CN112569179A

CN112569179A - Injectable hydrogel system and preparation method thereof

Info

Publication number: CN112569179A
Application number: CN202011585182.5A
Authority: CN
Inventors: 傅瑶; 陈良波; 庞燕; 严丹
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-03-30
Anticipated expiration: 2040-12-25
Also published as: CN112569179B

Abstract

The invention relates to the technical field of hydrogel, and particularly provides an injectable hydrogel system, wherein the hydrogel system takes injectable hydrogel as a continuous base phase, rosiglitazone is distributed in the continuous base phase, the injectable hydrogel is obtained by self-assembling amphiphile glycerin monostearate in a solvent, the method for preparing the injectable hydrogel system comprises the steps of heating a mixed solution containing rosiglitazone and amphiphile glycerin monostearate, standing and cooling to obtain the injectable hydrogel system, and the use of the injectable hydrogel system for preparing a product for treating meibomian gland dysfunction is provided. The injectable hydrogel system prepared by the invention has feasibility and high efficiency in the prevention and treatment of age-related meibomian gland dysfunction.

Description

Injectable hydrogel system and preparation method thereof

Technical Field

The invention relates to the technical field of hydrogel, in particular to an injectable hydrogel system and a preparation method thereof.

Background

Meibomian Gland Dysfunction (MGD), a "chronic diffuse abnormality of the meibomian gland, is often characterized by obstruction of the peripheral ducts and/or changes in the quality/quantity of glandular secretions. It may lead to changes in tear film, eye irritation symptoms, clinically significant inflammation and ocular surface disease. Meanwhile, meibomian gland dysfunction is also the major cause of dry eye, and more than 2000 million people in the united states have MGD as shown by an epidemiological study in 2001. At present, common means for treating meibomian gland dysfunction are divided into drug treatment and physical treatment, wherein the drug treatment comprises local azithromycin smearing, anti-inflammatory eye drops and the like, the physical treatment comprises eyelid margin cleaning and hot compress, strong pulse laser and the like, and the treatment means has unstable curative effect, long time consumption and high treatment cost. How to achieve the treatment, reduce the treatment frequency and improve the treatment effect has important significance for improving the life quality of the meibomian gland dysfunction patients.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to solve the problems of the prior art.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the present invention, there is provided an injectable hydrogel system having an injectable hydrogel as a continuous base phase, wherein rosiglitazone is distributed in the continuous base phase; the injectable hydrogel is obtained by self-assembly of an amphiphile glyceryl monostearate in a solvent.

The amphiphile glycerol monostearate and rosiglitazone are commercially available reagents.

In a second aspect of the present invention, there is provided a method for preparing the hydrogel system of the first aspect, wherein the method comprises heating a mixed solution containing rosiglitazone and amphiphilic triglyceride monostearate, and standing for cooling to obtain the hydrogel system.

In a third aspect of the invention, there is provided the use of the hydrogel system of the first aspect for the manufacture of a product for the treatment of meibomian gland dysfunction.

As described above, the present invention has the following advantageous effects:

the injectable hydrogel system prepared by the invention is introduced into the treatment of age-related meibomian gland dysfunction, and provides a new administration mode and a new treatment method for the treatment of the meibomian gland dysfunction. The biological safety of the hydrogel system is verified through in vivo and in vitro experiments, and the effect of the system on the prevention and treatment of age-related meibomian gland dysfunction is verified through a series of animal experiments; the technology of metabonomics, proteomics, molecular biology and the like is utilized to clarify the effect of the rosiglitazone medicine in the hydrogel system on the prevention and treatment of meibomian gland dysfunction, the specific effect of the rosiglitazone hydrogel in the process of intervening meibomian gland tissue atrophy is deeply explored, the potential action mechanism of the rosiglitazone hydrogel in the MGD treatment process is clarified, so that the feasibility and the high efficiency of the rosiglitazone hydrogel in the prevention and treatment of age-related meibomian gland dysfunction are clarified, and a new theoretical basis is provided for the clinical prevention and treatment of the age-related meibomian gland dysfunction in the future.

Drawings

FIG. 1 is a schematic diagram of a hydrogel system.

FIG. 2 is a flow chart of the preparation of a hydrogel system.

FIG. 3 is a graph showing the results of the characterization of the hydrogel system.

Figure 4 is a graph of the biocompatibility analysis of the hydrogel system.

FIG. 5 is a graph showing the effect of the hydrogel system on other parts of a rat.

