CN113262198A

CN113262198A - Microgel and preparation method and application thereof

Info

Publication number: CN113262198A
Application number: CN202110585929.5A
Authority: CN
Inventors: 闫昳姝; 吴青青; 任盼盼; 张娜; 刘秋熠
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-05-27
Filing date: 2021-05-27
Publication date: 2021-08-17
Also published as: WO2022246922A1

Abstract

The invention provides a microgel with an innovative structure and a preparation method and application thereof, belonging to the technical field of preparation and application of gel materials. The preparation method of the microgel comprises the following steps: uniformly mixing a hyaluronic acid aqueous solution and a chitosan aqueous solution, adding a cross-linking agent, and performing mixing reaction to obtain microgel; wherein the crosslinking agent is a metal salt. The microgel obtained by the invention can be applied to the preparation of drug carriers. The obtained microgel can also be applied to the preparation of a pulmonary drug delivery system. The microgel obtained by the invention has better biocompatibility.

Description

Microgel and preparation method and application thereof

Technical Field

The invention relates to the technical field of preparation and application of gel materials, in particular to a microgel and a preparation method and application thereof.

Background

Inhalation administration is the best mode of administration for treating various chronic lung diseases such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, chronic bronchitis, and the like. Compared with a systemic administration mode, the inhalation administration can directly deliver high-concentration medicines to a disease part, reduces the side effect of the medicines to the whole body, takes effect quickly, perfectly avoids treatment obstacles such as gastrointestinal absorption, first pass elimination of liver and the like generated by conventional administration routes such as oral administration, injection administration and the like, and can achieve the systemic administration effect only by small-dose administration. In addition, high compliance with inhalation is also an important factor affecting the trend of pharmaceutical preparations.

The lungs and the entire respiratory tract provide certain advantages for drug absorption, but administration by inhalation is not without hindrance. Inside the upper respiratory tract, the mucous layer is secreted by the goblet cells under the tracheal mucosa, and the lung mucus is driven by the movement of ciliated cells to be cleared at the flow rate of 0-5 mm/min. The major areas of gas exchange, such as the alveoli and the bronchial extremities, are protected by immune cells and a dense network of capillaries-the Air-blood Barrier (Air-blood Barrier), where the most prominent defense mechanism is macrophages particles deposited deep in the lung are phagocytosed by alveolar macrophages, which then slowly dislodge the particles along the bronchial-tracheal ladder or lymphatic system and migrate out of the lung. Especially for particles with a geometry between 1-3 μm, macrophage scavenging effect is best. Therefore, how to avoid phagocytosis of macrophages is a central issue to solve the efficiency of pulmonary drug delivery.

Respiratory mucus often changes in composition, thickness, physicochemical properties (e.g., viscosity, composition) and clearance in patients with airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. The above pathological conditions may further affect the efficacy of the pulmonary drug delivery system. In order to avoid pulmonary clearance of drugs, further understanding of the methods and improvements in the formulation of drugs is needed.

Current inhalation drug delivery systems are in need of further development and new pharmaceutical dosage forms suitable for pulmonary administration are still being developed. Therefore, it is the current research focus to closely combine the research of new drug carriers, feasible formulation technology and targeted sustained and controlled release medicament, and to explore a new controlled release technology and formulation.

Disclosure of Invention

In order to solve the technical problems, the invention provides a novel microgel and a preparation method and application thereof.

A novel microgel having the chemical structure:

wherein x is 4-10000, y is 4-100000, m is 1-500, n is 1-500, x, y, m and n are integers; and M is metal ions.

The metal ions are one or more of calcium ions, zinc ions, sodium ions, potassium ions, copper ions, barium ions, aluminum ions and iron ions;

further, x is 700-.

Further, the method comprises the following steps: uniformly mixing a hyaluronic acid aqueous solution and a chitosan aqueous solution, adding a cross-linking agent until the mass concentration is 0.05-6%, and performing a mixing reaction to obtain a microgel; wherein the crosslinking agent is a metal salt.

Further, the mass concentration of the hyaluronic acid aqueous solution is 0.025% -10%, and the mass concentration of the chitosan aqueous solution is 0.1% -10%.

Further, the final concentration of the cross-linking agent in the mixed solution is 4-6%;

further, the hyaluronic acid is one or more of phosphorylated oxidized hyaluronic acid, low sulfated oxidized hyaluronic acid LS, oxidized and high sulfated hyaluronic acid HS and oxidized hyaluronic acid; wherein said low sulfated oxidized hyaluronic acid LS refers to sulfated oxidized hyaluronic acid with a degree of substitution between 0.5 and 30%; the oxidized and highly sulfated hyaluronic acid HS refers to oxidized and sulfated hyaluronic acid with a substitution degree of 31-90%.

Further, the hyaluronic acid is oxidized hyaluronic acid, low sulfated oxidized hyaluronic acid LS and oxidized and high sulfated hyaluronic acid HS.

Further, the chitosan is selected from one or more of carboxymethyl chitosan, acetylated chitosan, N, O-alkylated chitosan, sulfated chitosan and phosphorylated chitosan.

