090592.6A CN202010090592A CN111437381B CN 111437381 B CN111437381 B CN 111437381B CN 202010090592 A CN202010090592 A CN 202010090592A CN 111437381 B CN111437381 B CN 111437381B
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- carboxymethyl
- cyclodextrin
- beta
- carboxymethyl chitosan
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 73
- 239000002245 particle Substances 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 92
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 7
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- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
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Classifications
-
- 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
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
Abstract
The invention discloses hydrogel particles and a preparation method thereof. The preparation method comprises the following steps: 1) Preparing carboxymethyl-beta-cyclodextrin solution; 2) Preparing carboxymethyl chitosan solution; 3) Preparing carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles. The hydrogel particles obtained by grafting and crosslinking carboxymethyl-beta-cyclodextrin and carboxymethyl chitosan mainly use two raw materials of carboxymethyl-beta-cyclodextrin and carboxymethyl chitosan, and the two raw materials are cheap and easy to obtain, so that the use of high-cost raw materials is avoided; the preparation steps are simple, the preparation process is safe and easy to operate; the hydrogel particles have almost no cytotoxicity and good biocompatibility, can be used for oral administration and gastric lavage, have no toxic or side effect, have good slow release effect after insulin is loaded, can avoid the damage and degradation of gastric acid and pepsin to insulin, can control the release of the medicine, prolong the curative effect of the medicine, keep the activity of the medicine unchanged and improve the bioavailability of the oral insulin protein medicine.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles and a method for preparing the hydrogel particles.
Background
Diabetes is one of the most common chronic diseases in modern people's lives and is difficult to radically cure, and insulin has been the main treatment mode. However, long-term injection is easy to cause side effects such as allergy, hypoglycemia, infection, subcutaneous fat atrophy and the like, which brings a lot of trouble and inconvenience to diabetics who need to take medicines for life. Oral administration is convenient and easy to accept by patients, so oral insulin is certainly an ideal and best treatment mode. At present, the oral insulin protein administration needs to overcome the problems of physiological disorder, enzyme disorder, physical and chemical stability and the like, so insulin needs to be prepared into a sustained and controlled release preparation by means of a carrier material with good biocompatibility and no cytotoxicity, thus not only avoiding the damage and degradation of gastric acid and pepsin to insulin, improving the bioavailability of insulin, but also delaying the action time of insulin in vivo and reducing the administration times.
The polysaccharide has the advantages of high stability, safety, no toxicity, hydrophilicity, biodegradability and the like, and has wide sources and low cost. The polysaccharide hydrogel is an important aspect of polysaccharide utilization, can be used as a slow release carrier, and has very broad application prospect.
Carboxymethyl chitosan is a water-soluble derivative of chitosan, has good biocompatibility, film forming property, adsorptivity, hydrophilicity, modifier and low physiological toxicity, is rich in storage and low in cost, and can wrap insulin and have good targeting property, so that the chitosan islet element material can avoid decomposition of insulin by digestive tract protease to a certain extent. Carboxymethyl-beta-cyclodextrin has the capacity of generating inclusion complex by a hydrophobic cavity, has low solubility in a solution with low pH, can be dissolved in any concentration due to carboxyl dissociation in a neutral and alkaline solution, has loose structure and obviously improves self-flowability. Therefore, the preparation has good solubility in the environment of pH6-7 in human intestinal tracts, can improve the stability and bioavailability of the medicine, and is used for slow release of the medicine and formulation improvement.
Disclosure of Invention
The invention discloses hydrogel particles used as carriers of oral insulin, which have a porous structure and an amorphous structure, and have good heat stability and slow release performance; and has better biocompatibility, can effectively encapsulate insulin drugs and resist the destructive effect of gastric acid and protect gastric mucosa from being stimulated by the drugs, thereby controlling the release of the drugs, prolonging the curative effect of the drugs and maintaining the activity of the drugs.
