Preparation and application of photopolymerization traditional Chinese medicine hydrogel system modified by DNA enzyme

Abstract

The invention provides preparation and application of a DNA enzyme modified photo-polymerized traditional Chinese medicine hydrogel system, and relates to the technical field of biological medicine, and the hydrogel system is prepared by loading effective components in a rheum officinale traditional Chinese medicine into DNA enzyme modified ultraviolet light polymerized HAMA hydrogel. The hydrogel system prepared by the invention has the following advantages: good biocompatibility, targeting property, slow release property and multi-target mechanism, specifically, the HAMA hydrogel has good biocompatibility, and the introduction of DNA enzyme and rheum officinale active ingredients does not significantly affect the biocompatibility of the hydrogel system. The hydrogel can be locally applied to an inflammation part and has targeting property. Effective components of rheum officinale and DNA enzyme are slowly released, so that the anti-inflammatory and immunoregulation effects can be continuously exerted, and the administration frequency is reduced. Through the synergistic effects of anti-inflammation and immunoregulation of the effective components of rheum officinale, degradation of DNA enzyme on own DNA, the physical barrier effect of hydrogel and the like, systemic inflammatory response in the SLE pathogenic process can be more effectively treated.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the preparation and application of a DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system. Background Art

[0002] Systemic lupus erythematosus (SLE) is a complex autoimmune disease, the pathogenesis of which involves the production of autoantibodies, the formation of immune complexes, complement activation, and abnormal activation of multiple immune cells. In the pathogenic process of SLE, systemic inflammatory response plays a key role, which can affect multiple organs and systems, such as the skin, kidneys, and joints, seriously affecting the quality of life of patients and threatening their health. Therefore, controlling systemic inflammation is one of the core goals of SLE treatment. Through comprehensive measures such as reasonable drug therapy and lifestyle adjustment, the systemic inflammatory response of SLE can be effectively controlled, thereby improving the quality of life and health of patients.

[0003] In the prior art, glucocorticoids or immunosuppressants are usually used to treat systemic inflammation of SLE, but the long-term use of the above two drugs may cause side effects of varying degrees. For example, long-term use of glucocorticoids may lead to Cushing's syndrome, diabetes, hypertension, etc., and long-term use of immunosuppressants may cause side effects such as infection and liver and kidney damage. Therefore, it is necessary to develop a new drug to achieve the purpose of safely treating systemic inflammation of SLE. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem that the long-term use of drugs for treating systemic inflammation of SLE in the prior art has certain side effects and it is urgent to develop a new drug.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A DNA enzyme modified photopolymerized traditional Chinese medicine hydrogel system is prepared by loading effective ingredients in rhubarb traditional Chinese medicine into DNA enzyme modified ultraviolet light polymerized HAMA hydrogel.

[0007] Preferably, the active ingredient in the rhubarb Chinese medicine is rhein, and its general structural formula is as follows:

[0008]

[0009] Preferably, the preparation method of the hydrogel system is as follows:

[0010] S1: Preparation of HAMA hydrogel;

[0011] S2: Loading DNA enzyme and rhein onto HAMA hydrogel by chemical coupling;

[0012] S3: UV curing of hydrogel system.

[0013] Preferably, the specific steps of S1 are: dissolving hyaluronic acid in a phosphate buffer solution, adjusting the pH value of the HA solution after complete dissolution, then slowly adding methacrylic anhydride, stirring the reaction, and centrifuging to remove unreacted MA after the reaction is completed to obtain a HAMA hydrogel.

[0014] Preferably, the concentration of hyaluronic acid dissolved in phosphate buffer solution in S1 is 2% (w / v).

[0015] Preferably, the centrifugation conditions in S1 are: 3500g, 3min.

[0016] Preferably, the specific steps of S2 are as follows: dissolving the HAMA solution prepared in S1 in a PBS solution, adding NHS and EDC and stirring after the solution is completely dissolved, and then adding DNA enzyme and rhein, stirring and reacting overnight;

[0017] The pH was adjusted and dialyzed in pure water with a molecular weight cutoff of >1 kDa. After the dialysis was completed, the hydrogel was pre-frozen at 20°C overnight and freeze-dried to obtain a HAMA hydrogel loaded with DNA enzyme and rhein.

[0018] Preferably, the specific steps of S3 are as follows: dissolve the HAMA hydrogel loaded with DNA enzyme and rhein obtained in S2 in a PBS solution, add HMPP high-efficiency photosensitive initiator, vortex and shake evenly, and irradiate under ultraviolet light to obtain a DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system.

