CN116239670A - Sulfonated albumin derivative and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and in particular relates to a sulfonated modified albumin derivative and application thereof. The derivatives introduce sulfonic acid groups into albumin molecular frameworks, so that the derivatives can inhibit M1 type macrophages from secreting inflammatory factors, and show anti-inflammatory activity. The protein derivative can be used as a functional material or a medicine for treating and/or preventing macrophage M1 type polarization related inflammatory diseases, and has application value.

Description

Sulfonated albumin derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to an albumin derivative modified by a sulfonated structure and application thereof.

Background

Macrophages, an important regulatory mediator of self-defense and repair of the body, can activate to form M1 and release pro-inflammatory substances through the classical pathway, develop and sustain inflammation to combat injury; m2 type macrophages are anti-inflammatory, and through secretion of cell surface molecules, anti-inflammatory active substances such as IL-10 and the like, inflammation is reduced to restore normal functions of the organism. However, when macrophages are in the M1 form for a long period of time, chronic inflammation is exhibited, and there is a great damage to the human body, such as rheumatoid arthritis and osteoarthritis. At present, the most widely used drugs in clinic are nonsteroidal anti-inflammatory drugs and glucocorticoids, and have more side effects after long-term administration, and are manifested by gastrointestinal damage, central obesity and the like. In addition, autoimmune diseases such as rheumatoid arthritis often use immunosuppressive drugs, but systemic immunosuppression and high price often place a great burden on patients and their families. Thus, there is a strong need for a safe, effective and inexpensive substance that inhibits macrophage M1 polarization, thereby treating and/or preventing the development of macrophage M1 polarization-related diseases.

Albumin, including human serum albumin, recombinant human serum albumin, bovine serum albumin, porcine serum albumin, etc., has been widely used because of its low immunogenicity, high biocompatibility, low cost, etc. Currently, the use of albumin is mainly focused on two aspects: 1) As a carrier, the medicine is encapsulated; 2) The chemical bonding medicine can reduce toxicity of the medicine and improve bioavailability. However, there have been few studies on structural modification thereof to impart a specific therapeutic effect to albumin itself.

Aiming at the problems, the invention firstly proposes that the albumin is subjected to sulfonation modification, and substitution reaction is carried out according to carboxyl, amino or sulfhydryl groups on an albumin molecular chain, so that albumin derivatives with different substitution degrees are obtained. In vivo and in vitro experiments show that the albumin derivatives can inhibit M1 type macrophages, so that proinflammatory substances released by the albumin derivatives, including active oxygen and proinflammatory cytokines, show remarkable anti-inflammatory activity, can be applied to various inflammatory diseases, and have great application prospects. The albumin derivatives have not been reported in any literature and patent.

Disclosure of Invention

It is an object of the present invention to provide an albumin derivative having a sulfonic acid group structure. The derivative can inhibit M1 type macrophage, exert anti-inflammatory activity, and can be used as a functional material or pharmaceutical adjuvant for inflammatory diseases related to macrophage M1 type polarization.

It is another object of the present invention to provide a process for the preparation of the above sulfonated albumin derivatives.

It is a further object of the present invention to provide the use of the above sulfonated albumin derivatives and compositions thereof in inflammatory diseases.

The sulfonated albumin derivatives include, but are not limited to, human serum albumin, recombinant human serum albumin, bovine serum albumin and porcine serum albumin.

The sulfonic acid group donor is characterized in that the sulfonic acid derivative is provided with a sulfonic acid group at one end and a group is coupled with albumin at the other end, and the sulfonic acid group donor comprises but is not limited to carboxyl, amino or aldehyde groups, preferably carboxyl. The chemical structure is as follows:

wherein n=0 to 20, preferably n=0 to 10.R is R ₁ Coupling groups that are substituted with albumin, including but not limited to carboxyl, amino, or aldehyde groups; y is a hydrogen ion, a metal ion or a mixture of a hydrogen ion and a metal ion, preferably sodium ion.