The preparation method of the hydrogel particles mainly comprises the following steps:
1) Preparing carboxymethyl-beta-cyclodextrin solution: dissolving carboxymethyl-beta-cyclodextrin in deionized water to obtain carboxymethyl cyclodextrin solution;
2) Preparing carboxymethyl chitosan solution: dissolving carboxymethyl chitosan in deionized water to obtain carboxymethyl chitosan solution;
3) Preparing carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles: mixing carboxymethyl-beta-cyclodextrin solution and carboxymethyl chitosan solution, adding proper amount of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to carry out graft crosslinking, and fully stirring for 24 hours at 25 ℃ to obtain grafted product solution; precipitating the grafted product solution with methanol, and filtering to obtain a precipitate; swelling the precipitate with distilled water, dialyzing with deionized water, precipitating with methanol, freeze drying, sieving with 20 mesh sieve, and collecting to obtain solid granular hydrogel.
Wherein, the molar ratio of the carboxymethyl-beta-cyclodextrin to the carboxymethyl chitosan to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to the N-hydroxysuccinimide is 3.3:17:19.2:19.2.
Further, each gram of carboxymethyl- β -cyclodextrin was dissolved in 20mL deionized water. Each gram of carboxymethyl chitosan was dissolved in 25mL deionized water. Preferably, the purity of the carboxymethyl-beta-cyclodextrin is more than or equal to 99.3%, and the carboxylation degree of the carboxymethyl chitosan is more than or equal to 80%.
Further, the mass of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride was 3.68g. The mass of the N-N-hydroxysuccinimide is 2.21g.
The carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles prepared by the method can be used for embedding and oral delivery of insulin. The carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles have a porous structure and belong to an amorphous structure. The porous structure is favorable for permeation of water molecules and storage and retention of drug molecules. Structural characteristics show that the swelling performance is good, and the thermal stability is good; after embedding insulin, the carboxymethyl chitosan hydrogel particles grafted with carboxymethyl-beta-cyclodextrin simulate human gastrointestinal tract environment, so that the insulin-carrying hydrogel particles can avoid the damage and degradation of gastric acid and pepsin to insulin, and keep the bioactivity of insulin unchanged. The cell survival rate of the hydrogel solution (12.5, 25, 50, 100, 200, 400, 800, 1600 mug/mL) cultured with different concentrations reaches more than 96%, and the hydrogel solution has no cytotoxicity; the insulin-carrying hydrogel particles (50, 75, 100 IU/kg) can maintain the low blood sugar level for 6-10h after being taken orally by the diabetes mice, and the insulin-carrying hydrogel particles have good and durable hypoglycemic activity effect on a type 2 diabetes mouse model.
Compared with the prior art, the hydrogel particles obtained by grafting and crosslinking the carboxymethyl-beta-cyclodextrin and the carboxymethyl chitosan have the following advantages:
(a) The invention mainly uses two raw materials of carboxymethyl-beta-cyclodextrin and carboxymethyl chitosan, and the two raw materials are cheap and easy to obtain, thereby avoiding the use of high-cost raw materials, having economic rationality and saving cost.
(b) The hydrogel particles have simple preparation steps, safe preparation process and easy operation.
(c) The hydrogel particles of the invention have almost no cytotoxicity and good biocompatibility, can be used for oral gastric lavage, have no toxic or side effect, and can be widely used for oral drug delivery.
(d) The hydrogel particles of the invention have good slow release effect after insulin is loaded, can avoid the damage and degradation of gastric acid and pepsin to insulin, can control the release of the medicine, prolong the curative effect of the medicine, keep the activity of the medicine unchanged and improve the bioavailability of the oral insulin protein medicine.
Drawings
FIG. 1 is a flow chart of a process for preparing carboxymethyl chitosan hydrogel microparticles grafted with carboxymethyl-beta-cyclodextrin;
FIG. 2 is a Fourier infrared analysis chart of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles;
FIG. 3 is an X-ray single crystal diffraction pattern of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles;
FIG. 4 is a scanning electron microscope image of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles;
FIG. 5 is a thermogravimetric analysis of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles;
FIG. 6 is a graph of swelling ratio of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles;
FIG. 7 is a graph showing the cumulative release of insulin from carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles loaded with insulin;
FIG. 8 is a graph showing fluorescence spectra of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles after release of insulin;
FIG. 9 is a circular dichroism spectrum of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles after release of insulin;
FIG. 10 is a graph of cytotoxicity of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles;
FIG. 11 is a graph showing the effect of reducing blood glucose activity of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles loaded with glucagon.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
Example 1 carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel microparticles were prepared.