[0019] Preferably, the wavelength of the ultraviolet light in S3 is 284 nm, and the irradiation time is 30 s.

[0020] The present application also provides the use of the above-mentioned DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system in the preparation of a drug for treating systemic inflammatory response in the pathogenic process of SLE.

[0021] Compared with the prior art, this application has the following beneficial effects:

[0022] Good biocompatibility: HAMA hydrogel itself has good biocompatibility. The introduction of DNA enzyme and rhubarb active ingredients does not significantly affect its biocompatibility, so that it will not cause serious immune response when used in vivo.

[0023] Targeting and sustained release: The hydrogel can be applied locally at the site of inflammation and has a certain degree of targeting. At the same time, the slow release of rhubarb active ingredients and DNA enzymes in the hydrogel can continuously exert anti-inflammatory and immunomodulatory effects and reduce the frequency of administration.

[0024] Multi-target mechanism of action: Through the synergistic effects of the anti-inflammatory and immunomodulatory active ingredients of rhubarb, the degradation of its own DNA by DNase, and the physical barrier effect of hydrogel, the systemic inflammatory response in the pathogenic process of SLE can be more effectively treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a scanning electron micrograph of a DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel prepared in one embodiment of the present invention;

[0026] Figure 2 This is a fluorescence image of the ability of the DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel to degrade NETs by DNA enzyme in one embodiment of the present invention;

[0027] Figure 3 This is the ROS flow cytometry result of the DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel in an in vitro anti-inflammatory experiment in one embodiment of the present invention.

[0028] Figure 4 The results are the experimental results of anti-inflammatory experiment on lupus mice using DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel in one embodiment of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments.

[0030] A DNA enzyme modified photopolymerized traditional Chinese medicine hydrogel system is prepared by loading effective ingredients in rhubarb traditional Chinese medicine into DNA enzyme modified ultraviolet light polymerized HAMA hydrogel.

[0031] The active ingredient in rhubarb is rhein, and its general structural formula is as follows:

[0032]

[0033] In addition, the present application also provides the role of the DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system in treating systemic inflammatory responses.

[0034] Example 1: Preparation of DNA enzyme modified photopolymerization traditional Chinese medicine hydrogel system:

[0035] The specific preparation method is as follows:

[0036] S1: Preparation of HAMA hydrogel:

[0037] 4 g of hyaluronic acid was dissolved in 200 mL of phosphate buffer solution (pH=7.2-7.4) to prepare a 2% (w / v) solution.

[0038] After complete dissolution, use 1M NaHCO3 The pH value of the HA solution was adjusted to 8.5, and then 12 mL of methacrylic anhydride was slowly added dropwise, and the mixture was stirred in an ice-water bath for 4 hours. Meanwhile, the pH value was monitored every half an hour to maintain the pH value at 7.5-8.5.

[0039] After the reaction was completed, the unreacted MA in the solution was removed by centrifugation at 3500g for 3 min to obtain HAMA supernatant.

[0040] After dilution, stir overnight and then dialyze against 12-14 kDa in 40°C pure water for 7 days. After dialysis, use 1M NaHCO 3 , adjust the pH to about 7.2, pre-freeze at -20℃ overnight, and freeze-dry.

[0041] S2: Loading DNA enzyme and rhein:

[0042] Dissolve 200 mg HAMA in 4 mL PBS solution, add 115.09 mg NHS (N-hydroxysuccinimide) and 218.5 μL EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) after complete dissolution, and stir for 30 minutes. Then add 400 μg DNase (10 μg / mL) and Rhein, and stir overnight to react.

[0043] Use 1M NaHCO 3 Adjust the pH to between 7-7.5, dialyze in pure water for 4 days to ensure that the small molecular weight molecules of NHS and EDC are dialyzed out, and the molecular weight cut-off is greater than 1kDa. After the dialysis is completed, pre-freeze at -20℃ overnight and freeze-dry.

[0044] S3: UV curing of HAMA hydrogel:

[0045] 200 mg of DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel freeze-dried product was taken and dissolved in PBS according to different concentrations (such as 0.5%, 1.5%, and 3%).

[0046] 1% HMPP high-efficiency photoinitiator (10 μL HMPP per 1 mL of liquid) was added, vortexed and evenly irradiated under ultraviolet light (wavelength of 284 nm) for 30 seconds to form a DNA enzyme-modified photopolymerized Chinese medicine hydrogel system. The scanning electron microscopy image of the DNA enzyme-modified photopolymerized Chinese medicine hydrogel system is shown in FIG. Figure 1 shown.