Further, the chitosan is carboxymethyl chitosan or acetylated chitosan.

Further, the mass ratio of the hyaluronic acid to the chitosan is 1:1-1:4, wherein the hyaluronic acid refers to solute hyaluronic acid in a hyaluronic acid solution, and the chitosan refers to solute chitosan in a chitosan solution.

Further, the mass ratio of the hyaluronic acid to the chitosan is 1:2-1: 4.

Further, the metal salt is one or more of calcium salt, zinc salt, sodium salt, potassium salt, copper salt, barium salt, aluminum salt and ferric ion salt.

Further, the metal salt is zinc salt, calcium salt, aluminum salt, copper salt and sodium salt.

Further, the metal salt is one or more of calcium chloride, zinc acetate, sodium chloride, sodium sulfate, zinc phosphate, potassium chloride, potassium sulfate, barium chloride and aluminum sulfate.

Further, the metal salt is calcium chloride, zinc acetate and sodium chloride.

Further, the time of the mixing reaction is 5min-8 h.

Further, the mixing reaction time is 1-3 h.

The microgel is applied to preparing a drug carrier.

The microgel is applied to preparing a pulmonary drug delivery system.

The reaction process of the invention is as follows:

wherein x is 4-10000, y is 4-100000, M is 1-500, n is 1-500, M is metal ion, and M, n, x and y are integers.

According to the invention, hyaluronic acid and chitosan amino form a compound through Schiff base, a metal salt cross-linking agent is coordinated with Schiff base outside the compound to form a metal salt-oxidized hyaluronic acid-carboxymethyl chitosan compound microgel structure, and the specific structure is shown in the figure.

The invention adopts carboxymethyl chitosan and hyaluronic acid as base materials, adopts metal ion solution as a precipitator, and prepares a novel pH-controllable degradable microgel for constructing a pulmonary drug delivery material by a precipitation polymerization method.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the material prepared by the invention is microgel particles with the diameter of 1-5 mu m, and has the characteristic of controllable degradation under low pH in vitro. The microgel particle material has higher histocompatibility with lung epithelial cells and common histiocytes, and cannot influence the growth of the cells. There was no significant toxicity to mice. In addition, the material can escape the phagocytosis of macrophages, the distribution of the medicine in the lung can reach 48h, and the aim of slow release of the medicine is fulfilled. The characteristic of low pH sensitivity can be applied to various chronic lung diseases, such as asthma, COPD, lung tumor and other local peracid cases, and the controlled release of the drug is realized.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

FIG. 1 is a schematic diagram of the present invention.

FIG. 2 is a graph showing the morphological structure characterization results of microgel prepared in example 1 of the present invention; wherein, (a) the macroscopic structure diagram of the microgel: the left figure shows the homogeneous particle dispersion solvent formed during the preparation of the microgel, and the right figure shows the gel-like aggregate formed after centrifugation; (b) - (c) is a TEM image of the microgel in aqueous solution; (d) determining the dynamic diameter of the microgel; (e) and (f) SEM determination of the microstructure of the microgel freeze-dried product.

FIG. 3 is an IR spectrum of a microgel obtained in example 1 of the present invention, wherein (a) is an IR spectrum of HA (hyaluronic acid) and A-HA (oxidized hyaluronic acid); (b) the IR spectra of CS (chitosan) and CMCS (carboxymethyl chitosan).

FIG. 4 is a graph showing in vitro degradation and swelling curves of microgel prepared in example 1 of the present invention, wherein (a) degradation curves of microgel in different buffers; (b) swelling curves for gels of different proportions.

FIG. 5 is a graph showing the survival of microgel cells described in test example 3 of the present invention; wherein (a) is the survival rate of 3T3 in different microgels, and (B) is the survival rate of the Beas-2B in different microgels.

FIG. 6 is a graph showing the survival of microgel cells described in test example 3 of the present invention; wherein (a) a survival plot of 3T3 in a 4:1 (6% zinc acetate) microgel; (b) survival of 3T3 in 4:1 (6% zinc chloride) microgel; (c) survival of Beas-2B in 4:1 (6% zinc acetate) microgel; (d) viability of Beas-2B in 4:1 (6% Zinc chloride) microgel.

FIG. 7 is the drug uptake of the microgel according to test example 4 of the present invention at various times; wherein, (a) and (d) represent the drug intake conditions at different times when the microgel concentration is 50 mug/mL; (b) and (e) the situation of the drug intake at different time when the concentration of the microgel is 100 mu g/mL; (c) and (f) the medicine intake at different times when the microgel concentration is 500 mu g/mL.

FIG. 8 is a laser confocal image of RAW cells incubated for 6h and 24h, respectively, after microgel was formulated to a drug concentration of 500. mu.g/mL in the present invention.

