WO2022246922A1

WO2022246922A1 - Microgel, preparation method therefor, and application thereof

Info

Publication number: WO2022246922A1
Application number: PCT/CN2021/099657
Authority: WO
Inventors: 闫昳姝; 吴青青; 任盼盼; 张娜; 刘秋熠
Original assignee: 江南大学
Priority date: 2021-05-27
Filing date: 2021-06-11
Publication date: 2022-12-01
Also published as: CN113262198A

Abstract

A microgel and a preparation method therefor. A hyaluronic acid aqueous solution and a chitosan aqueous solution are mixed uniformly, a crosslinking agent is added, and a mixing reaction is performed to obtain a microgel, wherein the crosslinking agent is a metal salt. The microgel can be used in the preparation of a drug carrier, can also be used in the preparation of a pulmonary drug delivery system, and has good biocompatibility.

Description

A kind of microgel and its preparation method and application

technical field

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

Background technique

Inhalation administration is the best way to treat various chronic lung diseases such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, and chronic bronchitis. Compared with the systemic drug delivery method, inhalation drug delivery can directly deliver high-concentration drugs to the disease site, reduce the side effects of the drug on the whole body, and have a fast onset of action, perfectly avoiding conventional drug administration routes such as oral administration and injection administration The resulting therapeutic barriers such as gastrointestinal absorption and liver first-pass elimination, etc., and only a small dose of administration can achieve the effect of systemic administration. In addition, the high compliance of inhalation administration is also an important reason affecting the trend of pharmaceutical preparations.

The lungs and the entire airway offer certain advantages for drug absorption, but inhalational administration is not without obstacles. Inside the upper respiratory tract, the goblet cells under the tracheal mucosa secrete a mucus layer, and the movement of ciliated cells drives the lung mucus to be cleared at a flow rate of 0-5mm/min. The respiratory mucus layer of healthy subjects is replaced every 20 minutes. Can effectively remove insoluble particles in the upper respiratory tract. Located in the main area of gas exchange, such as alveolar and bronchial terminal, it is protected by immune cells and dense capillary network-air-blood barrier (Air-blood Barrier), among which, the most prominent defense mechanism is macrophages. Deposition in the lungs Deep particles are phagocytosed by alveolar macrophages, which then slowly remove the particles and migrate out of the lung along the broncho-tracheal ladder or the lymphatic system. Especially for particles with a geometric size between 1-3 μm, macrophage clearance works best. Therefore, how to avoid the phagocytosis of macrophages is the core issue to solve the efficiency of lung drug delivery.

In patients with airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, the composition, thickness, physicochemical properties (eg, viscosity, composition), and clearance of respiratory mucus are often altered. 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 and modification of drug formulation methods are needed.

Current inhaled drug delivery systems still need further development, and new drug dosage forms suitable for pulmonary administration are still being developed. Therefore, it is the current research hotspot to combine the research and development of new drug carriers, the design of feasible preparation technology and the research of targeted sustained and controlled release drugs, and to explore a new controlled release technology and dosage form.

Contents of the invention

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

A novel microgel, the chemical structural formula of the microgel is:

Wherein, x=4-10000, y=4-100000, m is 1-500, n is 1-500, and x, y, m and n are all integers; the M is a metal ion.

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

Further, x=700-900, y=2000-4000, m is 2-100, and n is 2-100.

Further, it includes the following steps: mixing the aqueous solution of hyaluronic acid and the aqueous solution of chitosan, and adding a cross-linking agent to a mass concentration of 0.05%-6%, and mixing and reacting to obtain a microgel; wherein, the cross-linking agent is metal salts.

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 crosslinking 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 hypersulfated hyaluronic acid HS, and oxidized hyaluronic acid; Wherein the low sulfated oxidized hyaluronic acid LS refers to the sulfated oxidized hyaluronic acid with a substitution degree of 0.5-30%; the oxidized and highly sulfated hyaluronic acid HS refers to a substitution degree of 0.5-30%. 31-90% oxidized and sulfated hyaluronic acid.

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

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

Further, the chitosan is carboxymethyl chitosan, acetylated chitosan.

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

Further, the mass ratio of hyaluronic acid and 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 iron ion salt.

Further, the metal salts are zinc salts, calcium salts, aluminum salts, copper salts, and sodium salts.

Further, the metal salt is one or more of calcium chloride, zinc 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 chloride, zinc acetate, sodium chloride.

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

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

The application of the microgel in the preparation of drug carriers.

The application of the microgel in the preparation of a pulmonary drug delivery system.

In the present invention, reaction process is:

Wherein, x=4-10000, y=4-100000, m is 1-500, n is 1-500, M is a metal ion, and m, n, x and y are all integers.

The present invention utilizes hyaluronic acid and chitosan amino to form a complex through Schiff base, and the metal salt crosslinking agent coordinates with Schiff base outside the complex to form metal salt-oxidized hyaluronic acid-carboxymethyl chitosan complex The microgel structure, the specific structure is shown in the figure.