The sulfonated albumin derivative, wherein the coupling group connected with the sulfonic acid donor molecule in the albumin molecular chain is amino, carboxyl or sulfhydryl, preferably amino. The structure is schematically as follows:

where n=0 to 20 and m=1 to 60.Y is a hydrogen ion, a metal ion or a mixture of a hydrogen ion and a metal ion, preferably sodium ion.

The sulfonated albumin derivative is further preferably prepared by reacting an amino group on an albumin molecular chain with a carboxyl group in a sulfonic acid group donor molecule to provide a certain number of sulfonic acid groups, and inhibiting M1 type macrophages, which is schematically shown as follows:

where m=1 to 60.

The preparation method of the sulfonated albumin derivative comprises the following steps:

dissolving or dispersing albumin in water or mixed solvent of organic solvent, performing substitution reaction with amino group or carboxyl group or other groups in sulfonic acid group donor molecule and amino group, carboxyl group or sulfhydryl group in albumin molecular chain, removing impurities by dialysis bag, and lyophilizing. The reaction time and the feed ratio are controlled, and the albumin derivatives with different substitution degrees can be obtained.

The substitution degree calculating method is obtained by detecting the content change of free alpha-amino in albumin molecules before and after modification by an ninhydrin chromogenic method. The albumin molecule contains about 60 alpha-amino groups, and the number of sulfonic acid groups modified on the albumin molecule, namely the value of m, is determined according to the substitution percentage obtained by the ninhydrin method.

The sulfonated albumin derivative is characterized by being used for treating and/or preventing macrophage M1 type polarization related diseases, preferably inflammatory diseases including but not limited to atopic dermatitis and rheumatoid arthritis, preferably rheumatoid arthritis.

The substance for treating and/or preventing macrophage M1 type polarization-related disease includes, but is not limited to, a drug, a sulfonated albumin derivative preparation or a sulfonated albumin derivative pharmaceutical composition, preferably a sulfonated albumin derivative gel.

The sulfonated albumin derivative gel, preferably the gel matrix is hyaluronic acid, and is characterized by being of medical grade.

Advantageous effects

The invention surprisingly found that BSA itself can promote RAW 264.7 cells activated by LPS to release more active oxygen and pro-inflammatory cytokines, and the effect is obviously reversed after being modified by a sulfonated structure, namely, sBSA can obviously reduce the active oxygen level and the pro-inflammatory factor release level of cells in a positive model group and presents a dose-dependent characteristic. The importance of sulfonated structure modification is further illustrated by the ability to lower reactive oxygen and pro-inflammatory factor levels below the negative group following incubation with higher concentrations of sBSA (100. Mu.g/mL). Furthermore, there was no significant difference in the anti-inflammatory effect of sBSA from that of sBSA/HA, probably because the lower content of HA in the gel system was insufficient to reflect its anti-inflammatory activity, and there was no significant difference in the inflammatory level of the HA matrix control group from that of the positive group, which was also directly demonstrated. In addition, sulfonation is a protein substitution mode, and is not reported, and reversion occurs after sulfonation, and the reversion can be related to the formation of a derivative structure.

Drawings

FIG. 1 is a circular dichroism spectrum of sulfonated bovine serum albumin.

FIG. 2 scanning electron microscopy images of sulfonated bovine serum albumin and its hyaluronic acid gel (hyaluronic acid (a), sulfonated bovine serum albumin (b) and sulfonated bovine serum albumin hyaluronic acid gel (c)).

Fig. 3raw 264.7 intracellular reactive oxygen species levels (mean±sd, n=4, compared to positive group: ^ns p＞0.05， ^* p＜0.05， ^** p＜0.01， ^*** p is less than 0.001; comparison with the negative group: ^### p＜0.001)。

FIG. 4 proinflammatory factor expression levels (TNF-. Alpha. (a), IL-6 (b) and IL-1β (c), mean.+ -. SD, n=6) in RAW 264.7 cells compared to positive group: ^ns p＞0.05， ^* p＜0.05， ^** p＜0.01， ^*** p is less than 0.001; comparison with the negative group: ^## p＜0.01， ^### p＜0.001)。