FIG. 9 shows the distribution of microgel in mouse lung tissue in the application example of the present invention; wherein, (a) mice are dosed for 2h, 24h and 100h to obtain fluorescence imaging images; (b) graph of the change of fluorescence intensity of the gel in the mouse.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The experimental group and the control group in the embodiment of the invention are set as follows:

blank group: only PBS solution was added, without any addition of cells and drug groups.

Control group: cells cultured in media without drug were added.

Administration group: the cells cultured in the medium containing the drug are divided into groups according to the concentration and type of the drug contained therein.

The invention relates to cell culture:

(1) cell recovery

Placing the freezing tube into a water bath at 37 ℃ for rapid thawing, opening the freezing tube, taking out cell suspension, placing the cell suspension into a centrifuge tube, adding 5mL of corresponding culture medium, centrifuging at 1000rpm for 3min, discarding supernatant, re-suspending with culture solution, inoculating into a culture bottle, placing into a 95% air and 5% carbon dioxide incubator, standing and culturing at 35-37 ℃, replacing the recovery period culture medium every 12h, and then replacing the culture medium according to subculture.

(2) Culture of cells

3T3 line cell: adding DMEM high-sugar culture medium (containing 10% newborn calf serum), placing in 95% air and 5% carbon dioxide incubator, standing and culturing at 35-37 deg.C, and changing the culture solution every 2-3 days.

Beas-2B line cells: adding 1640 culture medium, placing in 95% air and 5% carbon dioxide incubator, standing at 35-37 deg.C, and changing the culture solution every 2-3 days.

RAW line cells: adding DMEM high sugar culture medium (containing 10% fetal calf serum), placing in 95% air and 5% carbon dioxide incubator, standing and culturing at 35-37 deg.C, and changing the culture solution every 2-3 days.

(3) Passage of cells

3T3 cells: when the fibroblast grows to more than 70% of the T75 culture flask, discarding the culture medium, adding PBS to wash for three times, discarding the PBS, adding 2mL of pancreatin to digest until the intercellular space becomes bigger and the cell morphology becomes round, stopping digestion with 10mL of DMEM high-sugar medium (containing 10% newborn calf serum), forming cell suspension, and separatingCentrifuging at 1000rpm for 3min, discarding the upper culture solution, adding fresh culture medium, resuspending cells, inoculating in a new culture flask at 37 deg.C and 5% CO at a ratio of 1:3₂Culturing in a constant temperature incubator.

Beas-2B cells: when lung epithelial cells grow to more than 70% of that in a T75 culture flask, removing the culture medium, adding PBS for cleaning for three times, removing the PBS, adding 2mL of pancreatin for digestion until cell gaps become larger and cell morphology becomes round, stopping digestion with 10mL of 1640 culture medium, forming cell suspension in a centrifuge, centrifuging at 1000rpm for 3min, removing upper layer culture solution, adding fresh culture medium, resuspending cells, inoculating in a new culture flask according to the proportion of 1:3, and culturing at 37 ℃ with 5% CO for 3min₂Culturing in a constant temperature incubator.

RAW cells: when the macrophage grows to more than 70% of the T75 culture bottle, the culture medium is discarded, PBS is added for washing for three times, PBS is discarded, 10mL of fresh DMEM high-sugar culture medium (containing 10% fetal calf serum) is added, adherent cells are gently scraped by a scraper to form cell suspension, and the ratio of the total weight of the adherent cells to the total weight of the cell suspension is calculated according to the following formula 1:3 in a new flask at 37 ℃ 5% CO₂Culturing in a constant temperature incubator.

(4) Cell cryopreservation

3T3 cells: digesting and centrifuging in the above steps, preparing cell suspension with newborn calf serum, counting cells according to 1-2 × 10⁴Adding the frozen stock solution into each cell/mL, subpackaging in 2mL frozen stock tubes, sealing, and marking the date and the holder. Storing at 4 deg.C for 5-15min in refrigerator, storing at-20 deg.C for 2h, storing at-80 deg.C for 2h, and storing at-196 deg.C for a long time.

Beas-2B cells: the procedure was as in 3T3 except that the serum used was fetal bovine serum.

RAW cells: after the resuspension in the steps, preparing cell suspension by using newborn calf serum, counting cells, adding the cryopreservation solution according to 1-2 × 104 cells/mL, subpackaging in 2mL cryopreservation tubes, sealing, marking the date and holder, storing for 5-15min in a refrigerator at 4 ℃, storing for 2h at-20 ℃, storing for 2h at-80 ℃, and then storing for a long time at-196 ℃.

Examples

Example 1

Respectively ultrasonically dissolving 200mg of carboxymethyl chitosan (CMCS) and 50mg of oxidized hyaluronic acid (A-HA) in 5mL of water at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the two solutions, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 2h, and rapidly adding ZnCl₂And (5) continuing stirring the solution until the final concentration is 6%, and finally preparing the milky microgel. The microgel obtained was lyophilized, then ground into powder and redissolved in water, and the structure and morphology thereof were observed by TEM and SEM, and the experimental results are shown in fig. 2. The microgel particles were found to be highly dispersed in water by TEM images. The solid powder exhibits a porous microstructure similar to a macroscopic gel crosslinked by small particles (diameter about 100 nm). These images further demonstrate the incorporation of Zn into aqueous systems²⁺A microgel system is formed, wherein the structural formula of the microgel is 900 ═ 700-.