Figure 6 is a graph of the therapeutic effect of the hydrogel system on meibomian glands.

FIG. 7 is a graph depicting the enhancement of PPAR γ expression and the inhibition of the NF-. kappa.B pathway by the hydrogel system.

Figure 8 is a graph of inflammation at the meibomian gland attenuation treatment with the hydrogel system.

FIG. 9 is a graph of MMP-9 activity inhibition following treatment with a hydrogel system.

FIG. 10 is a graph depicting inhibition of MMP-3 activity following treatment with a hydrogel system.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

In a first aspect of the present invention, there is provided a hydrogel containing rosiglitazone, wherein the hydrogel system uses an injectable hydrogel as a continuous base phase, and the rosiglitazone is distributed in the continuous base phase; the injectable hydrogel is obtained by self-assembly of an amphiphile glyceryl monostearate in a solvent.

The amphiphilic substance glycerin monostearate and the rosiglitazone are added into a solvent together, the amphiphilic substance glycerin monostearate is dissolved in the solvent and then self-assembled to obtain the injectable hydrogel, the injectable hydrogel has a layered structure, and the rosiglitazone is positioned in the middle of the layered structure.

In one embodiment, the solvent comprises water and dimethyl sulfoxide.

In one embodiment, the volume ratio of the dimethyl sulfoxide to the water is 1: 3 to 5. The volume ratio of the dimethyl sulfoxide to the water is selected by experimenters according to the use requirement, for example, the volume ratio can be 1: 3-4 or 1: 4 to 5.

Preferably, the volume ratio of the dimethyl sulfoxide to the water is 1: 4.

in one embodiment, the concentration of the amphiphile glyceryl monostearate in the solvent is 90-110 mg/mL. The concentration of the amphiphile monostearate triglyceride in the solvent is selected by experimenters according to the use requirement, and can be 90-95 mg/mL, 95-100 mg/mL, 100-105 mg/mL or 105-110 mg/mL.

Preferably, the concentration of the amphiphile glycerol monostearate in the solvent is 100 mg/mL. At this concentration, the injectable hydrogel obtained is suitable for microinjection into gel and can provide drug release for a suitable period of time.

In one embodiment, the addition amount of the rosiglitazone in the injectable hydrogel system is 9-11 mg/mL. The addition amount of the rosiglitazone can be selected according to the concentration of the lipid secreted by the meibomian gland epithelial cells stimulated by the rosiglitazone, and can be 9-9.5 mg/mL, 9.5-10 mg/mL, 10-10.5 mg/mL or 10.5-11 mg/mL, for example.

Preferably, the addition amount of the rosiglitazone in the injectable hydrogel system is 10 mg/mL. The addition of the rosiglitazone is safe, has better treatment effect and can not damage organisms.

In a second aspect of the present invention, there is provided a method for preparing the hydrogel system of the first aspect, wherein the method comprises heating a mixed solution containing rosiglitazone and amphiphilic triglyceride monostearate, and standing for cooling to obtain the hydrogel.

Adding the amphiphile monostearate triglyceride and the rosiglitazone into a solvent together to obtain a mixed solution, heating the mixed solution until the amphiphile monostearate triglyceride is completely dissolved, standing and cooling, wherein the amphiphile monostearate triglyceride forms the injectable hydrogel with a fiber layer structure through self-assembly, and the rosiglitazone is positioned between chains of the fiber layer amphiphile monostearate triglyceride.

In one embodiment, the mixed solution is heated to 60-70 ℃. The temperature of the mixed solution for heating is selected by experimenters according to the use requirement, and can be 60-65 ℃ or 65-70 ℃ for example.

Preferably, the mixed solution is heated to 65 ℃.

In one embodiment, the heating time is 14-16 min. The experimenter selects the heating time of the mixed solution according to the use requirement, for example, the heating time can be 4-15 min, and can also be 15-16 min.

Preferably, the heating time is 15 min.

The hydrogel system prepared by the invention has the characteristics of injectability and good biocompatibility, and the rosiglitazone contained in the hydrogel system has curative effects on various diseases including tissue repair and inflammation processes. Therefore, the hydrogel system prepared by the invention is used for preparing products for treating meibomian gland dysfunction.

In one embodiment, the hydrogel system is used for preparing a product for delaying atrophy of meibomian glands.