The process flow is shown in fig. 1, and mainly comprises the following steps:
(1) Preparing carboxymethyl-beta-cyclodextrin solution: 4.0g of carboxymethyl-beta-cyclodextrin is weighed and dissolved in 80mL of deionized water to prepare carboxymethyl-beta-cyclodextrin solution;
(2) Preparing carboxymethyl chitosan solution: weighing 4.0g of carboxymethyl chitosan, and dissolving the carboxymethyl chitosan in 100 mL deionized water to prepare carboxymethyl chitosan solution;
(3) Preparing carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles: mixing carboxymethyl chitosan solution and carboxymethyl-beta-cyclodextrin solution, adding 3.68g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2.21g of N-N-hydroxysuccinimide for graft crosslinking, and fully stirring at 25 ℃ for 24 hours to obtain a grafted product solution; precipitating the grafted product solution with methanol, and filtering to obtain a precipitate; swelling the precipitate with distilled water, dialyzing with deionized water, precipitating with methanol, freeze drying, sieving with 20 mesh sieve, and collecting to obtain solid granular hydrogel.
Example 2, structural characterization assay.
Fourier infrared spectrum detection: and respectively mixing and grinding the carboxymethyl-beta-cyclodextrin, carboxymethyl chitosan and a small amount of KBr uniformly, and pressing into slices. The gel sample after freeze drying is subjected to Fourier infrared spectroscopy at a wavelength of 500-4000 cm by means of an ATR accessory -1 The range is scanned.
The characterization of the carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles is shown in the following diagram, wherein the three are 3400cm in length as shown in figure 2 -1 The peak average of the positions is attributed to OH group vibration. Carboxymethyl-beta-cyclodextrin at 2930, 1601, 1420cm -1 The absorption peaks at the positions are respectively belonged to carboxymethyl aliphatic CH 2 Asymmetric stretching vibration of the group, asymmetric stretching vibration of the carboxylate group c=o, and symmetric stretching vibration. In the carboxymethyl chitosan spectrum, at 1597, 1414 cm -1 There is an absorption peak common to carboxymethyl-beta-cyclodextrin, namely asymmetric and symmetric stretching vibrations of carboxylate group c=o, but at 2875cm -1 Symmetrical CH with the absorption peak at the wave band belonging to the acetamido group 3 And (5) vibrating. The carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particle spectrum shows that the strength is up to 1634cm -1 This indication indicates successful amidation crosslinking between carboxymethyl chitosan and carboxymethyl-beta-cyclodextrin. Furthermore, due to vibration of the alpha (1.fwdarw.4) glucopyranose ring, carboxymethyl-beta-cyclodextrin is shown at 946cm -1 The wave band has absorption peak, and the hydrogel particles are 894cm by adding carboxymethyl-beta-cyclodextrin in the preparation process of the hydrogel -1 The absorption peak is stronger, which shows that the carboxymethyl-beta-cyclodextrin promotes the grafting of the carboxymethyl-beta-cyclodextrin to the carboxymethyl chitosan through the amide bond effect between the amino group of the carboxymethyl chitosan and the carboxymethyl of the carboxymethyl-beta-cyclodextrin, so that the grafting is successful.
Detecting by an X-ray single crystal diffractometer: grinding the freeze-dried gel sample; respectively placing gel powder, carboxymethyl-beta-cyclodextrin and carboxymethyl chitosan into a sample cell, and detecting by an X-ray single crystal diffractometer; the parameters are as follows: the radiation source Cu-K alpha (lambda=0.154 nm), the working voltage and the working current are respectively 40KV and 40mA, and the diffraction angle is 10 DEG to 45 deg.