In the present invention, carboxymethyl chitosan and hyaluronic acid are used as base materials, metal ion solution is used as precipitating agent, and a novel pH-controllable degradable microgel is prepared for pulmonary drug delivery by means of precipitation polymerization Materials of construction.

The above technical solution of the present invention has the following advantages compared with the prior art:

The material prepared by the invention is a microgel particle with a diameter in the range of 1-5 μm, and has the characteristic of controllable degradation at low pH in vitro. The microgel particle material has high histocompatibility with lung epithelial cells and general tissue cells, and will not affect cell growth. No significant toxicity to mice. In addition, the material can escape the phagocytosis of macrophages, and the drug distribution in the lung can reach 48 hours, achieving the goal of slow drug release. For its low pH sensitivity, it can be applied to a variety of chronic lung diseases, such as asthma, COPD, lung tumors and other local overacid conditions, to achieve controlled release of drugs.

Description of drawings

In order to make the content of the present invention more easily understood, the present invention will be described in further detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

Fig. 1 is the research schematic diagram of the present invention.

Fig. 2 is the microgel morphological and structural characterization result diagram prepared in Example 1 of the present invention; wherein, (a) microgel macroscopic structure diagram: the left diagram is the uniform particle dispersion solvent formed in the microgel preparation stage, and the right diagram The gel-like aggregates formed after centrifugation; (b)-(c) are TEM images of microgels in aqueous solution; (d) are the dynamic diameter determination of the obtained microgels; (e)-(f) are SEM images Determination of the microstructure of the microgel lyophilizate.

Fig. 3 is the microgel infrared spectrogram obtained in the embodiment of the present invention 1, wherein (a) is the infrared spectrum of HA (hyaluronic acid) and A-HA (oxidized hyaluronic acid); (b) is CS (chitosan) sugar) and CMCS (carboxymethyl chitosan) infrared spectra.

Fig. 4 is the in vitro degradation and swelling curves of microgels prepared in Example 1 of the present invention, wherein (a) degradation curves of microgels in different buffers; (b) swelling curves of gels with 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) the survival of Beas-2B in different microgels Rate.

Fig. 6 is the microgel cell viability figure described in Test Example 3 of the present invention; Wherein (a) 3T3 is in the viability figure of 4:1 (6% zinc acetate) microgel; (b) 3T3 is in 4: 1 (6% ZnCl) survival rate in microgel; (c) Beas-2B survival rate in 4:1 (6% ZnAcetate) microgel; (d) Beas-2B in 4:1 (6% Zinc Chloride) Survival in Microgels.

Fig. 7 is the drug uptake situation of the microgel described in Test Example 4 of the present invention at different times; wherein, (a) and (d) are the drug uptake situation at different times when the microgel concentration is 50 μg/mL; (b) and (e) is the drug uptake at different times when the microgel concentration is 100 μg/mL; (c) and (f) are the drug uptake at different times when the microgel concentration is 500 μg/mL.

Fig. 8 is the laser confocal images of the RAW cells incubated in the microgel for 500 μg/mL drug concentration for 6 hours and 24 hours respectively in the present invention.

Fig. 9 is the distribution of the microgel in the lung tissue of the mouse in the application example of the present invention; wherein, (a) the fluorescence imaging images of the mouse administered for 2h, 24h, and 100h; (b) the change of the fluorescence intensity of the gel in the mouse Situation diagram.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

The setting of experimental group and control group in the embodiment of the present invention is as follows:

Blank group: only adding PBS solution without adding any cells and drugs.

Control group: cells cultured in medium without drugs.

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

Regarding cell culture in the present invention:

(1) Recovery of cells

Put the frozen storage tube in a 37°C water bath to thaw quickly, open the frozen storage tube, take out the cell suspension and place it in a centrifuge tube, add 5mL of the corresponding medium, centrifuge at 1000rpm for 3min, discard the supernatant, resuspend with the culture medium, and inoculate in In a culture bottle, place it in a 95% air, 5% carbon dioxide incubator, and culture it statically at 35-37°C. During the recovery period, the medium is replaced every 12 hours, and then the medium is replaced according to subculture.

(2) Cell culture

3T3 cells: add DMEM high-glucose medium (containing 10% newborn bovine serum), place in 95% air, 5% carbon dioxide incubator, culture statically at 35-37°C, and change the medium every 2-3 days.

Beas-2B line cells: add 1640 medium, place in 95% air, 5% carbon dioxide incubator, culture statically at 35-37°C, change the medium every 2-3 days.

RAW cells: add DMEM high-glucose medium (containing 10% fetal bovine serum), place in 95% air, 5% carbon dioxide incubator, culture statically at 35-37°C, and change the medium every 2-3 days.

