阅读说明：本技术 磺酸化透明质酸类化合物、其制备方法及其应用 (Sulfonated hyaluronic acid compound, preparation method and application thereof ) 是由 王春明 陈佳羲 谢达平 张哲� 于 2021-10-19 设计创作，主要内容包括：本发明涉及医学,材料化学,糖生物学等交叉技术领域,具体而言,涉及磺酸化透明质酸类化合物、其制备方法及其应用。磺酸化透明质酸类化合物的结构式如下所示：其中,R为碱金属阳离子或氢,R1、R2、R3和R4分别独立地选自氢或磺酸盐离子,且R1、R2、R3和R4不能同时为氢,10<n<4000且n为整数。其与LTBP蛋白具有更强的相互作用力,从而阻止LTBP和ECM结合,使得机械力不足从而无法活化TGF-β,从信号传导的源头抑制纤维化。(The invention relates to the crossed technical fields of medicine, material chemistry, glycobiology and the like, in particular to a sulfonated hyaluronic acid compound, a preparation method and application thereof. The sulfonated hyaluronic acid compound has the following structural formula: wherein R is an alkali metal cation or hydrogen, R1, R2, R3 and R4 are each independently selected from hydrogen or a sulfonate ion, and R1, R2, R3 and R4 cannot be hydrogen at the same time, 10<n<4000 and n is an integer. It has a stronger interaction with LTBP protein, preventing LTBP and ECM binding, rendering the mechanical force insufficient to activate TGF- β, inhibiting fibrosis from the source of signaling.)

1. A sulfonated hyaluronic acid compound is characterized by having a structural formula as follows:

wherein R is an alkali metal cation or hydrogen, R1, R2, R3 and R4 are each independently selected from hydrogen or a sulfonate ion, and R1, R2, R3 and R4 cannot be hydrogen at the same time, 10<n<4000 and n is an integer.

2. The sulfonated hyaluronic acid-based compound of claim 1, wherein R is sodium or potassium ion or hydrogen, R1, R2, R3 and R4 are each independently selected from hydrogen or sulfonate ion, and R1, R2, R3 and R4 cannot be hydrogen at the same time.

3. The method for producing a sulfonated hyaluronic acid-based compound according to claim 1, wherein the sulfonated hyaluronic acid-based compound is synthesized by reference to the following synthetic route:

4. the production method according to claim 3, wherein the molecular weight of the raw material hyaluronic acid is 1500Kd or less;

preferably, the molecular weight of the raw material hyaluronic acid is any of <10Kd, 100-200Kd and 800Kd-1500 Kd;

preferably, the sulfonating agent is selected from pyridine sulfur trioxide.

5. The method according to claim 3 or 4, wherein the step of preparing the sulfonated hyaluronic acid compound comprises: dissolving a raw material hyaluronic acid, mixing the dissolved hyaluronic acid with TBAOH for reaction, freeze-drying to form hyaluronic acid intermediate powder, mixing the hyaluronic acid intermediate powder with a sulfonation reagent, adjusting the pH value of a reaction system to 8-9, and dialyzing.

6. A hyaluronic acid nanoparticle is characterized in that the structural formula is as follows:

wherein R1, R2, R3 and R4 are sulfonate ions, 10<n<4000 and n is an integer.

7. A method for producing a hyaluronic acid nanoparticle, characterized by synthesizing a hyaluronic acid nanoparticle by reference to the following synthesis route:

8. the method of claim 7, comprising: the sulfonated hyaluronic acid-based compound of claim 1, which is ion-exchanged to form a sulfonated hyaluronic acid intermediate, then mixed with an activating agent, then mixed with 4- (1-pyrenyl) butanamide, and dialyzed.

9. A sugar biomaterial comprising the sulfonated hyaluronic acid-based compound of claim 1 or the hyaluronic acid nanoparticle of claim 6.

10. Use of the sulfonated hyaluronic acid-based compound of claim 1 or the hyaluronic acid nanoparticle of claim 6 or the sugar biomaterial of claim 9 for the preparation of a medicament for inhibiting fibrosis;

preferably, the fibrosis comprises tissue fibrosis;

more preferably, fibrosis includes pulmonary fibrosis, liver fibrosis, cardiac fibrosis, pancreatic fibrosis and kidney fibrosis;

more preferably, the agent is an agent that inhibits the activation of TGF- β.

