Hydrogel loaded with cyclodextrin aggregate as well as preparation method and application of hydrogel

Abstract

The invention belongs to the technical field of medicine, and particularly relates to hydrogel loaded with cyclodextrin aggregates and a preparation method and application of the hydrogel loaded with the cyclodextrin aggregates. The hydrogel loaded with the cyclodextrin aggregates comprises a hydrogel matrix and the cyclodextrin aggregates loaded on the hydrogel matrix; the cyclodextrin aggregate is sulfobutyl beta-cyclodextrin modified by a hydrophobic group; the hydrophobic group is one of n-dodecyl and 2-methyl undecyl. The hydrogel adhesive tape has rich negative charges, the binding capacity of the hydrogel adhesive tape and polyamine with positive charges can be effectively enhanced, meanwhile, a cyclodextrin cavity is matched with the molecular size of polyamine, the binding efficiency of the hydrogel adhesive tape and the polyamine is improved, and excessive polyamine in a periodontal local inflammatory area is captured and removed in a hydrogel mode. Meanwhile, the hydrogel loaded with the cyclodextrin aggregate can remarkably improve the inflammatory microenvironment, effectively reduce the oxidative stress level of cells and maintain the immune homeostasis, and a new thought is provided for treatment of periodontitis areas.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a hydrogel loaded with cyclodextrin aggregates, a preparation method thereof, and applications thereof. Background Art

[0002] Periodontitis is a chronic inflammatory disease, mainly resulting from the stimulation of periodontal tissues by bacteria in dental plaque. During the onset of periodontitis, when pathogenic bacteria invade periodontal tissues, a series of immune responses will be triggered in the body, including infiltration of immune cells and up-regulation of the expression levels of inflammation-related factors, which will further lead to imbalance of immune homeostasis and cause chronic damage to periodontal tissues. During this process, there are many inflammatory stimulatory factors. Clearing these inflammatory stimulatory factors helps to maintain immune homeostasis and achieve the purpose of treating periodontitis.

[0003] Polyamines, as metabolites of bacteria and cells, are also one of the inflammatory stimulatory factors, which can promote the occurrence and development of local inflammatory reactions. Polyamines mainly include spermine, spermidine, putrescine, etc. They are a class of biological metabolites with two or more amino groups that are commonly present in eukaryotic and prokaryotic cells. They carry positive charges and are involved in life processes such as cell growth, tissue regeneration, and inflammation repair. Regulating the homeostatic balance of polyamine concentration in the microenvironment is closely related to maintaining the health of the body. During the occurrence and development of periodontitis, there are also changes in polyamine concentration. Studies have found that the polyamine concentration in the saliva of normal healthy people is only 10 - 20 μM, while the polyamine concentration in the gingival crevicular fluid of periodontitis patients is significantly increased. After systematic periodontal treatment, its concentration will decrease significantly, but it is still higher than the normal level. It can be seen that if the polyamine concentration can be regulated, the occurrence and development process of periodontitis can be controlled. In addition, in addition to mechanical treatment, the way of combining with auxiliary medication can be used to further remove the excessive polyamines in the periodontal pocket and maintain the balance of periodontal immune homeostasis.

[0004] In previous studies, the main way to remove polyamines was to inhibit the activity of their synthetic rate-limiting enzymes, thereby inhibiting polyamine synthesis. However, this method has disadvantages such as large side effects and narrow application range. Therefore, finding a class of small molecule materials with appropriate size, low cost, and high biosafety, and simply modifying them so that they can effectively capture local excessive polyamines to achieve the purpose of removing polyamines and treating periodontitis has become an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to provide a hydrogel loaded with cyclodextrin aggregates, a preparation method thereof, and applications thereof, so as to overcome the deficiencies of the prior art. The modified cyclodextrin has more negative charges, which can effectively enhance its binding ability with positively charged polyamines, and realize the capture and removal of excessive polyamines in the local inflammatory area of the periodontium in the form of a hydrogel, so as to achieve the purpose of removing polyamines and treating periodontitis.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a hydrogel loaded with cyclodextrin aggregates, comprising a hydrogel matrix and cyclodextrin aggregates loaded on the hydrogel matrix;

[0008] The cyclodextrin aggregates are sulfobutyl-β-cyclodextrin modified with a hydrophobic group;

[0009] The hydrophobic group is -(CH 2 ) 11 CH 3 and -(CH 2 ) 9 CHCH 3 CH 3 one of them.