Example 2

Respectively ultrasonically dissolving 200mg of carboxymethyl chitosan (CMCS) and 67mg of oxidized hyaluronic acid (A-HA) in 5mL of water with the same amount at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the two solutions, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 3:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 2h, and quickly adding ZnCl with the mass concentration₂And (3) continuing stirring the solution until the final concentration is 6%, and finally preparing the yellow microgel, wherein in the structural formula of the microgel, x is 700-4000, y is 2000-300, m is 200-300, and n is 100-300.

Example 3

Respectively ultrasonically dissolving 200mg of carboxymethyl chitosan (CMCS) and 100mg of oxidized hyaluronic acid (A-HA) in 5mL of water at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the two solutions, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 2:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 2h, and rapidly adding the mass concentrationDegree of ZnCl₂And (3) continuing stirring the solution until the final concentration is 6%, and finally preparing yellow microgel, wherein in the structural formula of the microgel, x is 700-4000, y is 2000-4000, m is 1-500, and n is 1-500.

Example 4

Respectively ultrasonically dissolving 100mg of carboxymethyl chitosan (CMCS) and 100mg of oxidized hyaluronic acid (A-HA) in 5mL of water at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the two solutions, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 1:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 2h, and rapidly adding ZnCl with the mass concentration₂And (3) continuing stirring the solution until the final concentration is 6%, and finally preparing the yellowish microgel, wherein the structural formula of the microgel comprises x is 10-500, y is 50-100, m is 200-500-and n is 300-500-in-500.

Example 5

Respectively ultrasonically dissolving 200mg of carboxymethyl chitosan (CMCS) and 50mg of oxidized hyaluronic acid (A-HA) in 5mL of water at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the two solutions, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 2h, and rapidly adding zinc acetate (Zn (Ac))₂) And (3) continuing stirring the solution until the final concentration is 6%, and finally preparing the yellowish microgel, wherein in the structural formula of the microgel, x is 1000-2000-containing-oil-water-oil-water-oil-.

Example 6

Respectively ultrasonically dissolving 200mg of carboxymethyl chitosan (CMCS) and 50mg of oxidized hyaluronic acid (A-HA) in equal amount of water at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the two solutions, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 2h, and rapidly adding CaCl₂The solution is stirred until the final concentration is 6 percent (mass concentration), and the microgel which is creamy yellow is finally preparedThe structural formula of the microgel is that x is 800-.

Examples 7 to 13

The experimental procedures, raw materials and proportions are the same as those in example 1, except that the crosslinking agent added in examples 7-13 is ZnCl₂Solution and ZnCl₂The final concentration of the solution was 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.05%, respectively.

Examples 14 to 15

The experimental procedures, raw materials and proportions are the same as those in example 5, except that CaCl is added as a crosslinking agent in examples 14-15₂The mass concentration of the solution is 1.5 percent and 0.05 percent respectively.

Example 16

Respectively ultrasonically dissolving 200g of carboxymethyl chitosan (CMCS) and 50g (HS, oxidized and highly sulfated hyaluronic acid, 31-90%) in 5mL of water with the same amount at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the carboxymethyl chitosan (CMCS) solution and the oxidized hyaluronic acid (A-HA) solution, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 4h, and rapidly adding ZnCl₂And (3) continuing stirring the solution until the final concentration is 6%, and finally preparing the milky microgel, wherein in the structural formula of the microgel, x is 1000-.

Example 17

Respectively ultrasonically dissolving 200g of carboxymethyl chitosan (CMCS) and 50g (LS, the substitution degree of oxidized and low sulfated hyaluronic acid is 0.5-30%) in 5mL of water at room temperature to obtain a carboxymethyl chitosan (CMCS) solution and an oxidized hyaluronic acid (A-HA) solution, mixing the carboxymethyl chitosan (CMCS) solution and the oxidized hyaluronic acid (A-HA) solution, wherein the mass ratio of the carboxymethyl chitosan (CMCS) to the oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 1h, and rapidly adding ZnCl₂And (3) continuing stirring the solution until the final concentration is 6%, and finally preparing the yellow microgel, wherein in the structural formula of the microgel, x is 5000-8000, y is 5000-8000, m is 400-500, and n is 400-500.

Example 18

Respectively ultrasonically dissolving 200g of phosphorylated chitosan and 50g of phosphorylated oxidized hyaluronic acid in 5mL of water with the same amount at room temperature to obtain a phosphorylated chitosan solution and a phosphorylated oxidized hyaluronic acid solution, mixing the phosphorylated chitosan solution and the phosphorylated oxidized hyaluronic acid solution, wherein the mass ratio of the phosphorylated chitosan to the phosphorylated oxidized hyaluronic acid in the mixed solution is 4:1, placing the mixed solution on a magnetic stirrer, mixing and incubating for 30min, rapidly adding a sodium chloride solution to a final concentration of 6%, and continuously stirring to finally prepare the yellow microgel, wherein in the structural formula of the microgel, x is 4000-plus 8000, y is 5000-plus 10000, m is 400-plus 500, and n is 4-500.