(3) Subculture of cells

3T3 cells: When the fibroblasts grow to more than 70% of the T 75 culture flask, discard the medium, add PBS to wash three times, discard the PBS, add 2 mL of trypsin to digest until the intercellular space becomes larger and the cell shape becomes round, use 10 mL DMEM high-glucose medium (containing 10% newborn bovine serum) was used to stop the digestion, and the cell suspension was formed in a centrifuge, centrifuged at 1000rpm for 3 minutes, the upper culture medium was discarded, and fresh medium was added to resuspend the cells at a ratio of 1:3. Inoculate in a new culture flask and culture in a constant temperature incubator at 37°C with 5% CO ₂ .

Beas-2B cells: When the lung epithelial cells grow to more than 70% of the T75 culture flask, discard the medium, add PBS to wash three times, discard the PBS, add 2 mL of trypsin to digest until the intercellular space becomes larger and the cell shape becomes round Use 10mL 1640 medium to stop the digestion, form a cell suspension in a centrifuge, centrifuge at 1000rpm for 3min, discard the supernatant culture medium, add fresh medium, resuspend the cells, and inoculate them in a new culture bottle at a ratio of 1:3 Cultured in a constant temperature incubator at 37°C with 5% CO ₂ .

RAW cells: When the macrophages grow to more than 70% of the T 75 culture flask, discard the medium, add PBS to wash three times, discard the PBS, and add 10 mL of fresh DMEM high-glucose medium (containing 10% fetal bovine serum) , Gently scrape the adherent cells with a scraper to form a cell suspension, inoculate them in a new culture bottle at a ratio of 1:3 and culture them in a 37°C 5% CO ₂ constant temperature incubator.

(4) Cell cryopreservation

3T3 cells: After digesting and centrifuging in the above steps, make a cell suspension with newborn bovine serum, count the cells, add freezing solution at 1-2× ¹⁰⁴ cells/mL, divide into 2mL cryopreservation tubes, seal, Mark the date and holder. Store in a refrigerator at 4°C for 5-15 minutes, at -20°C for 2 hours, at -80°C for 2 hours, and then store at -196°C for long-term storage.

Beas-2B cells: Except that the serum used is fetal bovine serum, other operations are the same as 3T3.

RAW cells: after resuspension in the above steps, make cell suspension with newborn bovine serum, count the cells, add freezing solution at 1-2×104 cells/mL, aliquot into 2mL cryopreservation tubes, seal and label Good date and holder. Store in a refrigerator at 4°C for 5-15 minutes, at -20°C for 2 hours, at -80°C for 2 hours, and then store at -196°C for long-term storage.

Example

Example 1

At room temperature, 200 mg of carboxymethyl chitosan (CMCS) and 50 mg of oxidized hyaluronic acid (A-HA) were ultrasonically dissolved in 5 mL of water, respectively, to obtain a solution of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid ( A-HA) solution, and the two are mixed, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, and the mixed solution is placed in a magnetic stirrer Mix and incubate for 2 hours, quickly add ZnCl ₂ solution to a final concentration of 6%, continue to stir, and finally prepare a milky white microgel. The obtained microgel was freeze-dried, ground into powder and redissolved in water, and its structure and shape were observed by TEM and SEM. The experimental results are shown in Figure 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 cross-linked by small particles (about 100 nm in diameter). These images further confirm the formation of a microgel system by introducing Zn ²⁺ into the aqueous solution system, and the structural formula of the microgel is x=700-900, y=2000-4000, m=100-200, n=100 -200.

Example 2

At room temperature, 200 mg of carboxymethyl chitosan (CMCS) and 67 mg of oxidized hyaluronic acid (A-HA) were ultrasonically dissolved in an equivalent amount of 5 mL of water, respectively, to obtain a solution of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid. acid (A-HA) solution, and mix the two, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 3:1, and the mixed solution is placed under magnetic force Mix and incubate on a stirrer for 2 hours, quickly add a _ZnCl solution with a mass concentration of 6% to a final concentration of 6%, continue to stir, and finally prepare a yellow microgel. The structural formula of the microgel is x=700-900, y =2000-4000, m=200-300, n=100-300.

Example 3

At room temperature, 200 mg of carboxymethyl chitosan (CMCS) and 100 mg of oxidized hyaluronic acid (A-HA) were ultrasonically dissolved in 5 mL of water, respectively, to obtain a solution of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid ( A-HA) solution, and the two are mixed, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 2:1, and the mixed solution is placed in a magnetic stirrer Mix and incubate for 2 hours, quickly add a _ZnCl solution with a mass concentration of 6% to a final concentration of 6%, continue to stir, and finally prepare a yellow microgel. The structural formula of the microgel is x=700-900, y=2000 -4000, m=1-500, n=1-500.