As shown in figure 3, the carboxymethyl-beta-cyclodextrin has the characteristic diffraction absorption peak of crystal face, strong peak intensity and good peak shape, which indicates that the carboxymethyl-beta-cyclodextrin has a regular structure and a good crystal form. The X-ray single crystal diffraction spectrum of carboxymethyl chitosan shows the crystal form of the crystal characteristic caused by the hydrogen bond of hydroxyl molecules at the reflection angle 2 theta value of 20 degrees. And the X-ray single crystal diffraction pattern of the carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles shows a wide single-peak mode at a reflection angle 2 theta value of 20 degrees, and compared with carboxymethyl chitosan, the X-ray single crystal diffraction pattern shows reduced crystallinity and weakened absorption peaks, and shows more amorphous forms. Thus, the grafting was completed and the X-ray single crystal diffraction results were also consistent with those of the infrared spectrum.
Scanning electron microscope detection: and freeze-drying the carboxymethyl chitosan hydrogel particles grafted with carboxymethyl-beta-cyclodextrin, attaching a freeze-dried gel sample on the conductive adhesive, and spraying gold for 20min in a vacuum environment. The microstructure of the section of the glass is observed by a scanning electron microscope under the voltage of 10 KV.
FIG. 4 is a scanning electron microscope image of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles, wherein more pores are observed in the cross section of the gel under different multiples, and the gel has an irregular loose porous structure.
Thermogravimetric analysis detection: detecting the freeze-dried hydrogel particles by a thermogravimetric analyzer; the sample was placed in a sealed aluminum sample basket and heated from 25 ℃ to 600 ℃ in nitrogen at a rate of 10 ℃/min.
FIG. 5 is a thermogravimetric analysis of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles, wherein the hydrogel is mainly water lost before 100 ℃, and the carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles have a chemical degradation time of 200-600 ℃ as a functional group, and the decomposition time of the carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles is earlier than that of carboxymethyl-beta-cyclodextrin and carboxymethyl chitosan, thus indicating that the thermal stability of the gel particles is better.
Example 3 physical and chemical properties measurement index evaluation.
Swelling experiment: taking a certain amountQuality (W) 0 ) Is placed in 5mL of 0.1M HCl solution at ph=1.2 and 0.1% (w/v) PBS phosphate solution at ph=6.8, ph=7.4, and is shaken at 37 ℃ for 2h at 100 rpm. The surface of the excess liquid was removed by gently absorbing with filter paper at 30, 60, 90, 120min, respectively, and weighing (W t ). The swelling ratio was calculated by the following formula, and a change curve of the swelling ratio with time was drawn.
Swelling ratio: sr= (W t -W 0 )/W 0 ×100％
The results demonstrate the rational properties of carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles, wherein figure 6 shows that the hydrogel particles have higher swelling rate and excellent swelling performance under different pH conditions. The hydrogel particles can absorb a large amount of water molecules and are beneficial to the mass storage of medicines.
Drug loading experiment: immersing frozen hydrogel with a certain mass into 5mg/mL insulin hydrochloride solution, carrying out adsorption medicine at the constant temperature of 37 ℃ and 100r/min, sampling and measuring the absorbance of the solution at intervals until the absorbance of the solution is unchanged, taking out the hydrogel, carrying out vacuum drying at normal temperature, measuring the absorbance values of the solution before and after adsorption by using an ultraviolet spectrophotometer, calculating the concentration of the hydrogel by using a standard curve method, and calculating the medicine carrying amount of the hydrogel. The drug loading rate is calculated according to the following formula:
drug loading rate: q= [ v× (C 0 -C e )]/W×100％
In vitro drug release performance assay: airing the hydrogel which reaches the balance of adsorption and swelling to constant weight at room temperature, weighing and placing the hydrogel in a conical flask, simulating the change of the pH value of the gastrointestinal tract, firstly placing the islet-carrying hydrogel particles in an HCl solution with the pH value of 1.2, continuously oscillating and releasing the medicament at constant temperature under the condition of 100r/min in a constant temperature culture oscillator with the temperature of 37 ℃, then placing the hydrogel in PBS with the pH value of 6.8 as a release medium, continuously releasing the medicament for 2 hours at the temperature of 37 ℃, finally placing the hydrogel in PBS with the pH value of 7.4 as the release medium, continuously releasing the medicament for 3 hours at the temperature of 37 ℃, transferring 2mL of slow-release liquid by a pipette every 0.5 hour, supplementing fresh 2mL of simulated gastrointestinal tract slow-release medium with corresponding volume, respectively taking the blanks of each as a reference, measuring the absorbance value of the medicament, obtaining the content of the medicament, and drawing an accumulated medicament release curve. The accumulated drug release rate is calculated according to the following formula, and a drug release curve is drawn.