FIG. 5 clinical index change (clinical score (a), knee diameter (b), paw thickness (c) of rats treated with sulfonated albumin derivatives and hydrogels thereof for rheumatoid arthritis by injecting the same into the joint cavity) And ankle diameter (d), mean±sd, n=6, ^ns p＞0.05， ^# p＜0.05， ^## p＜0.01， ^### p < 0.001 compared to disease group: ^* p＜0.05， ^** p＜0.01， ^*** p＜0.001； ^## p＜0.01， ^### p＜0.001)。

FIG. 6 knee joint histological analysis of rats treated with sulfonated albumin derivatives and hydrogels thereof (H & E staining (a) and type II collagen immunohistochemical staining (b)) by intra-articular injection.

Detailed Description

The present invention is further illustrated by the following specific examples, but the present invention is not limited to the following examples.

sHSA: sulfonated human serum albumin;

example 1

Respectively dissolving human serum albumin and 3-sulfonic group benzoic acid disodium salt monohydrate containing carboxyl into a proper amount of phosphate buffer solution (PBS, pH 7.2), dropwise adding the phosphate buffer solution into a human serum albumin solution, performing an amide reaction by utilizing amino in the human serum albumin and carboxyl in a sulfonic group donor, reacting for 10-30min, placing the solution into PBS (pH 7.2), dialyzing for 48h, and freeze-drying to obtain the final product.

Determination of the degree of substitution:

the substitution degree of the prepared human serum albumin sulfonic acid group is tested, and the specific method is as follows:

human serum albumin is used as a standard substance, and purified water is used for dissolving to prepare a series of standard protein solutions with different concentrations. 1mL of glacial acetic acid buffer solution (pH 5.4) is taken and mixed uniformly with 1mL of standard protein solution and 2% ninhydrin color development solution respectively, the mixture is heated in a water bath at 100 ℃ for 15min, and after the mixture is stood and cooled to room temperature, 5mL of 60% ethanol is added for dilution, the absorbance A of the solution is measured at 570nm, and the measurement is repeated for 3 times.

The free amino groups in the ninhydrin and the albumin can form a blue-violet substance under the heating condition, and the content of the free amino groups in the albumin and the sulfonated albumin derivative can be detected by utilizing a color reaction, so that the substitution degree is calculated.

The substitution degree formula (1) is as follows:

as is clear from the calculation of the formula (1), the degree of substitution of the sulfonated human serum albumin prepared in example 1 is 42.89.+ -. 8.23%.

The structure of the sulfonated human serum albumin derivative prepared in example 1 is schematically shown as follows:

wherein m is determined to be 21 to 31 based on the determined percentage of substitution of 42.89.+ -. 8.23% and the unmodified albumin molecule contains 60 α -amino groups.

Example 2

Respectively dissolving human serum albumin and 3-sulfonic acid disodium salt monohydrate containing carboxyl into a proper amount of phosphate buffer solution (PBS, pH 7.2), dropwise adding the former, reacting for 30-60min, dialyzing the solution in PBS (pH 7.2) for 48h, and freeze-drying.

Determination of the degree of substitution:

the measurement method is the same as that of example 1, and the sulfonation degree of the human serum albumin prepared in example 2 is 54.19 +/-8.02%.

The structure of the sulfonated human serum albumin derivative prepared in example 2 is schematically shown as follows:

wherein m is determined to be 28 to 37 based on the determined percentage of substitution of 54.19.+ -. 8.02% and the unmodified albumin molecule contains 60 α -amino groups.

Example 3

Respectively dissolving bovine serum albumin and 3-sulfonic acid disodium salt monohydrate containing carboxyl into a proper amount of phosphate buffer solution (PBS, pH 7.2), dropwise adding the phosphate buffer solution into the bovine serum albumin solution, performing an amide reaction for 20-40min, placing the solution into PBS (pH 7.2), dialyzing for 48h, and freeze-drying to obtain the product. Determination of the degree of substitution:

the measurement method was the same as in example 1, and the sulfonation degree of substitution of the bovine serum albumin obtained in example 3 was 63.37.+ -. 7.08%.