Example 4

At room temperature, 100 mg of carboxymethyl chitosan (CMCS) and 100 mg of oxidized hyaluronic acid (A-HA) were ultrasonically dissolved in 5 mL of water, respectively, to obtain a solution of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid ( A-HA) solution, and the two are mixed, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 1:1, and the mixed solution is placed in a magnetic stirrer Mix and incubate for 2 hours, quickly add a _ZnCl solution with a mass concentration of 6% to a final concentration of 6%, continue to stir, and finally prepare a slightly yellow microgel. In the structural formula of the microgel, x=10-500, y= 50-100, m=200-500, n=300-500.

Example 5

At room temperature, 200 mg of carboxymethyl chitosan (CMCS) and 50 mg of oxidized hyaluronic acid (A-HA) were ultrasonically dissolved in 5 mL of water, respectively, to obtain a solution of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid ( A-HA) solution, and the two are mixed, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, and the mixed solution is placed in a magnetic stirrer Mix and incubate for 2 hours, quickly add zinc acetate (Zn(Ac) ₂ ) solution to a final concentration of 6%, continue to stir, and finally prepare a slightly yellow microgel. The structural formula of the microgel is x=1000- 2000, y=2000-3000, m=400-500, n=400-500.

Example 6

At room temperature, 200 mg of carboxymethyl chitosan (CMCS) and 50 mg of oxidized hyaluronic acid (A-HA) were ultrasonically dissolved in an equal amount of water respectively to obtain a solution of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) solution, and the two are mixed, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 4:1, and the mixed solution is placed in magnetic stirring Mix and incubate on the container for 2 hours, quickly add _CaCl2 solution to a final concentration of 6% (mass concentration), continue to stir, and finally prepare a milky yellow microgel. In the structural formula of the microgel, x=800-2000, y=500-3000, m=300-400, n=300-400.

Example 7-13

The experimental steps, raw materials and proportioning are the same as in Example 1, the difference is that the cross-linking agent added in Examples 7-13 is _ZnCl2 solution, and the final concentration of _ZnCl2 solution is 4%, 3%, 2% respectively , 1.5%, 1%, 0.5%, 0.05%.

Example 14-15

The experimental steps, raw materials and proportions are the same as those in Example 5, except that the cross-linking agent added in Examples 14-15 is a CaCl ₂ solution, and the mass concentrations are 1.5% and 0.05%, respectively.

Example 16

At room temperature, 200 g of carboxymethyl chitosan (CMCS) and 50 g (HS, oxidized and hypersulfated hyaluronic acid, 31-90%) were ultrasonically dissolved in an equivalent amount of 5 mL of water to obtain carboxymethyl chitosan Sugar (CMCS) solution and oxidized hyaluronic acid (A-HA) solution, and the two are mixed, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 4 : 1, the mixed solution is placed on a magnetic stirrer to mix and incubate for 4h, quickly add _ZnCl solution to a final concentration of 6%, continue to stir, and finally prepare a milky white microgel, the structural formula of the microgel is x =1000-5000, y=4000-6000, m=400-500, n=300-500.

Example 17

At room temperature, 200 g of carboxymethyl chitosan (CMCS) and 50 g (LS, oxidized and low-sulfated hyaluronic acid with a substitution degree of 0.5-30%) were ultrasonically dissolved in 5 mL of water to obtain carboxymethyl chitosan Sugar (CMCS) solution and oxidized hyaluronic acid (A-HA) solution, and the two are mixed, wherein the mass ratio of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (A-HA) in the mixed solution is 4 : 1, the mixed solution is placed on a magnetic stirrer to mix and incubate for 1h, quickly add _ZnCl solution to a final concentration of 6%, continue to stir, and finally prepare a yellow microgel, the structural formula of the microgel is x =5000-8000, y=5000-8000, m=400-500, n=400-500.

Example 18

At room temperature, 200 g of phosphorylated chitosan and 50 g of phosphorylated oxidized hyaluronic acid were ultrasonically dissolved in an equivalent amount of 5 mL of water to obtain a phosphorylated chitosan solution and a phosphorylated oxidized hyaluronic acid solution, and the two were mixed, The mass ratio of phosphorylated chitosan and phosphorylated oxidized hyaluronic acid in the mixed solution is 4:1, the mixed solution is placed on a magnetic stirrer and incubated for 30 minutes, and sodium chloride solution is quickly added to a final concentration of 6%. After stirring, a yellow microgel is finally prepared. The structural formula of the microgel is x=4000-8000, y=5000-10000, m=400-500, n=4-500.

Example 19

At room temperature, ultrasonically dissolve 100 g of sulfated chitosan and 50 g of sulfated oxidized hyaluronic acid in 5 mL of water, respectively, to obtain a sulfated chitosan solution and a sulfated oxidized hyaluronic acid solution, and mix the two, The mass ratio of the sulfated chitosan solution and the sulfated oxidized hyaluronic acid in the mixed solution is 2:1, the mixed solution is placed on a magnetic stirrer and incubated for 4 hours, and the sodium chloride solution is quickly added to a final concentration of 6%. Stirring is continued, and a yellow microgel is finally prepared. The structural formula of the microgel is x=1000-2000, y=40000-50000, m=400-500, n=300-500.