Technical Field

The invention relates to the crossed technical fields of medicine, material chemistry, glycobiology and the like, in particular to a sulfonated hyaluronic acid compound, a preparation method and application thereof.

Background

Fibrosis is a main cause of disability and death of many diseases, and can occur in various organs, and particularly, fibrosis is related to various diseases such as liver cirrhosis, hepatitis, non-alcoholic steatohepatitis, chronic kidney disease, myocardial infarction, heart failure, idiopathic pulmonary fibrosis, diabetes, scleroderma and the like, and seriously threatens human health and life.

By 2020, no regulatory approved drug has been available to avoid or reverse the fibrosis process. At present, drugs which target metabolic processes and have the function of partially inhibiting or relieving fibrosis are mainly used clinically. Most anti-fibrosis drugs mainly comprising small-molecule drugs have anti-fibrosis, anti-inflammatory and anti-oxidation effects, can delay organ function reduction and disease progression caused by fibrosis, but the specific pharmacological basis is unclear and the effect is weak. Some medicines mainly act on the target points of proteins and receptors downstream of the TGF-beta-smad pathway, and the pharmacological action mechanism of the medicines is clear, but the medicines can only improve the fibrosis effect or are effective in the early stage in clinical use and have certain adverse reactions. Current research is mainly focused on the development of small molecule drugs and the inhibition of fibrosis downstream of the signaling pathway.

In the process of fibrosis occurrence and development, TGF-beta has the functions of activating HSC, promoting collagen gene expression, promoting ECM synthesis and deposition and the like, and is one of the most important initiation factors of fibrosis. TGF-. beta.can regulate physiological processes and function through the TGF-. beta.smad signaling pathway, and most studies are currently focused on the regulation of downstream signals of TGF-. beta.s. However, few TGF-beta activation processes are regulated and controlled, and inhibition is carried out at the source of a signal pathway, so that research on anti-fibrosis effect is realized, and no medicine can be inhibited at the source of the signal pathway, so that anti-fibrosis is realized.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a sulfonated hyaluronic acid compound, a preparation method and application thereof. The sulfonated hyaluronic acid compound has stronger interaction force with LTBP protein, thereby preventing LTBP and ECM from combining, preventing mechanical force from being out of order and being incapable of activating TGF-beta, and inhibiting fibrosis from a signal conduction source.

The invention is realized by the following steps:

in a first aspect, the present invention provides a sulfonated hyaluronic acid compound, which has a structural formula as follows:

wherein R is an alkali metal cation or hydrogen, R1, R2, R3 and R4 are each independently selected from hydrogen or a sulfonate ion, and R1, R2, R3 and R4 cannot be hydrogen at the same time, 10<n<4000 and n is an integer.

In a second aspect, the present invention provides a method for producing a sulfonated hyaluronic acid-based compound according to the above embodiment, wherein the sulfonated hyaluronic acid-based compound is synthesized by referring to the following synthesis route:

in a third aspect, the present invention provides a hyaluronic acid nanoparticle, which has a structural formula as follows:

wherein R1, R2, R3 and R4 are sulfonate ions, 10<n<4000 and n is an integer.

In a fourth aspect, the present invention provides a method for preparing a hyaluronic acid nanoparticle, wherein the hyaluronic acid nanoparticle is synthesized by reference to the following synthesis route:

in a fifth aspect, the present invention provides a sugar biomaterial comprising the sulfonated hyaluronic acid-based compound of the previous embodiment or the hyaluronic acid nanoparticle of the previous embodiment.

In a sixth aspect, the present invention provides a use of the sulfonated hyaluronic acid-based compound according to the foregoing embodiment, or the hyaluronic acid nanoparticle according to the foregoing embodiment, or the sugar biomaterial according to the foregoing embodiment, in the preparation of a medicament for inhibiting fibrosis;

preferably, the fibrosis comprises tissue fibrosis;

more preferably, fibrosis includes pulmonary fibrosis, liver fibrosis, cardiac fibrosis, pancreatic fibrosis and kidney fibrosis;

more preferably, the agent is an agent that inhibits the activation of TGF- β.