[0010] In some other embodiments, in the cyclodextrin aggregates, the molar ratio of sulfobutyl-β-cyclodextrin to the hydrophobic group is 1:(1 - 6).

[0011] In some other embodiments, the hydrogel matrix is one or more of methacrylated dextran hydrogel, methacrylated carboxymethyl chitosan hydrogel, methacrylated gelatin hydrogel, and methacrylated sodium alginate hydrogel.

[0012] In a second aspect, the present invention provides a preparation method of the hydrogel loaded with cyclodextrin aggregates described in the first aspect, comprising the following steps:

[0013] (1) React an alkanol, p-toluenesulfonyl chloride, and a catalyst in a first solvent to obtain an alkyl p-toluenesulfonate;

[0014] (2) After mixing sulfobutyl-β-cyclodextrin with a second solvent, add potassium iodide and sodium hydride and carry out a first reaction, then add the alkyl p-toluenesulfonate and carry out a second reaction to obtain alkyl-sulfobutyl-β-cyclodextrin;

[0015] (3) Add a photoinitiator to the phosphate buffer solution of alkyl-sulfobutyl-β-cyclodextrin to form a mixed solution, and add the hydrogel matrix to the mixed solution to carry out a photocrosslinking reaction to obtain the hydrogel loaded with cyclodextrin aggregates.

[0016] In some other embodiments, in step (1), the catalyst is triethylamine and 4-dimethylaminopyridine;

[0017] The molar ratio of the alkanol, p-toluenesulfonyl chloride, triethylamine, and 4-dimethylaminopyridine is (1.2 - 12):(1 - 10):(3 - 30):(0.1 - 1);

[0018] The alkanol is one of n-dodecanol and 2-methylundecanol;

[0019] The alkyl group is -(CH 2 ) 11 CH 3 or -(CH 2 ) 9 CHCH 3 CH 3 one of them;

[0020] The ratio of p-toluenesulfonyl chloride to the first solvent is (1 - 10) g : (10 - 50) mL;

[0021] The first solvent is one of dichloromethane, N,N-dimethylformamide and dimethyl sulfoxide;

[0022] The temperature of the reaction is 25 - 30 °C and the time is 15 - 30 h.

[0023] In some other embodiments, in step (1), after the reaction ends, purification treatment is further included, and the purification treatment is successively carried out with extraction, drying, filtration, and rotary evaporation;

[0024] The extraction is to add a saturated Na 2 S 2 O 3 solution to the organic phase for extraction, then separate the organic phase, extract the aqueous phase with dichloromethane, and then combine the organic phases;

[0025] The drying is to add MgSO 4 for drying;

[0026] The filtration is to add n-hexane and then carry out filtration.

[0027] In some other embodiments, in step (2), the mixed molar ratio of sulfobutyl-β-cyclodextrin, potassium iodide, sodium hydride and alkyl p-toluenesulfonate is 1 : (14 - 20) : (120 - 150) : (20 - 30);

[0028] The mixing ratio of sulfobutyl-β-cyclodextrin to the second solvent is (0.1 - 1) g : (20 - 50) mL;

[0029] The second solvent is one or two of N,N-dimethylformamide, dimethyl sulfoxide and acetonitrile;

[0030] The first step of the reaction is to react under an ice bath for 0.5 - 1.5 h, and then continue to react for 6 - 7 h after removing the ice bath;

[0031] The temperature of the second step of the reaction is 25 - 30 °C and the time is 30 - 35 h.

[0032] In some other embodiments, in step (2), after the second reaction is completed, a purification treatment is further included. The purification treatment is to concentrate and lyophilize after removing unreacted NaH and the solvent;

[0033] The method for removing NaH is to add methanol dropwise to the product until no bubbles are generated;

[0034] The method for removing the solvent is dialysis.