Drug release rate:
fig. 7 shows that there is no apparent burst effect of gel particles in the simulated gastric fluid environment for the first two hours, the release rate is only 8%, while the release rate is significantly increased to 55% to 70% in the simulated intestinal environment and a sustained release is achieved. Therefore, the hydrogel has good controlled release and sustained release performance on insulin, can avoid decomposition of insulin by gastric juice, has longer and more stable release time in the pH environment of intestinal tracts, and is favorable for absorption of insulin by human intestinal tracts.
Insulin biological Activity assay: the insulin standard solution and the insulin-carrying hydrogel are subjected to fluorescence spectrometer measurement and contrast analysis through insulin released by simulating human body environment, an excitation light monochromator is fixed at a selected excitation light wavelength, the fluorescence monochromator is adjusted to the selected fluorescence wavelength, and a signal obtained by a recorder is the fluorescence intensity of the sample solution. The excitation wavelength is set to 276nm, the width of the exciting and emitting slit is set to 2.5nm, and the emission wavelength is set to 280-450nm. Insulin standard solution and insulin-carrying hydrogel released by simulating human body environment are subjected to circular dichroism measurement and contrast analysis, and a cylindrical quartz cuvette with 1cm optical path difference is used for setting parameters of 0.2nm precision, 1.0nm bandwidth, 200nm/min scanning speed, 200-300nm scanning range and scanning measurement at constant temperature of 4 ℃.
FIG. 8 shows that characteristic peaks appear near 308nm in the fluorescence spectrum of insulin released by simulating human environment, which is consistent with the fluorescence spectrum of insulin standard solution, and shows that the secondary structure of the insulinolin is not changed in the process of embedding and releasing the insulinolin by gel particles, and the gel particles can keep the original bioactivity of insulin unchanged. FIG. 9 shows two negative peaks occurring near wavelengths 208nm and 222nm in a circular dichroism spectrum of insulin released through a simulated human environment, corresponding to characteristic peaks of alpha helix and antiparallel beta sheet of insulin, respectively. Therefore, the structure of the embedded insulin before and after release is not changed obviously, so that the biological activity of the embedded insulin is not changed, and the biological activity is consistent with the result of a fluorescence spectrum.
Example 4, evaluation of biocompatibility assay index.
Cytotoxicity experiment: caco-2 cells were seeded in 96-well plates at a density of 1X 10 5 Cells/well in 5% CO with 100. Mu.L MEM medium containing 20% high quality foetal calf serum, 1% diabody and 1% nonessential amino acids 2 Culturing at 37deg.C for 24 hr. The gel microparticles were added to MEM medium to make 12.5, 25, 50, 100, 200, 400, 800, 1600 μg/mL gel suspensions. After 24h, the medium in the 96-well plate was removed, 100. Mu.L of gel suspension of different concentrations was added, and the culture was continued for 24h, and then 10. Mu.L of CCK-8 solution was added per well. Wells with corresponding amounts of cell culture and CCK-8 solution but without cells added were used as blank controls. Incubation was continued for 0.5 hours in the cell incubator and absorbance was measured at 450nm. A control group was prepared with MEM medium without gel. 8 duplicate wells were run in parallel for each group and the average was taken.