The structure of the sulfonated bovine serum albumin derivative prepared in example 3 is schematically shown below:

wherein m is determined to be 34 to 42 based on the determined percentage of substitution of 63.37.+ -. 7.08% and the unmodified albumin molecule contains 60 α -amino groups.

Example 4

Respectively dissolving bovine serum albumin and 3-sulfonic acid disodium salt monohydrate containing carboxyl into a proper amount of phosphate buffer solution (PBS, pH 7.2), dropwise adding the phosphate buffer solution into the bovine serum albumin, reacting for 30-90min, dialyzing the solution in PBS (pH 7.2) for 48h, and freeze-drying to obtain the bovine serum albumin.

Determination of the degree of substitution:

the measurement method was the same as in example 1, and the sulfonation degree of substitution of the bovine serum albumin obtained in example 4 was 70.24.+ -. 7.29%.

The structure of the sulfonated bovine serum albumin derivative prepared in example 4 is schematically shown below:

wherein m is determined to be 38 to 47 based on the determined percentage of substitution of 70.24.+ -. 7.29% and 60 α -amino groups in the unmodified albumin molecule.

Example 5

Respectively dissolving bovine serum albumin and 3-sulfonic acid disodium salt monohydrate containing carboxyl into a proper amount of phosphate buffer solution (PBS, pH 7.2), dropwise adding the phosphate buffer solution into the phosphate buffer solution, reacting for 60-90min, dialyzing the solution in PBS (pH 7.2) for 48h, and freeze-drying to obtain the final product.

Determination of the degree of substitution:

the measurement method was the same as in example 1, and the sulfonation degree of substitution of bovine serum albumin obtained in example 5 was 73.12.+ -. 6.53%.

The structure of the sulfonated bovine serum albumin derivative prepared in example 5 is schematically shown below:

wherein m is determined to be 40 to 48 based on the determined percentage of substitution of 73.12.+ -. 6.53% and the unmodified albumin molecule contains 60 α -amino groups.

Example 6

37.5mg of the sulfonated human serum albumin (sHSA) lyophilized powder prepared in example 1 was weighed and dissolved in 1.0g of purified water. 0.1g carbomer was weighed, 9.9g purified water was added to fully swell, and triethylamine was used to adjust pH to 6-7. Taking 0.2g of swelled carbomer, adding 0.8g of sHSA solution, stirring uniformly to obtain sHSA carbomer gel, and freeze-drying to obtain sHSA carbomer gel freeze-drying agent.

Example 7

37.5mg of sHSA lyophilized powder prepared in example 2 was weighed and dissolved in 1.0g of phosphate buffer (pH 7.2). 0.1g of poloxamer 407 was weighed and added with 0.9g of phosphate buffer (pH 7.2) to swell well. Taking 0.2g of swelled poloxamer 407, adding 0.8g of sHSA solution, stirring uniformly to obtain sHSA poloxamer gel, and freeze-drying to obtain sHSA poloxamer gel freeze-drying agent.

Example 8

Weighing a proper amount of sulfonated bovine serum albumin (sBSA) freeze-dried powder and hyaluronic acid prepared in example 3 in a penicillin bottle according to a mass ratio of 10:1, adding a phosphate buffer (pH 7.2) for swelling to ensure that the concentration of sBSA is 30mg/mL and the concentration of hyaluronic acid is 3mg/mL, thus obtaining sBSA hyaluronic acid gel, and freeze-drying to obtain sBSA hyaluronic acid gel freeze-dried.

Example 9

Weighing a proper amount of sBSA lyophilized powder and hyaluronic acid prepared in example 4 according to a mass ratio of 6.7:1 in a penicillin bottle, adding a phosphate buffer (pH 7.2) for swelling to ensure that the concentration of sBSA is 30mg/mL and the concentration of hyaluronic acid is 4.5mg/mL, thus obtaining sBSA hyaluronic acid gel, and lyophilizing to obtain sBSA hyaluronic acid hydrogel lyophilized preparation.