[0035] In some other embodiments, in step (3), the concentration of alkyl-sulfobutyl-β-cyclodextrin in the mixed solution is 0-500 μM, and the concentration of the photoinitiator is 0.2-0.3 wt%;

[0036] The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphate;

[0037] The ratio of the hydrogel matrix to the mixed solution is (45-55) mg: 1 mL;

[0038] The hydrogel matrix is one or more of methacrylated dextran hydrogel, methacrylated carboxymethyl chitosan hydrogel, methacrylated gelatin hydrogel, and methacrylated sodium alginate hydrogel;

[0039] The photocrosslinking reaction is to cure at room temperature under ultraviolet light for 5-10 s;

[0040] The ultraviolet light has a wavelength of 405 nm and a light irradiance of 25 mW / cm 2 。

[0041] In a third aspect, the present invention provides the use of the hydrogel loaded with cyclodextrin aggregates described in the first aspect in the preparation of a drug for treating periodontitis.

[0042] Advantages of the present invention:

[0043] 1. The hydrogel loaded with cyclodextrin aggregates prepared by the present invention has abundant negative charges, effectively enhancing its binding ability with positively charged polyamines. At the same time, the cyclodextrin cavity matches the molecular size of polyamines, improving the binding efficiency with polyamines, and realizing the capture and clearance of excessive polyamines in the local inflammatory area of the periodontium in the form of a hydrogel;

[0044] 2. The hydrogel loaded with cyclodextrin aggregates prepared by the present invention can significantly improve the inflammatory microenvironment. By clearing excessive polyamines, it effectively reduces the level of cellular oxidative stress, maintains immune homeostasis, and achieves the purpose of treating periodontitis. Brief Description of the Drawings

[0045] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0046] Figure 1 Reaction route for preparing SCDC in Example 1 of the present invention 12 ;

[0047] Figure 2 ESI-MS results of SCDC prepared in Example 1 of the present invention 12 ;

[0048] Figure 3 One-dimensional proton nuclear magnetic resonance spectrum results of SCDC prepared in Example 1 of the present invention 12 ;

[0049] Figure 4 Zeta potential diagram of β-CD and SCDC in Example 1 of the present invention 12 ;

[0050] Figure 5 Scanning electron microscope structure of DexMA hydrogel and DexMA@SCDC hydrogel in Example 2 of the present invention 12 ;

[0051] Figure 6 Porosity of DexMA hydrogel and DexMA@SCDC hydrogel in Example 2 of the present invention 12 ;

[0052] Figure 7 Swelling ratio of DexMA hydrogel and DexMA@SCDC hydrogel in Example 2 of the present invention 12 ;

[0053] Figure 8 Rheological properties of DexMA hydrogel and DexMA@SCDC hydrogel in Example 2 of the present invention 12 ;

[0054] Figure 9 Degradation properties of DexMA hydrogel and DexMA@SCDC hydrogel in Example 2 of the present invention 12 ;

[0055] Figure 10 Effect diagram of polyamine scavenging ability of SCDC in artificial saliva in Example 1 of the present invention 12 ;

[0056] Figure 11 Effect diagram of the role of SCDC in scavenging intracellular ROS in Example 1 of the present invention 12 ;

[0057] Figure 12 For the DexMA hydrogel and DexMA@SCDC in Example 2 of the present invention 12 Statistical chart of the vertical distance between the cementoenamel junction (CEJ) and the alveolar crest (ABC) after the treatment of periodontitis rats with the hydrogel. Detailed implementation manners

[0058] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0059] The inventive concept adopted by the present invention is:

[0060] β-cyclodextrin, as a class of naturally occurring glucose derivatives, has a rigid cyclic structure, a size matching that of polyamines, and good biological safety and stability, and has been widely used in the drug development of various diseases. Sulfobutyl-β-cyclodextrin (no special requirement for the degree of substitution, and it can be a mixture of sulfobutyl-β-cyclodextrin with different degrees of substitution), as a class of derivatives of β-cyclodextrin, has good water solubility and biological safety, and is widely used in the fields of pharmaceutical excipients, vaccine carriers, etc. The internal cavity structure thereof matches the size of polyamine molecules and can be combined. In order to better capture polyamines, cyclodextrin aggregates are obtained by modifying sulfobutyl-β-cyclodextrin with hydrophobic groups. Then, the cyclodextrin aggregates are photocrosslinked and cured with a hydrogel matrix, which has abundant negative charges, effectively enhancing its binding ability with positively charged polyamines. At the same time, the cyclodextrin cavity matches the size of polyamine molecules, improving the binding efficiency with polyamines, and realizing the capture and clearance of excessive polyamines in the local inflammatory area of the periodontium in the form of a hydrogel.