In one embodiment, the hydrogel system is used for preparing a product for maintaining the lipid secretion function of meibomian glands.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Example 1

As shown in fig. 1 and fig. 2, 1g of TG-18 and 100mg of rosiglitazone were weighed and added to a solvent to obtain a mixed solution, the volume of the solvent was 10mL, and the volume ratio of DMSO to water in the solvent was 1: 4, then heating the mixed solution to 65 ℃ for 15 minutes until the TG-18 is completely dissolved, and then when the temperature is reduced to room temperature, an injectable hydrogel system is formed after 10 minutes.

TG-18 in this example was an amphiphile triglyceride monostearate

Example 2

As shown in fig. 3, the material properties of the rosiglitazone hydrogel. Specifically, a, b: preparing blank TG-18 hydrogel and rosiglitazone hydrogel (the preparation process is the same as the above), processing by a vacuum freeze dryer, plating gold on the sample, and shooting by a scanning electron microscope machine, wherein the blank TG-18 hydrogel is observed to be in a sheet layered structure, and the wrapped rosiglitazone drug is in a fibrous structure. c, d: rheological tests on the TG-18 hydrogel and the rosiglitazone hydrogel revealed that the elastic modulus G' rose rapidly as the temperature decreased from 65 ℃, the gelling temperature of the TG-18 hydrogel was 58.21 ℃ and that of the rosiglitazone hydrogel was 60.78 ℃. e: the viscosities of the TG-18 hydrogel and the rosiglitazone hydrogel decreased significantly with increasing shear rate, indicating that both gels had shear thinning behavior suitable for injection (shear rate from 0.1 to 100w/rad/s, test temperature 25 ℃). f: the in vitro drug release curve of the rosiglitazone hydrogel is that an experimental group is PBS containing MMP-9(1mg/mL), MMP-9 is added once every 7 days, a control group is pure PBS, sustained-release liquid is collected according to time points, and the content of rosiglitazone in the sustained-release liquid is detected by using High Performance Liquid Chromatography (HPLC), so that the MMP-9 can obviously promote the release of the rosiglitazone drug, and 50% of the total encapsulated quantity is released at the thirtieth day (P <0.05and P < 0.01). g: the in-vitro decomposition rate of the rosiglitazone hydrogel is observed by monitoring the weight of the rosiglitazone hydrogel, and the treatment of the experimental group and the control group is the same as that of the experimental group and the control group, so that MMP-9 can obviously accelerate the decomposition rate of the rosiglitazone hydrogel. h, i: by adding a fluorescent dye to the TG-18 hydrogel and then injecting the TG-18 hydrogel over the mouse eyelid, and monitoring the fluorescence intensity of the mouse eyelid site using in vivo animal imaging (IVIS) to reflect the residual fluorescent TG-18 hydrogel in the eyelid site, and comparing the decomposition rates of the TG-18 hydrogel in situ on the eyelids of mice of different ages (9M and 12M), it was found that the rosiglitazone hydrogel decomposition rate was significantly faster on the 12M mouse eyelid than on the 9M mouse eyelid site, and that approximately 15% of the initial hydrogel amount was detected on day 14 in the 9M mouse, and that mice of 12 months of age had significant differences from mice of 9 months of age ([ P ] 0.001).

In this example and the following examples, the TG-18 hydrogel is an injectable hydrogel, and the rosiglitazone hydrogel is an injectable hydrogel system wrapped with rosiglitazone drug. MMP-9 is matrix metalloproteinase 9(matrix metalloproteinasenine). 9M was 9 months of age, and 12M was 12 months of age.

Example 3

As shown in fig. 4, specifically, a, b: taking eyelid tissues of a C57/B6J mouse, removing skin, subcutaneous tissues and conjunctiva of the eyelid by using a microscope through microscopic scissors to obtain a simple mouse meibomian gland, then digesting the simple mouse meibomian gland by using collagenase and Dispase enzyme, inoculating the digested simple mouse meibomian gland to a culture dish, and culturing primary meibomian gland epithelial cells of the mouse; co-culturing meibomian gland epithelial cells with TG-18 hydrogel and rosiglitazone hydrogel for 2 days respectively, and then performing dead-live staining and photographing on the cells by using a dead-live staining kit, wherein green fluorescence shows that the cells are live cells, red fluorescence shows that the cells are dead cells, and the rosiglitazone hydrogel or the TG-18 hydrogel can not induce the death of the meibomian gland epithelial cells; c: obtaining eyelid tissues of each group of experimental mice, performing HE staining, and observing staining to find that no obvious inflammatory cell infiltration exists in meibomian gland areas of each group of mice, thereby confirming the in vivo biological safety of the rosiglitazone hydrogel injection.