Example 19

Respectively ultrasonically dissolving 100g of sulfated chitosan and 50g of sulfated oxidized hyaluronic acid in 5mL of water with the same amount at room temperature to obtain a sulfated chitosan solution and a sulfated oxidized hyaluronic acid solution, mixing the sulfated chitosan solution and the sulfated oxidized hyaluronic acid solution, placing the mixed solution on a magnetic stirrer for mixing and incubating for 4h, quickly adding a sodium chloride solution to the final concentration of 6%, and continuously stirring to finally prepare the yellow microgel, wherein in the structural formula of the microgel, x is 1000-.

Example 20

Respectively ultrasonically dissolving 100g of sulfated chitosan and 50g of sulfated oxidized hyaluronic acid in 5mL of water with the same amount at room temperature to obtain a sulfated chitosan solution and a sulfated oxidized hyaluronic acid solution, mixing the sulfated chitosan solution and the sulfated oxidized hyaluronic acid solution, placing the mixed solution on a magnetic stirrer, mixing and incubating for 2h, quickly adding a copper chloride solution until the final concentration is 3%, and continuously stirring to finally prepare the yellow microgel. The structural formula of the microgel is that x is 3000-4000, y is 50000-60000, m is 400-500, and n is 400-500.

Test example 1

Structural characterization: the freeze-dried sample obtained in example 1 was ground into powder, characterized by powder infrared diffraction spectroscopy, and set for detectionThe wavelength range is 500-4000cm^-1。

The experimental results are shown in the figure: in FIG. 3, FIG. 3(a) shows the IR spectra of HA and A-HA, which are very similar in FTIR spectra, probably due to hemiacetal formation and thus difficult to detect the signal of aldehyde group in the chain, but the overall reaction can be judged to have occurred due to the increased overall absorption intensity. FIG. 3(b) is an IR spectrum of CS and CMCS at 3138cm^-1The absorption peak is broadened, which shows that N-H and O-H form hydrogen bond, and the spectrum of the carboxymethyl chitosan is stretched due to the fact that one carboxyl group with the hydrogen bond is arranged in the carboxymethyl chitosan dimer, and the CS is shown in the graph at 1583cm^-1The peak is amino, and the characteristic peak of CMCS is 1557cm^-1And C is 1557cm^-lThe vibration absorption peak after stretching showed that the carboxyl group (COOH) added a C ═ O group, indicating that carboxymethyl chitosan had been formed.

Test example 2

1, testing of degradation Properties of microgels

The microgel prepared in example 1 was divided into two groups, 35mg of each group was dispensed into 1.5mL centrifuge tubes, 1mL of buffer solution (pH 7.4, 0.2M lysozyme solution and pH 4 sodium acetate buffer) was added to each group, the mixture was placed on a mixer and slowly rotated, and samples were taken at different time points (5min, 20min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, and 8 h). Centrifuging at 12000rpm/min for 10min, removing supernatant, and weighing precipitate. Let the mass after degradation be W_tInitial mass is W₀When the degradation rate is equal to (W)_t-W₀)/W₀X 100%. The experimental results are shown in FIG. 4 (a). Can be seen from the figure. FIG. 4(a) is a graph showing the degradation curve of microgel in sodium acetate buffer, the percent degradation of microgel rapidly increased from 0 to 64% in 0 to 3 hours and stabilized at that value without further change; in the lysozyme-containing buffer solution with the pH value of 7.4, the degradation curve of the microgel has no obvious change within 0-5 h. Therefore, the degradation of the prepared CMCS-A-HA microgel is influenced by the pH value of the environment, the degradation of the CMCS/A-HA microgel is rapid under the environment of pH 4, and the maximum degradation degree is reached within 5 hours; while the microgel is not substantially degraded under the condition of adding lysozyme, thereby proving thatObviously, the microgel has controllable degradation rate and acid sensitivity.

2, testing swelling degradation performance of microgel

After freeze-drying the microgel obtained in examples 1 to 3, the freeze-dried product was divided into 35mg portions and put into 1.5mL centrifuge tubes, 1mL of PBS solution (pH 7.4) was added, the mixture was placed on a mixer and slowly rotated, and samples were taken at different time points (5min, 20min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, and 8 h). Centrifuging at 12000rpm/min for 10min, removing supernatant, and weighing precipitate. The mass after water absorption is W_tInitial mass is W₀When the swelling ratio is%_t-W₀)/W₀X 100%. The experimental results are shown in FIG. 4 (b).