[0061] Example 1

[0062] A preparation method of a hydrogel loaded with cyclodextrin aggregates, as Figure 1 shown, includes the following steps:

[0063] (1) Using 30 mL of dichloromethane (CH 2 Cl 2 ) as a solvent, 12.74 g (90 mM) of triethylamine (Et 3 N) and 0.363 g (3 mM) of 4-dimethylaminopyridine (DMAP) as catalysts, adding 5.58 g (36 mM) of n-dodecanol (C 12 H 24 OH) and 6.86 g (30 mM) of p-toluenesulfonyl chloride (TsCl), and reacting at room temperature for 16 hours.

[0064] Adding Na 2 S 2 O 3The saturated solution was added to the organic phase. After phase separation extraction, the organic phase was separated. The aqueous phase was extracted with dichloromethane, and the organic phases were combined. A small amount of MgSO 4 was added for drying, filtration, and rotary evaporation to obtain a pale yellow oily substance with precipitate. n-Hexane was added, and a precipitate appeared. After filtration, the filtrate was obtained. After rotary evaporation, a pale yellow liquid was obtained, which was the product dodecyl p-toluenesulfonate (TsC 12 ).

[0065] (2) 0.3 g (0.1 mM) of sulfobutyl-β-cyclodextrin (SEB 6 -β-CD) was dissolved in 30 mL of dimethyl sulfoxide (DMSO) and 3 mL of N,N-dimethylformamide (DMF). 0.24 g (1.45 mM) of potassium iodide (KI) and 0.3 g (12.5 mM) of sodium hydride (NaH) were added successively. The reaction was carried out for 1 hour in an ice bath. After removing the ice bath, the reaction was continued for 6 - 7 hours. 0.8 g (2.35 mM) of TsC 12 was added, and the reaction was continued for 32 hours. During this period, if a solid precipitated, DMSO and DMF solvents were added to redissolve the precipitated solid. The final product dodecyl-sulfobutyl-β-cyclodextrin (SCDC 12 ) was obtained

[0066] (3) Post-treatment: Methanol was added dropwise to the final product until no bubbles were generated to remove the excess NaH. Methanol was removed by rotary evaporation. Inorganic small molecules and organic solvents were removed by dialysis for 48 hours. The liquid after dialysis was collected, and the solvent water was removed by rotary evaporation again for concentration. The concentrated product was collected and freeze-dried.

[0067] Example 2

[0068] A method for preparing a hydrogel loaded with cyclodextrin aggregates was as described in Example 1, except that after obtaining SCDC 12 according to step (3) of Example 1, it was added to a pre-gel solution of DexMA (model EFL-DexMA-500K) containing 0.25% photoinitiator LAP, and cured by irradiation with ultraviolet light (405 nm, 25 mW / cm 2 ) for 5 s. After curing, a DexMA@SCDC 12 hydrogel with stable performance could be formed.

[0069] Example 3

[0070] Different from Example 2, a methacrylated carboxymethyl chitosan hydrogel (CMCSMA,

[0071] model EFL-CMCSMA-200K) was used to replace the methacrylated dextran hydrogel (DexMA), and other preparation methods were the same as those in Example 2.

[0072] Example 4

[0073] Different from Example 2, methacrylated gelatin hydrogel (GelMA, model EFL-GM-90K) was used to replace methacrylated dextran hydrogel (DexMA), and other preparation methods were the same as those in Example 2.

[0074] EFL-GM-90K) to replace the methacrylated dextran hydrogel (DexMA), and other preparation methods were the same as those in Example 2.

[0075] Example 5

[0076] Different from Example 2, methacrylated sodium alginate hydrogel (AlgMA, model EFL-AlgMA-300K) was used to replace methacrylated dextran hydrogel (DexMA), and other preparation methods were the same as those in Example 2.