The invention has the following beneficial effects: the sulfonated hyaluronic acid compound provided by the embodiment of the invention has higher affinity with LTBP, so that the combination of LTBP and ECM is prevented, mechanical force is not available, TGF-beta cannot be activated, fibrosis is inhibited from a signal conduction source, and then fibrosis is improved or treated, and then fibrosis of a treatment substance is treated.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Figure 1 is a graph of the characterization results of the sulfonated hyaluronic acid compounds provided in examples 1-9 of the present invention;

FIG. 2 is a nuclear magnetic characterization result chart of 4- (1-pyrenyl) butanamide provided in example 10 of the present invention;

FIG. 3 is a graph showing the nuclear magnetic characterization results of hyaluronic acid nanoparticles provided in examples 10 to 12 of the present invention;

FIG. 4 is a graph showing the particle size and morphology of hyaluronic acid nanoparticles provided in examples 10 to 12 of the present invention;

FIG. 5 is a detection chart provided in Experimental example 1 of the present invention;

FIG. 6 is a detection chart provided in Experimental example 2 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

TGF-. beta.function requires that newly synthesized TGF-. beta.form inactive small dormant complexes with potentially related peptides (LAPs) in a non-covalent form, in the form of active TGF-. beta.s, which in turn form large dormant complexes with LTBPs (potential TGF-. beta.binding proteins) in a disulfide bond. Activation of TGF- β requires LTBP to interact with the extracellular matrix (ECM) and generate a certain amount of mechanical force to pull on the promotion of TGF- β to fall off the "tight" LAP to become active TGF- β. Therefore, the inventors studied that the formation of active TGF- β can be reduced by suppressing the activation by regulating the mechanical force deficiency during the TGF activation, and thereby the fibrosis can be suppressed from the root, and thus, the present invention provides a sulfonated hyaluronic acid-based compound having the structural formula shown below:

wherein R is an alkali metal cation or hydrogen, R1, R2, R3 and R4 are each independently selected from hydrogen or a sulfonate ion, and R1, R2, R3 and R4 cannot be hydrogen at the same time, 10<n<4000 and n is an integer. The sulfonated hyaluronic acid provided by the embodiment of the invention has strong affinity with LTBP, LTBP can be combined with ECM under normal physiological condition to provide sufficient mechanical force to promote TGF-beta activation, and the sulfonated hyaluronic acid can have higher affinity with LTBPThereby preventing LTBP from binding to ECM, rendering mechanical forces unavailable to activate TGF- β, inhibiting fibrosis from a source of signaling.

Specifically, R is a sodium ion or a potassium ion or hydrogen, R1, R2, R3 and R4 are each independently selected from hydrogen or a sulfonate ion, and R1, R2, R3 and R4 cannot be hydrogen at the same time.

In a second aspect, the present invention provides a method for producing a sulfonated hyaluronic acid-based compound according to the above embodiment, wherein the sulfonated hyaluronic acid-based compound is synthesized by referring to the following synthesis route:

specifically, a raw material hyaluronic acid is dissolved and then mixed with TBAOH for reaction, and then freeze-dried to form hyaluronic acid intermediate powder, and then the hyaluronic acid intermediate powder is mixed with a sulfonation reagent, the pH value of a reaction system is adjusted to 8-9, and then dialysis is performed. The raw material hyaluronic acid may be hyaluronic acid having any molecular weight, for example, the molecular weight of the raw material hyaluronic acid is 1500Kd or less; preferably, the molecular weight of the raw material hyaluronic acid is any of <10Kd, 100-200Kd and 800Kd-1500 Kd. The sulphonation agent used may also be an existing sulphonation agent, for example, selected from pyridine sulphur trioxide.

In a third aspect, embodiments of the present invention provide hyaluronic acid nanoparticles, which have a structural formula as follows:

wherein R1, R2, R3 and R4 are sulfonate ions, 10<n<4000 and n is an integer. According to the embodiment of the invention, 4- (1-pyrenyl) butyramide and sulfonated hyaluronic acid are covalently combined to form hydrophilic and hydrophobic ends, so that the dust-like nanoparticles with hyaluronic acid chains around can be formed by self-assembly, and the sizes and compaction degrees of different nanoparticles can be adjusted by adjusting the amount of the 4- (1-pyrenyl) butyramide. Hyaluronic acid polysaccharide chains can enrich L in tissueLC large complex and prevents its ECM from contacting and thereby activating TGF-beta, and acts to inhibit TGF-beta activation.