Cell viability (%) =od 490 (sample) -OD 490 (blank) /OD 490 (control) -OD 490 (blank) ×100％
The carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles have good biocompatibility, wherein, as shown in figure 10, after the gel suspension (12.5, 25, 50, 100, 200, 400, 800 and 1600 mug/mL) is cultured for 24 hours, the survival rate of Caco-2 cells reaches over 96 percent, which indicates that the gel particles are nontoxic and have good biocompatibility.
Example 5, pharmacological activity assay index assessment.
Blood glucose level lowering detection in mice: 60 male Kunming mice were taken for 2-3 weeks and model type 2 diabetes mice were established using Streptozotocin (STZ) induction. After the model is successfully constructed (the blood sugar level is more than or equal to 16.7 mmol/L), the model is used as a diabetes mouse model for further experiments. The diabetic mice were divided into six groups of 10 animals each, which were a model group (oral administration of 0.9% physiological saline), a positive group (subcutaneous injection of 5 IU/kg), a negative group (oral administration of 100IU/kg of insulin solution), a high dose group (oral administration of 100IU/kg of insulin-loaded gel particles), a medium dose group (oral administration of 75 IU/kg of insulin-loaded gel particles), and a low dose group (oral administration of 50IU/kg of insulin-loaded gel particles). After administration according to the different modes of the pre-design, blood glucose values were obtained by collecting blood samples (once per hour) at the tail end and the change law of blood glucose with time was detected for the different modes of administration.
As shown in FIG. 11, the insulin-loaded gel particles have a good hypoglycemic activity. The model group and the negative group did not play any role in the blood glucose levels of the mice; the positive group can ensure that the serum insulin concentration reaches the peak value 2 hours after injection, but the action time is short, the blood glucose value is rapid and the blood glucose value is quickly raised; in contrast, oral gavage-loaded insulin hydrogel particles (50, 75, 100 IU/kg) all exhibited serum insulin concentration peaks around 6 hours, and mice could be controlled to maintain low blood glucose levels for long periods of time with sustained release of insulin for approximately about 10 hours. This shows that the hydrogel particles can realize slow release effect after embedding insulin and have longer-lasting hypoglycemic activity effect on a type 2 diabetes mouse model.
Claims (6)
1. Use of hydrogel microparticles in the entrapment of insulin;
the preparation method of the hydrogel particles mainly comprises the following steps:
1) Preparing carboxymethyl-beta-cyclodextrin solution: dissolving carboxymethyl-beta-cyclodextrin in deionized water to obtain carboxymethyl cyclodextrin solution;
2) Preparing carboxymethyl chitosan solution: dissolving carboxymethyl chitosan in deionized water to obtain carboxymethyl chitosan solution;
3) Preparing carboxymethyl-beta-cyclodextrin grafted carboxymethyl chitosan hydrogel particles: mixing carboxymethyl-beta-cyclodextrin solution and carboxymethyl chitosan solution, adding proper amount of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to carry out graft crosslinking, and fully stirring for 24 hours at 25 ℃ to obtain grafted product solution; precipitating the grafted product solution with methanol, and filtering to obtain a precipitate; swelling the precipitate with distilled water, dialyzing with deionized water, precipitating with methanol, freeze drying, sieving with 20 mesh sieve, and collecting to obtain solid granular hydrogel;
the molar ratio of the carboxymethyl-beta-cyclodextrin to the carboxymethyl chitosan to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to the N-hydroxysuccinimide is 3.3:17:19.2:19.2.
2. The use according to claim 1, wherein each 1.0 g carboxymethyl- β -cyclodextrin in step 1) is dissolved in 20mL deionized water.
3. The use according to claim 1, wherein each 1.0. 1.0 g carboxymethyl chitosan in step 2) is dissolved in 25mL deionized water.
4. The use according to claim 3, wherein the carboxylation degree of carboxymethyl chitosan is not less than 80%.
5. The use according to claim 1, wherein the mass of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 3.68g.
6. The use according to claim 1, wherein the mass of N-hydroxysuccinimide is 2.21g.
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