[0077] Example 6

[0078] Different from Example 2, 2-methylundecanol was used to replace n-dodecanol in Example 1, and other preparation methods were the same as those in Example 2.

[0079] Comparative Example 1

[0080] Different from Example 2, sulfobutyl-β-cyclodextrin was not modified, that is, sulfobutyl-β-

[0081] cyclodextrin was directly reacted with methacrylated dextran hydrogel, and other preparation methods were the same as those in Example 2.

[0082] Comparative Example 2

[0083] Different from Example 2, β-cyclodextrin was used to replace sulfobutyl-β-cyclodextrin, and other preparation methods were the same as those in Example 2.

[0084] Comparative Example 3

[0085] Different from Example 2, butanol was used to replace n-dodecanol, and other preparation methods were the same as those in Example 2

[0086] consistent.

[0087] Performance Test

[0088] (1) Performance Test of Cyclodextrin Aggregates

[0089] Figure 2 For the ESI-MS result of SCDC 12 , Figure 3 For the one-dimensional 1H NMR spectrum result of SCDC 12 . It can be seen from Figure 2 and Figure 3 that dodecyl-sulfobutyl-β-cyclodextrin (SCDC) was successfully prepared12 )。

[0090] Figure 4 This is the Zeta potential diagram of β-CD and SCDC in Example 1 of the present invention 12 ; where β-CD is β-cyclodextrin and SCDC 12 is a cyclodextrin aggregate. As can be seen from Figure 4 , after β-cyclodextrin is sulfobutyl-modified, SCDC 12 has a stronger negative charge, proving that the cyclodextrin aggregate is more likely to bind to the positively charged polyamine.

[0091] (2) Structural performance test of DexMA hydrogel and DexMA@SCDC 12 hydrogel

[0092] Figure 5 This is the scanning electron microscope structure of the DexMA hydrogel and DexMA@SCDC 12 hydrogel in Example 2 of the present invention. As can be seen from Figure 5 , both have a loose and porous structure, and loading SCDC 12 has no significant effect on the internal structure of the hydrogel.

[0093] Figure 6 This is the porosity of the DexMA hydrogel and DexMA@SCDC 12 hydrogel in Example 2 of the present invention. As can be seen from Figure 6 , the porosity of both is about 45%, and loading SCDC 12 has no significant effect on the porosity of the hydrogel.

[0094] Figure 7 This is the swelling ratio of the DexMA hydrogel and DexMA@SCDC 12 hydrogel in Example 2 of the present invention. The test method for the swelling ratio is: calculate the swelling ratio through the mass change of the hydrogel. The dry weight of the freeze-dried hydrogel is W 0 . At 37 °C, the freeze-dried hydrogel sample is immersed in PBS solution and weighed at different time points, denoted as W 1 . Swelling ratio (%) = (W 1 - W 0 ) / W 0 ×100%. As can be seen from Figure 7 , both reach a relatively high swelling ratio within 4 hours, and the swelling ratios at different time points are similar, indicating that loading SCDC 12 has no significant effect on the swelling performance of the hydrogel.

[0095] Figure 8 This is the DexMA hydrogel and DexMA@SCDC in Example 2 of the present invention12 Rheological properties of the hydrogel. The test method for rheological properties was as follows: Disc-shaped hydrogel samples with a diameter of about 10 mm and a thickness of about 2 mm were prepared. At room temperature, a rheometer was used to measure the storage modulus (G') and loss modulus (G") of the hydrogel under different stress-strains at an angular velocity of 5 rad / s. From Figure 8 it can be seen that the change trends of G' and G" of both under different stress-strains are similar, indicating that loading SCDC 12 has no significant effect on the rheological properties of the hydrogel.

[0096] Figure 9 This is for the DexMA hydrogel and DexMA@SCDC in Example 2 of the present invention 12 Degradation properties of the hydrogel. The test method for degradation properties was as follows: The prepared hydrogel samples were weighed, and the initial mass of the hydrogel was recorded. The samples were immersed in artificial saliva, oscillated at 70 rpm at 37 °C, and the solution was changed every 8 hours. The samples were taken out at different time points and weighed again to record the remaining mass of the hydrogel. The in vitro degradation rate was calculated by calculating the ratio of the remaining mass of the hydrogel to the initial mass. From Figure 9 it can be seen that both have good degradation properties, and the degradation time in artificial saliva is about 15 days, indicating that loading SCDC 12 has no significant effect on the degradation properties of the hydrogel.