Example 20

At room temperature, ultrasonically dissolve 100 g of sulfated chitosan and 50 g of sulfated oxidized hyaluronic acid in 5 mL of water, respectively, to obtain a sulfated chitosan solution and a sulfated oxidized hyaluronic acid solution, and mix the two, The mass ratio of sulfated chitosan and sulfated oxidized hyaluronic acid in the mixed solution is 2:1, the mixed solution is placed on a magnetic stirrer and incubated for 2 hours, and the copper chloride solution is quickly added to a final concentration of 3%. Continue After stirring, a yellow microgel was finally prepared. In the structural formula of the microgel, x=3000-4000, y=50000-60000, m=400-500, n=400-500.

test case 1

Structural characterization: The lyophilized sample obtained in Example 1 was ground into powder, and characterized by powder infrared diffraction spectroscopy, and the detection wavelength range was set to 500-4000cm ^-1 .

The experimental results are shown in the figure: in Figure 3, Figure 3(a) is the infrared spectrum of HA and A-HA, and the FTIR spectra of HA and A-HA are very similar, which may be due to the formation of hemiacetal, so it is difficult to detect The signal of the aldehyde group in the chain, but due to the increase in the absorption intensity of the whole, it can be judged that the whole has reacted. Figure 3(b) is the infrared spectrum of CS and CMCS, and the absorption peak of CMCS becomes broad at 3138cm ^-1 , indicating that NH and OH form hydrogen bonds, because there is a hydrogen bond in the carboxymethyl chitosan dimer Carboxyl group, so the spectrum of carboxymethyl chitosan is stretched, as shown in the figure, the peak of CS at 1583cm ^-1 is amino group, while the characteristic peak of CMCS is at 1557cm ^-1 , C=O after stretching at 1557cm ^-1 Vibration absorption peaks showed that carboxyl groups (COOH) added C=O groups, indicating that carboxymethyl chitosan had been formed.

test case 2

1. Degradation performance test of microgel

The microgels prepared in Example 1 were divided into two groups, and 35 mg in each group were divided into 1.5 mL centrifuge tubes, and 1 mL of buffer solution (pH=7.4, 0.2M lysozyme solution and pH=4 sodium acetate buffer), placed on a mixer and rotated slowly, and samples were taken at different time points (5min, 20min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h). After centrifugation at 12000 rpm/min for 10 min, the supernatant was removed and the weight of the precipitate was weighed. Assuming that the mass after degradation is W _t and the initial mass is W ₀ , the degradation rate=(W _t -W ₀ )/W ₀ ×100%. The experimental results are shown in Figure 4(a). There are pictures to see. Fig. 4 (a) is the degradation curve of microgel. In sodium acetate buffer solution, microgel degradation percentage rises rapidly from 0 to 64% in 0 to 3 hours, and stabilizes at this value no longer changes; In the lysozyme-containing buffer of 7.4, the degradation curve of the microgel did not change significantly within 0-5h. It can be seen that the degradation of the prepared CMCS-A-HA microgel is affected by the pH of the environment, and the CMCS/A-HA microgel degrades rapidly under the environment of pH 4, and reaches the maximum degree of degradation within 5 hours; Under the condition of adding lysozyme, the microgel will not be degraded basically, which proves that the degradation rate of the microgel is controllable and has acid sensitivity.

2. Swelling and degradation performance test of microgel

After freeze-drying the microgels obtained in Implementation 1-3, the freeze-dried products were divided into 1.5mL centrifuge tubes according to 35mg, and 1mL of PBS solution (pH=7.4) was added, and placed on the mixer to rotate slowly. Samples were taken at different time points (5min, 20min, 30min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h). After centrifugation at 12000 rpm/min for 10 min, the supernatant was removed and the weight of the precipitate was weighed. Assuming that the mass after water absorption is W _t and the initial mass is W ₀ , then the swelling rate %=(W _t -W ₀ )/W ₀ ×100%. The experimental results are shown in Figure 4(b).