In a fourth aspect, embodiments of the present invention provide a method for preparing hyaluronic acid nanoparticles, the hyaluronic acid nanoparticles being synthesized by reference to the following synthesis pathway:

specifically, the sulfonated hyaluronic acid-based compound is ion-exchanged to form a sulfonated hyaluronic acid intermediate, and then mixed with an activating agent, and then mixed with 4- (1-pyrenyl) butanamide, followed by dialysis.

In a fifth aspect, the present invention provides a sugar biomaterial comprising the sulfonated hyaluronic acid-based compound of the previous embodiment or the hyaluronic acid nanoparticle of the previous embodiment.

In a sixth aspect, the present invention provides a use of the sulfonated hyaluronic acid-based compound according to the foregoing embodiment, or the hyaluronic acid nanoparticle according to the foregoing embodiment, or the sugar biomaterial according to the foregoing embodiment, in the preparation of a medicament for inhibiting fibrosis; wherein the fibrosis comprises tissue fibrosis;

for example, fibrosis includes pulmonary fibrosis, liver fibrosis, cardiac fibrosis, pancreatic fibrosis, and renal fibrosis; the drug is a drug that inhibits the activation of TGF-beta.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:wherein, R1 ═ SO₃ ^-,R2＝H,R3＝H,R4＝H；10<n<30。

The embodiment of the invention provides a preparation method of a sulfonated hyaluronic acid compound, which comprises the following steps:

dissolving 3g of low molecular weight hyaluronic acid (the molecular weight of the hyaluronic acid is less than 10Kd) in 300ml of deionized water, reacting with 6ml of 25% tetrabutyl sodium ammonium hydroxide at normal temperature under stirring for 2 hours, and freeze-drying to obtain hyaluronic acid intermediate powder soluble in organic reagents. Dissolving 300mg of hyaluronic acid intermediate powder in anhydrous dimethylformamide, stirring and dispersing, dissolving pyridine sulfur trioxide with low substitution degree (0.9g) in dimethylformamide, adding a hyaluronic acid solution in ice bath, reacting for 1h, adding water to terminate the reaction, adjusting pH to 8.5 by using sodium hydroxide, dialyzing in water for 2 days, and freeze-drying to obtain the sulfonated hyaluronic acid compound, which is recorded as S-HA-1.

Note that the low substitution sulfonated hyaluronic acid: r1 ═ SO₃ ^-R2 ═ H, R3 ═ H, R4 ═ H; degree of substitution sulfonated hyaluronic acid: r1 ═ SO₃ ^-,R2＝SO₃ ^-R3 ═ H, R4 ═ H; high degree of substitution sulfonated hyaluronic acid: r1 ═ SO₃ ^-,R2＝SO₃ ^-,R3＝SO₃ ^-,R4＝SO₃ ^-。

Low molecular weight hyaluronic acid: (<10Kd) <10 < n < 30; medium molecular weight hyaluronic acid: (100 Kd-200 Kd) 260< n < 530; high molecular weight hyaluronic acid: (800 Kd-1500 Kd) < 2100< n < 4000.

Example 2

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:wherein, R1 ═ SO₃ ^-,R2＝SO₃ ^-,R3＝H,R4＝H；10<n<30。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that pyridine sulfur trioxide, namely the substitution degree (2.28g) of hyaluronic acid, is prepared from the following raw materials: low molecular weight hyaluronic acid, denoted as S-HA-2.

Example 3

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:

wherein, R1 ═ SO₃ ^-,R2＝SO₃ ^-,R3＝SO₃ ^-,R4＝SO₃ ^-；10<n<30。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that pyridine sulfur trioxide, namely a high-substitution-degree (3.66g) hyaluronic acid raw material, is as follows: low molecular weight hyaluronic acid, denoted as S-HA-3.

Example 4

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:

wherein, R1 ═ SO₃ ^-,R2＝H,R3＝H,R4＝H；260<n<530。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that the molecular weight of the adopted hyaluronic acid is 100-200Kd, and pyridine sulfur trioxide, namely low substitution degree, is marked as S-HA-4.

Example 5

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:

wherein, R1 ═ SO₃ ^-,R2＝SO₃ ^-,R3＝H,R4＝H；260<n<530。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that the molecular weight of the adopted hyaluronic acid is 100-200Kd, and the substitution degree (2.28g) of pyridine sulfur trioxide is marked as S-HA-5.