[0097] (3) Effect of cyclodextrin aggregates on the polyamine scavenging ability in artificial saliva

[0098] At room temperature, using artificial saliva (pH 6.8, Leagene, CZ0190) as the solvent, 20 μM of spermine, spermidine, and putrescine were respectively mixed with 20, 40, 100, 200 μM of SCDC 12 for 1 hour, and a polyamine assay kit (Abcam, ab239728) was used to measure the residual polyamine amount. Residual ratio (%) = final polyamine concentration / initial polyamine concentration × 100%.

[0099] Figure 10 This is for SCDC in Example 1 of the present invention 12 Effect diagram of the polyamine scavenging ability of SCDC in artificial saliva; among them, Spermine is the spermine group, Spermidine is the spermidine group, Putrescine is the putrescine group; Residual ratio (%) is the remaining ratio; NC is the blank control group, and 1:1, 1:2, 1:5, 1:10 are the concentration ratios of polyamine to SCDC 12 From Figure 10 it can be seen that in artificial saliva, the ability of SCDC 12 to bind and scavenge polyamines is related to the polyamine type and concentration ratio. Spermine and putrescine are more easily scavenged by SCDC12 Combined with clearance, and along with SCDC 12 The efficiency of polyamine binding increases with the increase in relative concentration.

[0100] In Example 2, after loading SCDC 12 onto DexMA hydrogel, it will be beneficial for the intrasulcular injection operation during the treatment of periodontitis. Since DexMA hydrogel has stable physicochemical properties and good biocompatibility, and can be slowly degraded by biological enzymes in the human body, after loading, the slow release process of SCDC 12 in the periodontal pocket can be achieved. At the same time, the loading has little effect on the polyamine scavenging ability of SCDC 12 before and after loading.

[0101] In Examples 3, 4, and 5, similar to Example 2, SCDC 12 was loaded into CMCSMA, GelMA, or AlgMA hydrogels. The above hydrogels all have stable physicochemical properties and good biocompatibility, can be degraded in vivo, and have no obvious effect on the polyamine scavenging ability of SCDC 12 before and after loading.

[0102] In Example 6, 2-methylundecanol was used to replace n-dodecanol in Example 1, and the properties of the prepared product were similar.

[0103] In Comparative Example 1, since sulfobutyl-β-cyclodextrin does not have a hydrophobic chain, its self-assembly performance in solution is not strong. Compared with the product in Example 1, its specific surface area, reaction activity, and dispersibility in solution are poor, which is not conducive to the binding with polyamines.

[0104] In Comparative Example 2, since β-cyclodextrin does not have a sulfobutyl group containing negative charges, its electrostatic attraction with positively charged polyamines is weak. Therefore, its polyamine binding ability is weak, and compared with the product in Example 1, its polyamine scavenging ability is poor.

[0105] In Comparative Example 3, butanol was used to replace n-dodecanol, and the self-assembly performance of the obtained product was poor. The length of the hydrophobic chain affects the self-assembly performance of the product. The longer the length, the stronger the hydrophobic interaction between molecules, the higher the aggregation ability and the stability of the self-assembly body. However, when the carbon chain length is greater than 12, the solubility will be reduced, restricting self-assembly. Therefore, an appropriate hydrophobic chain length can balance hydrophobicity and hydrophilicity to achieve efficient self-assembly.

[0106] (4) The role of cyclodextrin aggregates in scavenging intracellular ROS

[0107] An active oxygen detection kit (S0033S, Beyotime) was used to evaluate the production of ROS in human gingival fibroblasts (HGFs) stimulated by spermine. The HGFs cells were seeded at 2×10 4Cells were seeded at a density of [[ID=]] cells / well in 6-well plates and cultured for 24 hours. After stimulating the cells with 10 μM spermine for 24 hours, SCDC at concentrations of 0, 10, 20, and 40 μM were used respectively 12 to culture the cells for 24 hours. An ROS detection kit was used to evaluate the production of intracellular ROS, and a fluorescence microscope (Leica, Germany) was used to observe the fluorescence intensity of ROS in each group of cells.