It can be seen from Figure 4(b) that the microgel prepared by the present invention has a three-dimensional interpenetrating network structure, contains multiple hydrophilic groups such as hydroxyl groups inside, can absorb a large amount of water, has the property of swelling and insolubility, and can be used as a drug delivery system good drug carrier. Figure 4(b) is the curve of the swelling degree of the microgel prepared in the solution with the mass ratio of CMCS and HA at 2:1, 3:1, and 4:1 versus time: as time increases, within 0-2h, The swelling degree of CMCS and HA 4:1 microgel rises rapidly to 5% and then the line tends to be stable, and the swelling degree of CMCS and HA 3:1 microgel rapidly rises to 4% and then reaches stable swelling Spend. The swelling degree of CMCS and HA 2:1 microgel increases slowly, and basically reaches a stable value, that is, 4% after 6 hours; the swelling degree of CMCS and HA 4:1 microgel is the largest. It can be seen that the prepared microgel is a limited swelling reaction, and the swelling degree of adding different proportions of CMCS and HA in the reaction is stable in the range of 400%-500%, the swelling property is good, and there is no obvious and regular swelling degree difference. The results showed that the addition of different ratios of CMCS and HA had no effect on the swelling properties of the microgels.

Test case 3

MTT experiment

(1), the microgels obtained in Examples 1 to 3 and Examples 14 and 15 are respectively configured into drug groups with a concentration of 100 μg/mL of 4:1, 3:1, and 2: 1. 4:1 (HS), 4:1 (LS) solution. At the same time, a blank control group was set up. The specific experimental operation is as follows:

a, Bears-2B cells were seeded in a 96-well plate at a density of 6×10 ⁴ /mL (about 100 μL per well), and cultured for 12 hours. Aspirate the supernatant, add new DMEM high-glucose medium (containing 10% newborn bovine serum), set the drug concentration to 100 μg/mL, add the above-mentioned microgel drugs respectively, set 5 duplicate wells for each group, and set up 5 replicate wells at 37°C After culturing for 24 hours, the supernatant was sucked off, 100 μL of MTT was added, and cultured at 37° C. in the dark for 3-4 hours. After incubation in the dark, 100 μL DMSO was added to each well, and the absorbance was measured at a wavelength of 570 nm with a microplate reader.

b, 3T3 cells were seeded in a 96-well plate at a density of 6×10 ⁴ cells/mL (about 100 μL per well), and cultured for 12 hours. Aspirate the supernatant, add 1640 medium, set the drug concentration to 100 μg/mL, add the above-mentioned microgel drugs respectively, set up 5 multiple wells in each group, and set up 5 multiple wells in each group, and culture at 37°C for 24 hours, Aspirate the supernatant, add 100 μL MTT, and incubate at 37°C for 4 hours in the dark. After incubation in the dark, 100 μL DMSO was added to each well, and the absorbance was measured at a wavelength of 570 nm with a microplate reader.

The experimental results are shown in Figure 5, wherein 2:1, 3:1, 4:1, 4:1HS and 4:1LS correspond to the microcoagulation obtained in Example 3, Example 2, Example 1 and Example 14 and Example 15 respectively The viability of cells in the gel solution, NC represents the result of the blank control group.

Conclusion: Figure 5(a) shows the survival rate of Beas-2B cells. It can be seen from the figure that: horizontal comparison, the data of the experimental group and the control group have no significant difference, both are around 0.6, and there is no significant difference between the survival rate and the feed ratio of reactants Therefore, it can be considered that when the drug concentration is set to 100 μg/mL, the microgels prepared with different feeding ratios have no or low cytotoxicity to Beas-2B cells, and have good biocompatibility to Beas-2B cells .

Figure 5(b) shows the survival rate of 3T3 cells. It can be seen from the figure that: including the control group, the cell survival rate is close to 1 under various environmental conditions. From this analysis, under the culture conditions of microgels with different components of 100 μg/mL microgel drug concentration, the growth and reproduction of 3T3 cells were not significantly inhibited by activity, which proved that the prepared microgels had less cytotoxicity, Excellent biocompatibility on normal fibroblasts.

(2) Dilute the microgels obtained in Example 1 and Example 5 with PBS buffer solution to 10 μg/mL, 20 μg/mL, 50 μg/mL, 80 μg/mL, 100 μg/mL, 250 μg/mL, 500 μg/mL mL. The above microgel solution was used for MTT experiments in 3T3 cells and Beas-2B cells, and a blank control group was set at the same time. The specific steps are as follows:

a, 3T3 cells were seeded in a 96-well plate at a density of 6×10 ⁴ cells/mL (about 100 μL per well), cultured for 12 hours, the supernatant was sucked off, and new DMEM high-glucose medium (containing 10% newborn bovine Serum), according to the gel drug concentration contained in the 10μg/mL, 20μg/mL, 50μg/mL, 80μg/mL, 100μg/mL, 250μg/mL, 500μg/mL gradient grouping for drug addition, each group set 5 multiple holes. After culturing at 37°C for 24 hours, the supernatant was sucked off, 100 μL of MTT was added, and cultured at 37°C in the dark for 3 hours. After incubation in the dark, 100 μL DMSO was added to each well, and the absorbance was measured at a wavelength of 570 nm with a microplate reader.

b. Seed Beas-2B cells in a 96-well plate at a density of 6×10 ⁴ cells/mL (about 100 μL per well), culture for 12 hours, remove the supernatant, add new 1640 medium, and press the contained condensate The gel concentrations were 10 μg/mL, 20 μg/mL, 50 μg/mL, 80 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL for drug addition in groups, and 5 replicate wells were set up for each group. After culturing at 37°C for 24 hours, the supernatant was aspirated, and 100 μL of MTT was added, and incubated at 37°C in the dark for 3 hours. After incubation in the dark, 100 μL DMSO was added to each well, and the absorbance was measured at a wavelength of 570 nm with a microplate reader.