Example 10

Weighing a proper amount of sBSA lyophilized powder and hyaluronic acid prepared in example 5 according to a mass ratio of 4.8:1 in a penicillin bottle, adding a phosphate buffer (pH 7.2) for swelling to ensure that the concentration of sBSA is 30mg/mL and the concentration of hyaluronic acid is 6.25mg/mL, thus obtaining sBSA hyaluronic acid gel, and lyophilizing to obtain sBSA hyaluronic acid hydrogel lyophilized preparation.

Example 11

The following are assays for cellular anti-inflammatory activity of a partial series of sulfonated albumin derivatives and compositions thereof.

The experimental method comprises the following steps:

1. active oxygen level determination

Taking RAW 264.7 cells in logarithmic growth phase according to 2×10 ⁵ The cells were inoculated into 24-well plates and cultured for 24 hours to attach the cells to the wall and enter the logarithmic phase, and each of the other groups except the negative control group was replaced with a culture medium containing Lipopolysaccharide (LPS) at a concentration of 1. Mu.g/mL for 4 hours. The culture medium in the experimental group is replaced by a complete culture medium containing 100 mug/mL of sulfonated albumin derivative, and specifically, the lyophilized preparation of the sulfonated albumin derivative or hydrogel thereof is diluted by the complete culture medium according to the actual mass ratio. The positive control group was replaced with complete medium. The negative control group was supplemented with cells only and complete medium. After 24h of culture in the incubator, the average fluorescence intensity in the cells was detected by flow cytometry using an active oxygen kit, thereby reflecting the level of active oxygen, and the lower the average fluorescence intensity, the lower the level of active oxygen in the cells, and repeating 4 times per group.

2. Measurement of expression level of proinflammatory factor

Taking RAW 264.7 cells in logarithmic growth phase according to 2×10 ⁵ The cells were inoculated into 48-well plates and cultured for 24 hours to adhere the cells to the walls and enter the logarithmic phase, and each of the other groups except the negative control group was replaced with a culture solution containing LPS at a concentration of 1. Mu.g/mL for 4 hours. The culture medium in the experimental group is replaced by a complete culture medium containing sulfonated albumin derivative with the concentration of 100 mug/mLIs prepared by diluting freeze-dried preparations of sulfonated albumin derivatives or hydrogels thereof with complete culture medium according to the actual mass ratio. The positive control group was replaced with complete medium. The negative control group was supplemented with cells only and complete medium. After culturing in an incubator for 24 hours, cell supernatants were taken, and the expression levels of three pro-inflammatory factors TNF-alpha, IL-6 and IL-1 beta were detected using ELISA kit, and each group was repeated 6 times.

The following table is data on the anti-inflammatory activity of partially sulfonated albumin derivatives and compositions thereof.

TABLE 1 anti-inflammatory Activity of sulfonated Albumin derivatives and compositions thereof (mean+ -SD)

As shown in Table 1, the sulfonated albumin derivatives with different substitution degrees all show obvious functions of inhibiting the M1 type macrophages activated by LPS, show good antioxidant activity and capability of inhibiting the secretion of proinflammatory cytokines, especially inhibit the secretion of IL-6, and show that the structural modification of the sulfonic acid groups can endow albumin with the function of inhibiting the M1 type macrophages, show obvious anti-inflammatory activity and have great application value.

To investigate the presence of dose dependency of the sulphonated albumin derivatives, we therefore randomly selected examples 4 and 9 for intensive studies, the group of additional experiments was: BSA solution (100. Mu.g/mL), BSA solution (50. Mu.g/mL), example 4 and example 9, which correspond to sBSA of 50. Mu.g/mL, were additionally provided with a hyaluronic acid control group, and the intracellular active oxygen and the levels of pro-inflammatory factors in the cell supernatants were examined, and the results are shown in FIGS. 3 and 4.