Example 6

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:

wherein, R1 ═ SO₃ ^-,R2＝SO₃ ^-,R3＝SO₃ ^-,R4＝SO₃ ^-；260<n<530。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that the molecular weight of the adopted hyaluronic acid is 100-200Kd, and the substitution degree (2.28g) of pyridine sulfur trioxide is recorded as S-HA-6.

Example 7

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:

wherein, R1 ═ SO₃ ^-,R2＝H,R3＝H,R4＝H；2100<n<4000。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that the molecular weight of the adopted hyaluronic acid is 800Kd-1500Kd of high molecular weight hyaluronic acid, and pyridine sulfur trioxide, namely low substitution degree, is marked as S-HA-7.

Example 8

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:

wherein, R1 ═ SO₃ ^-,R2＝SO₃ ^-,R3＝H,R4＝H；2100<n<4000。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that the molecular weight of the adopted hyaluronic acid is 800Kd-1500Kd, and the substitution degree in pyridine sulfur trioxide is recorded as S-HA-8.

Example 9

The embodiment of the invention provides a sulfonated hyaluronic acid compound, which has the following structural formula:

wherein, R1 ═ SO₃ ^-,R2＝SO₃ ^-,R3＝SO₃ ^-,R4＝SO₃ ^-；2100<n<4000。

The preparation method of the sulfonated hyaluronic acid compound provided by the embodiment of the invention is basically the same as that provided by the embodiment 1, and the difference is that the molecular weight of the adopted hyaluronic acid is 800Kd-1500Kd of high molecular weight hyaluronic acid, and pyridine sulfur trioxide, namely high substitution degree, is marked as S-HA-9.

The sulfonated hyaluronic acid compounds synthesized in examples 1 to 9, i.e., S-HA-1 to S-HA-9, were characterized by nmr techniques, and the results are shown in fig. 1 and specifically analyzed as follows: the arrowed marks indicate the peak shift of the methylene proton in C-6, and the triangles indicate the peak shift of the adjacent hydroxyl groups, indicating that the sulfosubstitution degree of S-HA-1, S-HA-2 and S-HA-3 is gradually increased compared with HA-1 with the molecular weight of less than 10 Kd; shows that the sulfosubstitution degree of S-HA-4, S-HA-5 and S-HA-6 is gradually increased compared with HA-2 with the molecular weight of 100Kd-200 Kd; indicating that the sulfosubstitution degree of S-HA-7, S-HA-8 and S-HA-9 is gradually increased compared with HA-3 with the molecular weight of 800Kd-1500 Kd.

The sulfonated hyaluronic acid compounds synthesized in examples 1 to 9, i.e., the degrees of sulfonated substitution of S-HA-1 to S-HA-9, were characterized by a potentiometer. The results of the tests are shown in FIG. 1. Specific analysis shows that the gradual reduction of each group of potential indicates that the sulfonation substitution degree is higher and higher.

Example 10

The embodiment of the invention provides a hyaluronic acid nano particle, which has the following structural formula:

2100<n<4000。

the embodiment of the invention provides a hyaluronic acid nano particle, which comprises:

s1, preparing 4- (1-pyrenyl) butyramide;

500mg of pyrenebutyric acid (1.73eq) was dissolved in 10ml of anhydrous dimethylformamide, and 1970mg (3eq) of 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide Hexafluorophosphate (HATU) and 903mg (5.19eq) of propylenediamine and 669mg (5.19eq) of Diisopropylethylamine (DIPEA) were added to the solution and reacted at ordinary temperature with stirring for 16 hours to obtain an ester of carboxy-linked ethylenediamine. And the side reaction products and the raw materials which are not completely reacted are removed by extraction with ethyl acetate and dichloromethane in a ratio of 1:1, and pure 4- (1-pyrenyl) butyramide is obtained by using a reverse phase silica gel chromatographic column.

The characterization is carried out by using a nuclear magnetic resonance technology 4- (1-pyrenyl) butyramide. The results are shown in FIG. 2, and analyzed in detail as follows: chemical shifts 8-8.5 show significant nine benzene ring hydrogens, and two hydrogens at chemical shift 3.5 indicate successful covalent attachment of ethylenediamine, indicating successful synthesis of PBA.