Example 4

As shown in fig. 5, a: the body weights of the mice in each group have no obvious difference; b: important organs of each group of mice including heart, liver, spleen, lung and kidney are obtained, slicing and HE staining are carried out, the fact that the whole body organ of the mice of the rosiglitazone hydrogel injection group has no obvious lesion is found, and the fact that the rosiglitazone hydrogel injected into the eyelid in situ has no toxicity to the whole body organ of the mice is verified.

Example 5

And (3) verifying the curative effect of the rosiglitazone hydrogel on treating ARMGD. Mice were divided into 4 groups, each of which was a blank Control group (Control), a simple hydrogel injection group (BlankGel), an oral rosiglitazone group (ROSI) and a rosiglitazone hydrogel group (ROSIGel), and normally bred under the same conditions. As shown in fig. 6, a: injection schematic diagram of rosiglitazone hydrogel and TG-18 hydrogel, eyelid in situ injection once every two weeks; b, c: acquiring the upper eyelids of each group of mice, and taking pictures of the meibomian glands by using an eye surface comprehensive analyzer, wherein the meibomian glands of the upper eyelids of the mice in the Control and blank gel groups are obviously fallen or irregular with the age, while the meibomian glands of the mice in the ROSI group and the ROSIGel group are relatively kept intact, and meanwhile, according to the meibomian gland pictures, the meibomian glands of the mice in the ROSIGel group are scored by an eye surface professional doctor, so that the average score of the meibomian glands of the mice in the ROSIGel group is relatively kept at a low level, and the rosiglitazone hydrogel is proved to be capable of delaying the atrophy of the meibomian glands; d, e: OCT embedding and oil red staining are carried out on meibomian gland tissues of the mice, oil in the meibomian gland tissues can be displayed, then the size of a single meibomian gland is sketched and calculated by using ImageJ software, and the fact that the size of the single meibomian gland of the mice is reduced and the atrophy of the single meibomian gland of the ROSIGel group is obviously slowed down is found in the aging process; f, g: the mouse cornea surface is dripped with sodium fluorescein staining solution and washed by PBS, then the fluorescence staining condition of the mouse cornea is observed by using cobalt blue light and photographed, the sodium fluorescein staining condition can reflect the integrity of the cornea epithelium and indirectly reflect the lipid secretion function of the meibomian gland, the mouse cornea epithelium of the ROSIGel group is found to be basically intact, and the spot or sheet sodium fluorescein staining does not occur, so that the rosiglitazone hydrogel can maintain the lipid secretion function of the meibomian gland.

In this example, ARMGD refers to age-related meibomian gland dysfunction.

An oct (optical cutting temperature) embedding medium is a water-soluble mixture of polyethylene glycol and polyvinyl alcohol, and is widely used in immunohistochemical laboratories at present, and the purpose of the embedding medium is to support tissues during frozen sections so as to increase continuity of the tissues and reduce wrinkles and fragmentation.

Example 6

As shown in the experimental results of FIG. 7, the rosiglitazone hydrogel could maintain the PPAR γ expression level of meibomian glands and inhibit NF-. kappa.B signaling pathway. Specifically, a, b: obtaining meibomian gland tissues of each group of mice and carrying out OCT embedding to obtain frozen sections of the meibomian gland tissues of the mice, and then detecting the expression quantity of PPAR gamma in the meibomian gland tissues by using an immunofluorescence staining technology (the frozen sections are unfrozen for 5 minutes at room temperature; PBS is soaked for 10 minutes; an immunohistochemical pen is circled; sealing liquid (10% donkey serum + 0.3% Triton X-100+ PBS) is added), 1 hour at room temperature, primary antibody liquid is added, 4 ℃ is incubated overnight, the PBS is washed for three times, 5 minutes each time; secondary antibody developing liquid is added, 1 hour at room temperature, the PBS is washed for three times, 5 minutes each time; Dapi (nuclear dye)) is added, so that the rosiglitazone hydrogel can effectively maintain the PPAR gamma of the meibomian glands of the mice in the aging process, and the PPAR gamma of the mice in the Control group is obviously reduced; c-f: after the mouse meibomian gland tissue frozen section is obtained, the expression quantity of NF-kB and p-NF-kB in the meibomian gland tissue is detected by immunofluorescence staining, the two proteins of the mouse meibomian gland of the Control group are obviously increased in the aging process, and the expression of NF-kB and p-NF-kB is effectively inhibited by the rosiglitazone hydrogel injection; g: the Westernblot technology is utilized to further prove that the rosiglitazone hydrogel can effectively maintain the expression condition of meibomian gland PPAR gamma in the aging process and inhibit the NF-kB signal channel of meibomian gland tissues.