The literature reports that albumin has both "positive" and "negative" effects in disease progression, possibly contributing to the release of more pro-inflammatory mediators by inflammatory cells, exacerbating inflammation ^[1,2] . The results in figures 3 and 4 show that BSA itself can promote the release of more reactive oxygen species and pro-inflammatory cytokines by LPS-activated RAW 264.7 cells. However, this effect is significantly reversed after modification with the sulphonated structure, i.e. sBSA significantly reduces positivityReactive oxygen species and pro-inflammatory factor release levels in the model group cells and exhibit dose-dependent characteristics. The importance of sulfonated structure modification is further illustrated by the ability to lower reactive oxygen and pro-inflammatory factor levels below the negative group following incubation with higher concentrations of sBSA (100. Mu.g/mL). Furthermore, there was no significant difference in the anti-inflammatory effect of sBSA from that of sBSA/HA, probably because the lower content of HA in the gel system was insufficient to reflect its anti-inflammatory activity, and there was no significant difference in the inflammatory level of the HA matrix control group from that of the positive group, which was also directly demonstrated. EXAMPLE 12 establishment of murine atopic dermatitis model and evaluation of therapeutic Effect of sulfonated Albumin derivative and composition thereof

BALB/c mice of 6-8 weeks of age were randomly divided into 4 groups of 6 animals each, each of which was a blank group, model group, sHSA group (example 2), sHSA gel group (example 7). An acetone/olive oil=4:1 (v/v) solution was prepared as a matrix solution, and 1% and 0.5% Dinitrochlorobenzene (DNCB) was prepared as an atopic dermatitis inducer solution. All mice were depilated (2 cm. Times.2 cm) on the back using depilatory cream prior to modeling and administration. On days 1,3 and 7, 200 μl of 1% DNCB was applied to the back of the mice, 20 μl of 1% DNCB was applied to the right ear of the mice, and the blank group was applied with matrix solution; normal rearing was not treated on days 8 to 14; two days between day 15 and 28, mice were smeared with 0.5%50 μl DNCB on their backs, and 0.5%10 μl DNCB treated right ears for disease symptoms, with the blank group smeared with matrix solution.

Two weeks after the start of molding, 200 μl of sHSA (30 mg/mL) solution (example 2), sHSA gel (example 7), blank and model groups were given the same amount of solvent every two days on the backs of the mice. Until the end of the experiment on day 28. The extent of swelling of the right ear of the mice was measured on days 14, 21 and 28, respectively, data were recorded while the extent of skin damage on the back of the mice was scored, and the extent of skin damage was evaluated for each mouse from four symptoms of dryness/desquamation, rash/hemorrhage, ulceration, edema, respectively. Each symptom was scored from four classes of no (0 score), mild (1 score, symptom identified), moderate (2 score, symptom identified easily) and severe (3 score, symptom identified), the scores obtained for the four symptoms added together, ranging from 0 to 12 total score.

Tables 2 and 3 below show the therapeutic effects of sHSA and sHSA gels on atopic dermatitis mice.

Table 2 changes in ear swelling in atopic dermatitis mice (mean±sd, n=6)

Table 3 change in back skin loss score of atopic dermatitis mice (mean±sd, n=6)

From tables 2 and 3 above, it is clear that sHSA and its poloxamer gel also have significant anti-inflammatory activity when applied topically to treat atopic dermatitis in mice, and the symptoms such as dry back, erythema, ulceration and edema in mice are all significantly alleviated, while the simple sHSA has significant anti-inflammatory effects, and the gel may have better anti-inflammatory effects in the initial stage of treatment than the solution due to its adhesion, while the sHSA solution and its gel both can alleviate the symptoms of atopic dermatitis to a considerable extent in the final stage of treatment, further demonstrating the anti-inflammatory activity and clinical application value of sHSA. EXAMPLE 13 evaluation of therapeutic effects of collagen-induced rheumatoid arthritis rats injected with sulfonated Albumin derivatives and compositions thereof

The bovine type II collagen (2 mg/mL) and Freund's complete adjuvant are mixed uniformly to form an emulsion which is injected subcutaneously to the root of SD rat tail twice according to the ratio of 1:1 (v/v), the initial injection is 0.2mL, and the immunity is enhanced by 0.1mL after one week, so that a rat rheumatoid arthritis model is induced, after 14 days of initial molding, the rat shows obvious foot swelling, the ankle and knee joint diameters are increased, and the clinical score is increased, namely the molding is successful.