As shown in FIG. 4(b), the microgel prepared by the invention has a three-dimensional interpenetrating network structure, contains multiple hydrophilic groups such as hydroxyl groups and the like in the interior, can absorb a large amount of water, has swelling and insolubility properties, and can be used as a good drug carrier of a drug delivery system. FIG. 4(b) is a graph showing the change of swelling degree of microgel prepared in a solution with time according to the mass ratio of CMCS to HA of 2:1, 3:1 and 4: 1: with the increase of time, within 0-2h, the swelling degree of the microgel reacting the CMCS and the HA in a ratio of 4:1 rapidly rises to 5%, then the walk tends to be stable, and the swelling degree of the microgel reacting the CMCS and the HA in a ratio of 3:1 rapidly rises to 4%, then the stable swelling degree is reached. The swelling degree of the microgel reacting the CMCS and the HA in a ratio of 2:1 is relatively slow to rise, and the microgel basically reaches a stable value (4%) after 6 hours; wherein the CMCS and HA have the largest swelling degree of 4:1 microgel. Therefore, the prepared microgel is in limited swelling reaction, the swelling degrees of CMCS and HA which are added in different proportions in the reaction are all stable within the range of 400-500%, the swelling property is good, and obvious and regular swelling degree difference does not occur. The results show that the addition of CMCS in a different ratio to HA HAs no effect on the swelling properties of the microgel.

Test example 3

MTT assay

(1) The microgel obtained in examples 1 to 3 and examples 14 and 15 were prepared into 4:1, 3:1, 2:1, 4:1(HS), 4:1(LS) solutions of drug groups at a concentration of 100 μ g/mL using respective media. A blank control group was also set. The specific experimental operations were as follows:

a, 6X 10 of Bears-2B cells⁴The cells were seeded at a density of one/mL in 96-well plates (approximately 100. mu.L per well) and cultured for 12 h. And (3) sucking a supernatant, adding a new DMEM high-glucose medium (containing 10% newborn calf serum), setting the concentration of the medicine to be 100 mu g/mL, respectively adding the prepared microgel medicine into each group to arrange 5 compound holes, culturing at 37 ℃ for 24h, sucking the supernatant, adding 100 mu L MTT, and culturing at 37 ℃ in a dark place for 3-4 h. After incubation in the dark, 100. mu.L of DMSO was added to each well, and the absorbance was measured at a wavelength of 570nm using a microplate reader.

b, 6X 10 cells of 3T3⁴The cells were seeded at a density of one/mL in 96-well plates (approximately 100. mu.L per well) and cultured for 12 h. And (3) sucking a supernatant, adding a 1640 culture medium, setting the drug concentration to be 100 mu g/mL, respectively adding the prepared microgel drugs into each group, setting 5 multiple holes in each group, culturing at 37 ℃ for 24 hours, sucking the supernatant, adding 100 mu L MTT, and culturing at 37 ℃ in a dark place for 4 hours. After incubation in the dark, 100. mu.L of DMSO was added to each well, and the absorbance was measured at a wavelength of 570nm using a microplate reader.

The results of the experiment are shown in FIG. 5, in which 2:1, 3:1, 4:1, 4:1HS and 4:1LS correspond to the cell viability of the microgel solution obtained in example 3, example 2, example 1 and example 14 and example 15, respectively, and NC represents the results of the blank control group.

And (4) conclusion: the viability of the Beas-2B cells is shown in FIG. 5(a), which shows that: in transverse comparison, the experimental group and the control group have no obvious difference, and are all about 0.6, and no obvious correlation exists between the survival rate and the reactant feeding ratio, so that the drug concentration is set to be 100 mu g/mL, the microgel prepared by different feeding ratios has no or lower cytotoxicity to the Beas-2B cells, and has good biocompatibility to the Beas-2B cells.

FIG. 5(b) is a graph showing the survival rate of 3T3 cells, showing that: cell viability was similar and around 1 for each environmental condition, including the control group. Therefore, the analysis proves that the growth and the multiplication of the 3T3 cells are not obviously inhibited by activity under the culture condition of the microgel with different components and 100 mu g/mL microgel medicament concentration, and the prepared microgel has low cytotoxicity and excellent biocompatibility on normal fibroblasts.

(2) The microgel obtained in example 1 and example 5 was diluted to a concentration of 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 80. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL, respectively, using a PBS buffer solution. Respectively carrying out MTT experiments on the microgel solution in 3T3 cells and Beas-2B cells, and simultaneously setting a blank control group, wherein the method comprises the following specific steps:

a, 6X 10 cells of 3T3⁴Each/mL of the cells was inoculated into a 96-well plate (about 100. mu.L per well), cultured for 12 hours, aspirated to remove the supernatant, and added with fresh DMEM high-glucose medium (containing 10% newborn calf serum) in a gradient containing 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 80. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, and 500. mu.g/mL of gel drug, and each set was provided with 5 duplicate wells. After culturing at 37 ℃ for 24 hours, the supernatant was aspirated, 100. mu. LMTT was added, and the mixture was cultured at 37 ℃ for 3 hours in the dark. After incubation in the dark, 100. mu.L of DMSO was added to each well, and the absorbance was measured at a wavelength of 570nm using a microplate reader.