The experimental results are shown in Fig. 6. Fig. 6(a)-(b) and Fig. 6(c)-(d) are respectively the results of the MTT assay after the 3T3 and Beas-2B cells were incubated with different concentrations and different types of microgels for 24 hours. Compared with the blank control group, there was no significant difference in the absorbance value. It was further demonstrated that the material had no effect on cell growth.

Therefore, in the microgel preparation process, within the concentration range of 10-500 μg/mL, the addition of the two cross-linking agent groups will not cause obvious organic damage to human lung epithelial cells and fibroblasts. It can be concluded that the microgel prepared by the method explored in this experiment has good biocompatibility and can be used as an excellent drug carrier in the lung-targeted drug delivery system.

Test Example 4 Cell Phagocytosis Experiment

(1), Sorting fluorescently labeled cells by flow cytometry

Preparation of microgel: the experimental procedure is the same as in Example 5, the difference is that Annexin V-FITC is used to oxidize hyaluronic acid (wherein, A-HA: Annexin V-FITC=50:1), and Annexin V-FITC-labeled microgel. The microgels prepared above were centrifuged to remove the supernatant, and then the microgels were respectively placed in fresh DMEM high-glucose medium (containing 10% fetal bovine serum), and the drug concentration of the microgels was adjusted to 50 μg/mL, 100 μg /mL, 500μg/mL. The configured microgel drug is placed on a mixer and rotated slowly overnight for thorough mixing (the above steps are all performed in a sterile light-proof environment). At the same time, a blank control group was set up.

The RAW cells were seeded in a 12-well plate at a density of 150,000/well, and the cells were cultured in a 37°C incubator for 12 hours. The microgels with the above three concentrations were set into three groups, and the administration time of each group was 0.5 hours. , 1h, 2h, 6h, 12h, 24h, two parallel wells were set up at each time point, and the plated cells were cultured in a 37°C incubator for 12h (the cell density was about 50%), administered in groups according to the above time, and cultured at 37°C box culture. After the administration time, collect the supernatant into a 2mL EP tube, add PBS to the well plate and wash it twice gently, then add 1mL PBS, blow with gun suction, blow off the adherent cells, and then suspend the cells The solution was pipetted into a 1.5mL EP tube, centrifuged at 1200rpm for 5min, and the supernatant was discarded. Add PBS to resuspend the cells, centrifuge at 1200rpm for 5min, and discard the PBS. Add 200 μL PBS and place on ice.

Use a flow cytometer to detect and mix again before detection. The experimental results are shown in Figure 7, in which Figure 7(a) and Figure 7(d) are microgel drug uptake at different times at 50 μg/mL; Figure 7(b) and Figure 7(e) are at different times at 100 μg/mL Microgel drug uptake; Figure 7(c) and Figure 7(f) show microgel drug uptake at different times at 500 μg/mL.

The key to the success of the microgel drug delivery system in lung tissue is whether the microgel can be taken up by macrophages in the lung. Figure 7 shows the uptake of RAW264.7 cells at different times and different microgel concentrations of drugs. It can be seen that, compared with the blank group, when the drug concentration is 50 μg/mL, the cells are less sensitive to fluorescent drugs within 0.5-24 hours. There was no significant change in the uptake of the fluorescent drug. When the drug concentration was 100 μg/mL, the uptake of the fluorescent drug by the cells increased a little within 0.5-24 hours, but it was not obvious. Only when the microgel drug concentration was 500 μg/mL, the uptake of the fluorescent drug by the cells within 0.5-24 h increased with time, and the cells had obvious phagocytosis of the microgel drug. Therefore, when the concentration ranges from 50 μg/mL to 100 μg/mL, the uptake rate of microgel drugs by RAW cells is low, which helps the accumulation of microgel drugs at the lesion site, thereby improving bioavailability.

(2), Confocal Laser Microscopy Sorting Fluorescently Labeled Cells

The preparation of the microgel material is the same as the above 1. The obtained microgel is centrifuged to remove the supernatant, and then the microgel is weighed in fresh DMEM high-glucose medium (containing 10% fetal bovine serum), and the concentration of the microgel drug is Dubbed 500μg/mL. The prepared medicines were placed on a mixer and rotated slowly overnight for thorough mixing (the above steps were all performed in a sterile light-proof environment).