The sBSA and sBSA/HA lyophilized powders prepared in examples 4 and 9, respectively, were reconstituted with an appropriate amount of PBS (pH 7.2) to prepare a solution (3% sBSA) and gel (3% sBSA/HA) having a sBSA concentration of 30mg/mL, and a gel (1.5% sBSA/HA) having a sBSA concentration of 15 mg/mL. Meanwhile, an HA matrix group and a Methotrexate (MTX) group, which were consistent with the HA concentration in the 3% bsa/HA gel group, were set as controls for exploring the therapeutic effect of collagen-induced rheumatoid arthritis rats. Specifically, rats were randomly divided into 7 groups of 6 animals each, which were respectively a disease group, a healthy group, an HA matrix group, a 3% sBSA gel group, a 1.5% sBSA gel group, a 3% sBSA solution group and an MTX group (dose of 1 mg/kg), each group was injected with 0.1mL of the corresponding formulation every two weeks, and the disease group and the healthy group were injected with phosphate buffer 2 times in total. Clinical scores of rats were recorded every 3 days from the first day of molding (scoring rules see table 4), while foot thickness of both hind limbs, ankle diameter and knee diameter of rats were recorded, and the average value was taken for recording.

TABLE 4 collagen-induced rheumatoid arthritis rats clinical score

The clinical scores, foot thickness, ankle diameter and knee diameter for the period of 6 weeks total from molding to end of dosing are shown in figure 5. At the end of treatment, the data for each clinical index are shown in Table 5, and the results of H & E sections and type II collagen (Col II) immunohistochemical sections of the femoral tissue of each group of rats are shown in FIG. 6.

Table 5 clinical scores for each group of treatment terminals, foot thickness, ankle diameter and knee diameter (mean±sd, n=6)

The results show that the 3% sBSA/HA gel can significantly improve the disease state of rats, the clinical score is significantly reduced after the first joint cavity injection, the conditions of foot swelling and ankle and knee joint enlargement are also significantly reversed, and the treatment end and healthy rats have no significant difference. The therapeutic effect of sBSA/HA gel is dose-dependent, and the 1.5% sBSA/HA gel group HAs obvious arthritis therapeutic effect, but the treatment end still HAs slight degreeArthritis. The anti-inflammatory effect of the 3% sBSA solution was lower than that of the gel group, indicating that the viscoelastic properties of the hyaluronic acid hydrogel system provide lubrication and buffering effects, which are important in the treatment of rheumatoid arthritis by intra-articular injection. Although HA matrices can alleviate disease conditions to some extent, there is no obvious therapeutic effect. MTX belongs to the first-line therapeutic drug in clinical treatment guidelines, and for improving the disease state, continuous administration of antirheumatic drugs (DMARDs) is required for 6 weeks to 6 months to see significant disease remission ^[3,4] . In experiments, MTX was administered as a solution and at longer intervals. The therapeutic effect in the experiments was poor due to the characteristics of short in vivo half-life and long-time sustained administration. In contrast, the 3% sBSA/HA gel shows more effective treatment, HAs a longer anti-inflammatory effect, and further directly shows the powerful anti-inflammatory advantage of sBSA and the treatment prospect of a hydrogel system.