B, 6X 10 of Beas-2B cells⁴Each/mL of the cells was inoculated into a 96-well plate (about 100. mu.L per well), cultured for 12 hours, the supernatant was aspirated, and a new 1640 medium was added to the cells in groups of 5 duplicate wells, each containing a gradient of 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 80. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, or 500. mu.g/mL of gel. After culturing at 37 ℃ for 24 hours, the supernatant was aspirated, 100. mu. LMTT was added, and the mixture was cultured at 37 ℃ for 3 hours in the dark. After incubation in the dark, 100. mu.L of DMSO was added to each well, and the absorbance was measured at a wavelength of 570nm using a microplate reader.

The experimental results are shown in FIG. 6, and the light absorption values of the MTT measurement are not significantly different from those of the blank control group after 3T3 and the Beas-2B cells are respectively incubated with different concentrations and different types of microgels for 24h in FIGS. 6(a) - (B) and FIGS. 6(c) - (d). It is further demonstrated that the material has no effect on cell growth.

Therefore, in the preparation process of the microgel, within the concentration range of 10-500 mu g/mL, the two crosslinking agent groups are added, and no obvious organic damage is caused to lung epithelial cells and fibroblasts of a human body. Therefore, the experiment researches that the microgel prepared by the method has good biocompatibility and can be used as an excellent drug carrier in a lung targeting drug delivery system.

Test example 4 cell phagocytosis assay

(1) Flow cytometry sorting of fluorescently labeled cells

Preparing microgel: the experimental procedure is the same as in example 5, except that Annexin V-FITC is used to oxidize hyaluronic acid (wherein, A-HA: Annexin V-FITC ═ 50:1), and Annexin V-FITC labeled microgel is prepared. Centrifuging and precipitating the prepared microgel to remove supernatant, then respectively taking the microgel into fresh DMEM high-sugar culture medium (containing 10% fetal calf serum), and preparing the microgel medicament concentration into 50 mu g/mL, 100 mu g/mL and 500 mu g/mL. The prepared microgel medicine is placed on a mixing instrument to slowly rotate overnight, and is fully mixed (the steps are operated under the sterile and dark environment). A blank control group was also set.

The method comprises the steps of inoculating RAW cells into a 12-hole plate at a density of 15 ten thousand per hole, culturing the cells in an incubator at 37 ℃ for 12h, setting the three concentrations of microgel into three groups, wherein the administration time of each group of cells is 0.5h, 1h, 2h, 6h, 12h and 24h respectively, arranging two parallel holes at each time point, performing group administration according to the time after plating the cells in the incubator at 37 ℃ for 12h (the cell density is about 50%), and culturing in the incubator at 37 ℃. After the administration time, the supernatant was collected into a 2mL EP tube, PBS was added to the well plate and gently washed twice, 1mL PBS was added, the adherent cells were blown down by gun, the cell suspension was then sucked into a 1.5mL EP tube, centrifuged at 1200rpm for 5min, and the supernatant was decanted. Add PBS to resuspend the cells, centrifuge at 1200rpm for 5min, discard PBS. Add 200. mu.L PBS and place on ice.

Detecting with flow cytometer, and mixing well again before detection. The experimental results are shown in FIG. 7, in which the microgel drug uptake at different times is shown in FIG. 7(a) and FIG. 7(d) at 50 μ g/mL; FIGS. 7(b) and 7(e) are graphs showing the microgel drug uptake at different times at 100 μ g/mL; FIGS. 7(c) and 7(f) show the microgel drug uptake at different times at 500. mu.g/mL.

The key to the success of the microgel delivery system in lung tissue is whether the microgel is taken up by macrophages in the lung. FIG. 7 shows the uptake of the microgel concentration drugs into the RAW264.7 cells at different times, and it can be seen that, compared with the blank group, the uptake of the fluorescent-containing drugs into the cells is not significantly changed within 0.5-24h when the drug concentration is 50 μ g/mL, and the uptake of the fluorescent-containing drugs into the cells is slightly increased but is not significantly increased within 0.5-24h when the drug concentration is 100 μ g/mL. Only when the concentration of the microgel medicine is 500 mu g/mL, the uptake of the medicine containing the fluorescence by cells increases along with the increase of time within 0.5-24h, and the cells obviously phagocytose the microgel medicine. Therefore, the concentration is between 50 mu g/mL and 100 mu g/mL, the uptake rate of the microgel medicine by RAW cells is low, the accumulation of the microgel medicine at a focus part is facilitated, and the bioavailability is further improved.

(2) Laser confocal microscopy sorting of fluorescently labeled cells

The preparation of the microgel material is the same as that in the step 1, the obtained microgel is centrifugally precipitated to remove supernatant, then the microgel is weighed into a fresh DMEM high-sugar culture medium (containing 10% fetal calf serum), and the microgel drug concentration is prepared into 500 mu g/mL. The prepared medicine is placed on a blending instrument to slowly rotate overnight, and is fully mixed (the steps are all operated under the sterile and dark environment).