S2, synthesizing hyaluronic acid nano particles;

the sulfonated hyaluronic acid compound prepared in example 9 was passed through an ion exchange resin, sodium hyaluronate was replaced with sulfonated hyaluronic acid, and lyophilized to obtain a sulfonated hyaluronic acid intermediate, 100mg was dissolved in 10ml of dimethyl sulfoxide, 50mg of carbodiimide and 30mg of n-hydroxysuccinimide were added after dissolution, 60mg of 4- (1-pyrenyl) butanamide was added after 30min of activation, ultrasonic dissolution was performed, stirring was performed at normal temperature for 16h, dialysis was performed in water for 2 days, and lyophilized to obtain hyaluronic acid nanoparticles, which were designated as S-HA-PBA-1.

Example 11

The hyaluronic acid nanoparticles were prepared according to the preparation method provided in example 10, except that the hyaluronic acid nanoparticles obtained using 50mg of 4- (1-pyrenyl) butanamide were designated as S-HA-PBA-2.

Example 12

The hyaluronic acid nanoparticles were prepared according to the preparation method provided in example 10, except that 4- (1-pyrenyl) butanamide was used in an amount of 90, and the obtained hyaluronic acid nanoparticles were designated as S-HA-PBA-3.

The hyaluronic acid nanoparticles prepared in examples 10 to 13, i.e., S-HA-PBA-1 to S-HA-PBA-3, were characterized by nuclear magnetic resonance and infrared spectroscopy. Referring to fig. 3, the detailed analysis is as follows: chemical shift 8-8.5ppm indicates the position of benzene ring hydrogen, and proves that successful covalent connection of S-HA-PBA-1, S-HA-PBA-2 and S-HA-PBA-3 is achieved, the binding rate is calculated by the ratio of pyrene ring hydrogen to amino hydrogen, the grafting rate of S-HA-PBA-1 is 17.5%, the grafting rate of S-HA-PBA-2 is 53.2%, and the grafting rate of S-HA-PBA-3 is 86.2%.

The particle size and morphology of the hyaluronic acid nanoparticles prepared in examples 10 to 13 were characterized by a particle sizer and a transmission electron microscope. Referring to fig. 4, the detailed analysis is as follows: both the particle sizer and transmission electron microscope showed that the nanoparticles were successfully synthesized and had particle sizes of about 100nm to 200 nm.

Experimental example 1 affinity assay

The method comprises the following steps: preparing biotinylated LTBP and preparing five samples to be detected with different concentrations (0.5mol/L,1mol/L,2mol/L,5mol/L,7mol/L) by using the sulfonated hyaluronic acid compounds of examples 1-9, respectively, obtaining a binding dissociation curve by using a procedure of buffer solution 60s, loading protein 60s, buffer solution 60s binding 180s and dissociation 180s on a biological membrane interferometer, and calculating a dissociation constant KD value according to the molecular weight, wherein the smaller the dissociation constant, the stronger the affinity.

The results are shown in FIG. 5. As can be seen from FIG. 5, the nine sulfonated hyaluronic acid compounds of examples 1 to 9, which have the highest affinity for S-HA-9, were sulfonated.

Experimental example 2 TGF-beta Activity reporter cell assay

The method comprises the following steps: the CAGA-TGF-beta activity report cells are plated (12-hole plates) until the fusion degree reaches 65%, PBS, S-HA-9 and S-HA-PBA-1 are added into different holes respectively to stimulate overnight, and TGF-beta (50ng/ml) is added into different holes; pro-TGF-. beta.1 (200 ng/ml); LLC at a suitable concentration (200 ng/ml). Collecting a sample: obtaining cell lysate for luciferase detection.

The results of the test are shown in FIG. 6. According to the figure 6, the sulfonated hyaluronic acid nano-particles can inhibit the release of TGF-beta, and the figure shows that TGF-beta stimulates to generate active TGF-beta, proTGF-beta stimulates to generate a small amount of active TGF-beta, which indicates that a report cell is available, and the cell can prove the release behavior of the active TGF-beta from a biological experiment level, and the result is credible; the LLC + S-HA-9 group released less active TGF-beta than the LLC + PBS group, suggesting that S-HA-9 regulation results in insufficient mechanical force for LLC to release TGF-beta, i.e., inhibition of TGF beta activation from LLC. The LLC + S-HA-PBA-1 group HAs lower activity TGF-beta amount than the LLC + S-HA-9 group, which indicates that the nano particles are more favorable for inhibiting the release of the activity TGF-beta.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.