[0108] Figure 11 This is the effect diagram of SCDC 12 in Example 1 of the present invention for scavenging intracellular ROS; where Control is the blank control group, 0 μM is the spermine-stimulated simulated inflammation group, and 10 μM, 20 μM, and 40 μM are the experimental groups of adding different concentrations of cyclodextrin aggregates after spermine stimulation of cells; Merged is the merged state, DCFH is the DCFH fluorescence probe signal channel, and Bright Field is the bright field image. From Figure 11 it can be seen that compared with the Control group, the fluorescence intensity increased in the 0 μM group under high-concentration polyamine stimulation, the intracellular oxidative stress response in HGFs cells was enhanced, and the intracellular ROS level increased significantly. However, in the 10 μM, 20 μM, and 40 μM groups, after treatment with different concentrations of SCDC 12 the fluorescence intensity decreased significantly and the ROS level decreased significantly, indicating that the capture and scavenging of polyamines by cyclodextrin aggregates enhanced the ROS scavenging ability.

[0109] (5) Statistical analysis of the vertical distance between the cementoenamel junction (CEJ) and the alveolar crest apex (ABC) after treating periodontitis rats with the hydrogel loaded with cyclodextrin aggregates

[0110] A rat periodontitis model was constructed by ligating the second molars of rats and injecting Porphyromonas gingivalis (P. gingivalis) into the gingival sulcus (the bacterial suspension was injected once every 3 days for 2 weeks, and the bacterial concentration was about 1×10 9 CFU / mL). Then, drugs were injected into the gingival sulcus or periodontal pocket (PBS was injected in the healthy group and the periodontitis group, DexMA gel was injected in the simple gel group, and DexMA@SCDC 12 gel was injected in the material group) for an intervention treatment for 28 days (twice a week, 10 μL each time). The alveolar bone loss (the vertical distance between CEJ and ABC) of the second molars of rats in each group was measured by Micro-CT to analyze the improvement of periodontal inflammation by DexMA@SCDC 12 .

[0111] Figure 12 This is the DexMA hydrogel and DexMA@SCDC in Example 2 of the present invention 12Statistical chart of the vertical distance between the cementoenamel junction (CEJ) and the alveolar crest apex (ABC) after the treatment of periodontitis rats with hydrogel; among them, Normal is the normal group, Periodontitis is the periodontitis group, DexMA is the dextran methacrylate hydrogel group, and DexMA@SCDC 12 is the hydrogel experimental group loaded with cyclodextrin aggregates; Mesial is the mesial surface, and Distal is the distal surface; CEJ-ABC (μm) is the vertical distance between the cementoenamel junction and the alveolar crest apex. From Figure 12 it can be seen that compared with the Normal group, the vertical distance between the cementoenamel junction (CEJ) and the alveolar crest apex (ABC) in the Periodontitis group and the DexMA group increased significantly, while that in the DexMA@SCDC 12 group was significantly reduced compared with the Periodontitis group, indicating that the hydrogel loaded with cyclodextrin aggregates can effectively relieve the bone tissue reduction caused by periodontal inflammation, reduce the bone resorption height, and relieve inflammatory bone loss.

[0112] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A hydrogel loaded with cyclodextrin aggregates, characterized in that: It includes a hydrogel matrix and cyclodextrin aggregates loaded on the hydrogel matrix; The cyclodextrin aggregate is sulfobutyl β-cyclodextrin modified with a hydrophobic group; The hydrophobic group is -(CH2) 11 One of CH3 and -(CH2)9CHCH3CH3.

2. The hydrogel loaded with cyclodextrin aggregates according to claim 1, characterized in that: In the cyclodextrin aggregates, the molar ratio of sulfobutyl β-cyclodextrin to the hydrophobic group is 1:(1-6).

3. The hydrogel loaded with cyclodextrin aggregates according to claim 1, characterized in that: The hydrogel matrix is ​​one or more of methacryloyl dextran hydrogel, methacryloyl carboxymethyl chitosan hydrogel, methacryloyl gelatin hydrogel and methacryloyl sodium alginate hydrogel.