Preparation of Lyso-Tracker Red working solution: a small amount of Lyso-Tracker Red (1mM) was added to the cell culture medium at a ratio of 1:20000 to give a final concentration of 75nM (the Lyso-Tracker Red working solution was preincubated at 37 ℃ before use and was completely protected from light).

The RAW cells are inoculated in a laser confocal culture dish at the density of 5 ten thousand per hole, the cells are cultured for 12 hours in an incubator at 37 ℃, the administration time of the cells is 6 hours and 24 hours, and each group is provided with two parallel holes. After the administration time, collecting the supernatant into a 2mL EP tube, adding PBS (phosphate buffer solution) into the cells, gently washing the cells twice, adding 1mL of Lyso-Tracker Red working solution, culturing and incubating for 45min in an incubator at 37 ℃, adding PBS, gently washing twice, adding 4% paraformaldehyde, fixing for 15-20min, sucking out the paraformaldehyde solution after fixing, adding PBS, gently washing twice, adding 200 mu L of PBS, and storing at-20 ℃. Laser confocal imaging. The results of the experiment are shown in FIG. 8. As can be seen, the FITC-stained material was not coincident with the Lyso-Tracker fluorescence, demonstrating that the material was not degraded by the lysosomal phagocytosis of macrophages.

Application example

Microgel distribution in lung tissue

Mice were anesthetized with tribromoethanol (anesthetic dose 240mg/kg) and the microgel drug was delivered into the mice by intratracheal administration, and mice at different time points (2 hours, 24 hours, 100 hours) were sacrificed and mouse lung tissue was taken. Fluorescence intensity of mouse lung tissue was measured by an IVIS Spectrum μ CT (Perkin Elmer) three-dimensional multimodal imaging System (PE small animal in vivo).

The microgel medicine is prepared by the following steps: preparation of fluorescently labeled microgel: mixing 75mg of A-HA and 25mg of Cy 3-labeled bovine serum albumin for 2h, adding 100mg of CMCS, mixing, reacting for 2h, and adding 1mL of 6% Zn²⁺Solution, overnight reaction. After the reaction is finished, 1mL of microgel is taken and centrifuged at 12000rpm for 2min, the supernatant is removed, ultrapure water is used for resuspension, the microgel is centrifuged at 12000rpm for 2min, 500 mu L of normal saline is added for resuspension, and the microgel is preserved at 4 ℃).

Meanwhile, a blank control group is set as a drug-free group, and an experimental group is set as a microgel drug administration group.

The results of the experiment are shown in FIG. 9.

As is clear from FIG. 9, the fluorescence intensity gradually decreased with time, and the fluorescence intensity was reduced by about 20% after 24 hours and very weak after 100 hours. It can be seen that the efficiency of microgel drug removal is relatively slow. On one hand, the microgel has better expansion performance and porous space structure, and on the other hand, due to the slow degradation efficiency of the CMCC lung, the material stays and degrades slowly, so that the controllability of the slow release of the medicine can be ensured.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A microgel, wherein the microgel has a chemical structural formula:

2. The microgel as claimed in claim 1, wherein the metal ions are one or more of calcium ions, zinc ions, sodium ions, potassium ions, copper ions, barium ions, aluminum ions and iron ions.

3. A method for preparing a microgel as claimed in claim 1, comprising the steps of: mixing a hyaluronic acid aqueous solution and a chitosan aqueous solution uniformly, adding a cross-linking agent to obtain a reaction solution, and reacting to obtain the microgel, wherein the cross-linking agent is a metal salt, and the mass concentration of the cross-linking agent in the reaction solution is 0.05-6%.

4. The method according to claim 3, wherein the hyaluronic acid is selected from one or more of phosphorylated oxidized hyaluronic acid, low sulfated oxidized hyaluronic acid LS, oxidized and high sulfated hyaluronic acid HS and oxidized hyaluronic acid; wherein the degree of substitution of the low sulfated oxidized hyaluronic acid LS is from 0.5 to 30%; the degree of substitution of the oxidized and highly sulfated hyaluronic acid HS is from 31 to 90%.

5. The method according to claim 3, wherein the chitosan is selected from one or more of carboxymethyl chitosan, acetylated chitosan, N, O-alkylated chitosan, sulfated chitosan, and phosphorylated chitosan.

6. The method according to claim 3, wherein the mass ratio of hyaluronic acid to chitosan is 1:1 to 1: 4.

7. The method according to claim 3, wherein the metal salt is one or more of a calcium salt, a zinc salt, a sodium salt, a potassium salt, a copper salt, a barium salt, an aluminum salt, and an iron ion salt.

8. The method according to claim 7, wherein the metal salt is one or more of calcium chloride, zinc acetate, sodium chloride, sodium sulfate, zinc phosphate, potassium chloride, potassium sulfate, barium chloride, and aluminum sulfate.

9. Use of a microgel as claimed in claim 1 in the preparation of a pharmaceutical carrier.

10. Use of a microgel as claimed in claim 1 in the preparation of a pulmonary delivery system.