In this example, PPAR γ is a lipid-sensitive nuclear receptor protein, and plays an important role in regulating differentiation of meibomian gland epithelial cells and lipid metabolism.

NF-kB refers to a nuclear factor kB (nuclear factor kappa-B) protein which can be selectively combined with a B cell kappa-light chain enhancer to regulate the expression of a plurality of genes, and an NF-kB signal channel is reported to be an important channel related to ocular surface inflammation.

Example 7

Collecting eyelid tissues of each group of mice, immersing the eyelid tissue in a protein lysate after separating to obtain meibomian glands, carrying out protein quantification by using a BCA method, providing 30 mu g of the meibomian gland protein sample of each mouse, providing 3 independent samples for each experimental group, and detecting the expression conditions of 6 proinflammatory factors in the meibomian gland tissues of each group of mice by using an inflammatory factor protein chip technology, wherein the experimental result is shown in figure 8, the rosiglitazone hydrogel can effectively inhibit the inflammation of the meibomian gland tissues, and the rosiglitazone hydrogel injection is found to effectively inhibit the secretion of the 6 proinflammatory factors.

Example 8

As shown in the experimental results of FIG. 9, the glitazone hydrogel can inhibit the secretion of MMP-9 in the meibomian gland region. Specifically, a, b: obtaining meibomian gland tissues of all groups of mice, carrying out OCT embedding, obtaining mouse meibomian gland tissue frozen sections, analyzing the expression condition of MMP-9 in meibomian gland regions of all groups of mice by using an immunofluorescence staining technology, and finding that MMP-9 in the meibomian glands of the mice can be obviously accumulated in the aging process, and the rosiglitazone hydrogel can effectively inhibit the secretion of MMP-9.

Example 9

As shown in the experimental results of FIG. 10, the rosiglitazone hydrogel could inhibit the secretion of MMP-3 in the meibomian gland region. Specifically, a, b: obtaining meibomian gland tissues of all groups of mice, carrying out OCT embedding, obtaining mouse meibomian gland tissue frozen sections, analyzing the expression condition of MMP-3 in meibomian gland regions of all groups of mice by using an immunofluorescence staining technology, and finding that MMP-3 in the meibomian glands of the mice can be obviously accumulated in the aging process, and the rosiglitazone hydrogel can effectively inhibit the secretion of MMP-3.

In this example, MMP-3 is referred to as matrix metalloproteinase 3(matrix metalloproteinasethree).

While the foregoing is directed to the preferred embodiment of the present invention, rather than to any specific form or essential limitation thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined in the appended claims. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims

1. An injectable hydrogel system characterized by: the hydrogel system takes injectable hydrogel as a continuous base phase, and rosiglitazone is distributed in the continuous base phase; the injectable hydrogel is obtained by self-assembly of an amphiphile glyceryl monostearate in a solvent.

2. The injectable hydrogel system of claim 1, wherein: the solvent includes water and dimethyl sulfoxide.

3. An injectable hydrogel system according to claim 2 wherein: the volume ratio of the dimethyl sulfoxide to the water is 1: 3 to 5.

4. The injectable hydrogel system of claim 1, wherein: the concentration of the amphiphile monostearate triglyceride in the solvent is 90-110 mg/mL.

5. The injectable hydrogel system of claim 1, wherein: the addition amount of the rosiglitazone in the injectable hydrogel system is 9-11 mg/mL.

6. A method of preparing an injectable hydrogel system according to any of claims 1 to 5, characterized in that: the method comprises the steps of heating a mixed solution containing rosiglitazone and amphiphilic substance glycerin monostearate, standing and cooling to obtain the injectable hydrogel system.

7. The method of claim 6, wherein the mixed solution is heated to 60-70 ℃.

8. The method of claim 6, wherein the heating time is 14-16 min.

9. Use of the injectable hydrogel system according to any one of claims 1 to 5 for the preparation of a product for the treatment of meibomian gland dysfunction.

10. Use of the injectable hydrogel system according to any one of claims 1 to 5 for the preparation of a product having at least one of the following effects:

delay atrophy of meibomian glands;

maintaining the lipid secretion function of meibomian glands.