Preparation of Lyso-Tracker Red working solution: Take a small amount of Lyso-Tracker Red (1mM) and add it to the cell culture medium at a ratio of 1:20000, the final concentration is 75nM (the Lyso-Tracker Red working solution needs to be pre-incubated at 37°C before use) , the whole process is protected from light).

The RAW cells were seeded in a laser confocal culture dish at a density of 50,000/well. The cells were cultured in a 37°C incubator for 12 hours, and the cells were administered for 6 hours and 24 hours. Two parallel wells were set for each group. After the administration time, collect the supernatant into a 2mL EP tube, add PBS to gently wash the cells twice, then add 1mL Lyso-Tracker Red working solution, incubate for 45min in a 37°C incubator, add PBS and gently wash Twice, then add 4% paraformaldehyde to fix for 15-20min, suck out the paraformaldehyde solution after fixation, add PBS to wash gently twice, then add 200μL PBS and store at -20°C. Laser confocal photography. The experimental results are shown in Figure 8. It can be seen from the figure that the FITC-stained material does not overlap with Lyso-Tracker, which proves that the material cannot be phagocytized and degraded by lysosomes of macrophages.

Application example

Microgel Distribution in Lung Tissue

Tribromoethanol was used to anesthetize the mice (the anesthesia dose was 240mg/kg), and the microgel drugs were injected into the mice by intratracheal administration, and then the mice were taken at different time points (2 hours, 24 hours, 100 hours). Mice were sacrificed, and mouse lung tissues were collected. The fluorescence intensity of mouse lung tissue was measured by PE small animal in vivo three-dimensional multimodal imaging system (IVIS Spectrum μCT (Perkin Elmer)).

Among them, the above-mentioned microgel drug is prepared by the following method: preparation of fluorescently labeled microgel: mix 75mg A-HA and 25mg Cy3-labeled bovine serum albumin for 2 hours, then add 100mg CMCS, mix for 2 hours, then add 1mL 6 % Zn ²⁺ solution, overnight reaction. After the reaction is complete, take 1 mL of microgel and centrifuge at 12,000 rpm for 2 min, remove the supernatant, resuspend in ultrapure water, centrifuge at 12,000 rpm for 2 min, add 500 μL of normal saline to resuspend, and store at 4°C).

At the same time, the blank control group was set as the non-administration group, and the experimental group was the microgel drug administration group.

The experimental results are shown in Figure 9.

It can be seen from Fig. 9 that the fluorescence intensity gradually weakens as time goes by, the fluorescence intensity decreases by about 20% after 24 hours, and the fluorescence intensity is very weak after 100 hours. It can be seen that the clearance efficiency of the microgel drug is relatively slow. On the one hand, the microgel of the present invention has better expansion performance and porous space structure; on the other hand, due to the slow degradation efficiency of CMCC lungs, the material stays and degrades slowly, which can ensure the controllability of drug sustained release.

Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims

A kind of microgel, is characterized in that, the chemical structural formula of described microgel is:

Wherein, x=4-10000, y=4-100000, m is 1-500, n is 1-500, and x, y, m and n are all integers; the M is a metal ion.
The microgel according to 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 .
A preparation method of microgel as claimed in claim 1, is characterized in that, comprises the following steps: mixing hyaluronic acid aqueous solution and chitosan aqueous solution, adding cross-linking agent to obtain reaction solution, reacting to obtain microgel Glue, wherein the cross-linking agent is a metal salt, and the mass concentration of the cross-linking agent in the reaction liquid is 0.05%-6%.
The preparation method according to claim 3, wherein the hyaluronic acid is selected from phosphorylated oxidized hyaluronic acid, low sulfated oxidized hyaluronic acid LS, oxidized and hypersulfated hyaluronic acid HS and One or more of oxidized hyaluronic acid; wherein the degree of substitution of the low sulfated oxidized hyaluronic acid LS is 0.5-30%; the degree of substitution of the oxidized and highly sulfated hyaluronic acid HS is 31-90%.
preparation method according to claim 3, is characterized in that, described chitosan is selected from carboxymethyl chitosan, acetylated chitosan, N, O-alkylated chitosan, sulfated chitosan and one or more of phosphorylated chitosan.
preparation method according to claim 3, is characterized in that, the mass ratio of described hyaluronic acid and chitosan is 1:1-1:4.
The preparation method according to claim 3, wherein the metal salt is one or more of calcium salt, zinc salt, sodium salt, potassium salt, copper salt, barium salt, aluminum salt and iron ion salt .
The preparation method according to claim 7, wherein the metal salt is calcium chloride, zinc chloride, zinc acetate, sodium chloride, sodium sulfate, zinc phosphate, potassium chloride, potassium sulfate, barium chloride and one or more of aluminum sulfate.
Application of the microgel as described in claim 1 in the preparation of drug carriers.
Application of the microgel as described in claim 1 in the preparation of a pulmonary drug delivery system.