In addition, H & E staining (fig. 6) was performed on knee joints of each treatment group, and the degree of injury and osteoclast secretion state thereof were preliminarily observed. It is apparent that there was a clear lesion in femoral head tissue of rats in the disease group and HA matrix group, the number of osteoclasts formed increased (at black arrow in fig. 6), and the number of cartilage decreased. The 3% sBSA/HA groups, 1.5% sBSA/HA groups and 3% sBSA groups were relatively intact in bone morphology and did not exhibit significant upregulation of osteoclast secretion. The expression level of knee bone tissue Col II is positively correlated with the amount of chondrocytes, and the degree of tissue cartilage destruction can be reflected by measuring the content thereof. The results show that the expression level of Col II in the disease group and the HA matrix group is obviously reduced compared with that of the healthy group, and the cartilage surface is obviously rough, which indicates that the articular cartilage of the two groups of rats HAs obvious lesions. The cartilage surface was rough in the MTX and 3% sBSA solution groups, but the cartilage damage was slightly lower than in the disease and HA groups. There was only slight disruption of cartilage in the 3% sBSA/HA group compared to healthy groups, and less in the 1.5% sBSA/HA group. The degree of cartilage destruction in the 3% bsa solution treatment group further demonstrates the powerful anti-inflammatory activity of bsa, which needs to be achieved with the aid of a hydrogel system, by alleviating the arthromeningitis state, preventing disease progression, and delaying cartilage and bone destruction.

Experiments prove that the sulfonated albumin derivative and the composition thereof prepared by the invention can be widely used for treating and/or preventing macrophage M1 type polarization related diseases, preferably inflammatory diseases, including but not limited to atopic dermatitis and rheumatoid arthritis, preferably rheumatoid arthritis.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Reference to the literature

[1]LeVine SM.Albumin and multiple sclerosis.BMC Neurol.2016；16:47.doi:10.1186/s12883-016-0564-9.

[2]da Silva NIO,Salvador EA,Rodrigues Franco I,et al.Bovine serum albumin nanoparticles induce histopathological changes and inflammatory cell recruitment in the skin of treated mice.Biomed Pharmacother.2018；107:1311-1317.doi:10.1016/j.biopha.2018.08.106.

[3]Smolen JS,Aletaha D,McInnes IB.Rheumatoid arthritis.Lancet.2016；388(10055):2023-2038.doi:10.1016/S0140-6736(16)30173-8.

[4]Radu AF,Bungau SG.Management ofRheumatoid Arthritis:An Overview.Cells.2021；10(11):2857.doi:10.3390/cells10112857.

Claims (10)

1. A sulfonated albumin derivative characterized by introducing sulfonic acid groups into the albumin molecular chain.

2. A sulfonated albumin derivative according to claim 1, characterized in that the chemical structure of the sulfonic acid group is:

wherein n=0 to 20; r is R ₁ A coupling group for attachment to albumin, comprising a carboxyl, amino or aldehyde group; y isHydrogen ions, metal ions or mixtures of hydrogen ions and metal ions.

3. The sulfonated albumin derivative according to claim 1, wherein said albumin comprises human serum albumin, recombinant human serum albumin, bovine serum albumin or porcine serum albumin.

4. The sulfonated albumin derivative according to claim 1, characterized in that the group on the albumin molecular chain coupled to the sulfonic acid group donor molecule comprises an amino group, a carboxyl group or a mercapto group.

5. The sulfonated albumin derivative according to any one of claims 1 to 4, characterized by the structure of the sulfonated albumin derivative being as follows:

wherein n=0 to 20; m=1 to 60; y is hydrogen ion, metal ion or mixture of hydrogen ion and metal ion.

6. The sulfonated albumin derivative according to claim 5, wherein the sulfonated albumin derivative has the structure:

where m=1 to 60.

7. The process for producing a sulfonated albumin derivative according to any one of claims 1 to 6, characterized by comprising the steps of:

the albumin is dispersed in a water phase or a mixed solvent of organic solvents, and a sulfonic acid group donor molecule and amino, carboxyl or sulfhydryl on an albumin molecule chain are subjected to substitution reaction, so that the albumin derivative has a sulfonic acid group structure.

8. Use of a sulfonated albumin derivative according to any one of claims 1 to 6 or a composition of said sulfonated albumin derivative and a pharmaceutically acceptable adjuvant for the preparation of a medicament for the treatment or prevention of a macrophage M1 polarization-related disease.

9. The use according to claim 8, wherein macrophage M1 polarization-related disease comprises atopic dermatitis and rheumatoid arthritis.

10. Use according to claim 8 or 9, characterized in that the auxiliary material is hyaluronic acid.

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