4. A method for preparing a hydrogel loaded with cyclodextrin aggregates according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) adding an alkanol, p-toluenesulfonyl chloride and a catalyst into a first solvent for reaction to obtain an alkyl p-toluenesulfonate; (2) mixing sulfobutyl-β-cyclodextrin with a second solvent, adding potassium iodide and sodium hydride to carry out a first step reaction, and adding alkyl p-toluenesulfonate to carry out a second step reaction to obtain alkyl-sulfobutyl-β-cyclodextrin; (3) Adding a photoinitiator to a phosphate buffer solution of alkyl-sulfobutyl-β-cyclodextrin to prepare a mixed solution, adding a hydrogel matrix to the mixed solution, and performing a photocrosslinking reaction to obtain a hydrogel loaded with cyclodextrin aggregates.

5. The method for preparing a hydrogel loaded with cyclodextrin aggregates according to claim 4, characterized in that: In step (1), the catalyst is triethylamine and 4-dimethylaminopyridine; The molar ratio of the alkanol, p-toluenesulfonyl chloride, triethylamine and 4-dimethylaminopyridine is (1.2-12): (1-10): (3-30): (0.1-1); The alkanol is one of n-dodecanol and 2-methylundecanol; The alkyl group is -(CH2) 11 One of CH3 and -(CH2)9CHCH3CH3; The ratio of p-toluenesulfonyl chloride to the first solvent is (1-10) g: (10-50) mL; The first solvent is one of dichloromethane, N,N-dimethylformamide and dimethyl sulfoxide; The reaction temperature is 25-30°C and the reaction time is 15-30h.

6. The method for preparing a hydrogel loaded with cyclodextrin aggregates according to claim 4, characterized in that: In step (1), after the reaction is completed, a purification process is further performed, wherein the purification process is sequentially performed by extraction, drying, filtering, and rotary evaporation; The extraction is to add a saturated solution of Na2S2O3 to the organic phase for extraction, separate the organic phase, extract the aqueous phase with dichloromethane, and combine the organic phases; The drying is performed by adding MgSO4; The filtration is performed after adding n-hexane.

7. The method for preparing a hydrogel loaded with cyclodextrin aggregates according to claim 4, characterized in that: In step (2), the mixing molar ratio of sulfobutyl-β-cyclodextrin, potassium iodide, sodium hydride and alkyl p-toluenesulfonate is 1:(14-20):(120-150):(20-30); The mixing ratio of sulfobutyl-β-cyclodextrin and the second solvent is (0.1-1) g: (20-50) mL; The second solvent is one or two of N,N-dimethylformamide, dimethyl sulfoxide and acetonitrile; The first step of the reaction is to react for 0.5-1.5 hours in an ice bath, and then continue to react for 6-7 hours after removing the ice bath; The temperature of the second step reaction is 25-30°C and the time is 30-35h.

8. The method for preparing a hydrogel loaded with cyclodextrin aggregates according to claim 4, characterized in that: In step (2), after the second step reaction is completed, a purification process is further included, wherein the purification process is to remove unreacted NaH and the solvent, and then concentrate and freeze-dry; The method for removing NaH is to drop methanol into the product until no bubbles are generated; The method for removing the solvent is dialysis.

9. The method for preparing a hydrogel loaded with cyclodextrin aggregates according to claim 4, characterized in that: In step (3), the concentration of alkyl-sulfobutyl-β-cyclodextrin in the mixed solution is 0-500 μM, and the concentration of the photoinitiator is 0.2-0.3 wt %; The photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphate; The ratio of the hydrogel matrix to the mixed solution is (45-55) mg:1 mL; The hydrogel matrix is ​​one or more of methacryloyl dextran hydrogel, methacryloyl carboxymethyl chitosan hydrogel, methacryloyl gelatin hydrogel and methacryloyl sodium alginate hydrogel; The photo-crosslinking reaction is cured at room temperature under ultraviolet light for 5-10 seconds; The ultraviolet light has a wavelength of 405nm and a light irradiance of 25mW / cm 2 .

10. Use of the hydrogel loaded with cyclodextrin aggregates according to any one of claims 1 to 3 in the preparation of a drug for treating periodontitis.

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