[0047] Example 2: DNA enzyme modified photopolymerized traditional Chinese medicine hydrogel degradation of NETs by DNA enzyme. The specific steps are as follows:

[0048] Neutrophils were extracted from human peripheral blood and inoculated into 24-well plates at a density of approximately 5 × 10 5 cells / well and place a 14mm cell slide at the same time.

[0049] Blank hydrogel, DNAse-modified photopolymerized traditional Chinese medicine hydrogel (extract obtained by immersing the solidified hydrogel in an appropriate solvent and centrifuging) and PMA (10 μmol / L) were added to the cell culture wells, and a positive control (addition of pure DNAse and PMA to stimulate cells) and a negative control (culture medium containing only PMA stimulation) were set up.

[0050] After 4 hours of culture, the culture supernatant was aspirated, the cell slides were washed with PBS, 200 μL DAPI (1:2000) was added to each well for staining in the dark for 5 minutes, and then washed with PBS and observed under a fluorescence microscope.

[0051] Experimental results: Figure 2 As shown, the photopolymerized traditional Chinese medicine hydrogel modified with DNA enzyme can degrade NETs formed by neutrophil PMA stimulation, and the formation of NETs is significantly reduced.

[0052] Example 3: In vitro anti-inflammatory experiment of DNA enzyme modified photopolymerized traditional Chinese medicine hydrogel

[0053] Experimental steps: Cultivate macrophages (iBMDM cells are used in this example), inoculate the cells in a 12-well plate, and culture them to the logarithmic growth phase.

[0054] Different concentrations (such as 0.5%, 1.5%, and 3%) of DNA enzyme-modified photopolymerized rhubarb traditional Chinese medicine HAMA hydrogel (the extract is obtained by soaking the solidified hydrogel in an appropriate solvent and centrifuging it) are added to the cell culture wells, and a positive control (hydrogen peroxide-stimulated cells) and a negative control (culture medium containing only cells) are set up.

[0055] After 5 hours of culture, the cell culture supernatant was collected and the content of inflammatory factors (such as TNF-α and IL-6) was detected by ELISA; cells were collected to detect ROS content, etc.

[0056] Experimental results: Figure 3 As shown, the solidified hydrogel product of the present invention can significantly reduce the ROS results in the experiment, and is dose-dependent.

[0057] Example 4: Application of DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel in anti-inflammatory experiment in lupus mice

[0058] Experimental steps: 10-week-old MRL / LPR mice were selected for animal experiments, and MRL / MPJ mice were used as healthy controls. After subcutaneous injection of 150 μL of 3% DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel and 3% blank HAMA hydrogel at the back of the mouse neck, UV light was polymerized for 30 seconds. The drug was administered once a week for 4 weeks.

[0059] After one month of treatment, 24-hour urine protein and spleen cells of each group were collected for flow cytometry monitoring of TH1 cells, macrophages, etc.

[0060] Experimental results: Figure 4 As shown, the solidified hydrogel product of the present invention can improve and reduce the inflammatory response of lupus mice.

[0061] The materials and sources used in the above examples are:

[0062] Material source DNaseⅠ Thermo Fisher PMA Selleck DAPI Blue Sky Reactive oxygen species detection kit Solebao

[0063] The mechanism of systemic inflammatory response during the treatment of the hydrogel prepared in this application is as follows:

[0064] Local anti-inflammatory effect: When the hydrogel is applied subcutaneously, the three-dimensional network structure of the hydrogel itself can act as a physical barrier to reduce the spread of inflammatory factors. At the same time, the active ingredients of rhubarb are slowly released from the hydrogel and directly act on local inflammatory cells (such as macrophages, lymphocytes, etc.), inhibiting the production of inflammatory factors (such as TNF-α, IL-6, etc.).

[0065] Immunomodulatory effect: The active ingredients of rhubarb can also regulate the functions of immune cells, such as inhibiting the overactivation of T cells and regulating the antibody production of B cells. The presence of DNA enzymes can degrade abnormal self-DNA in local tissues, reduce the stimulation of these self-DNA on immune cells, and thus alleviate the autoimmune response.

[0066] Systemic anti-inflammatory effect: Due to the reduction of local inflammatory response, the amount of inflammatory factors entering the blood circulation is reduced, thereby reducing the systemic inflammatory response. In addition, the active ingredients in the hydrogel may enter the blood circulation through local tissue absorption, and have a regulatory effect on the immune cells and inflammatory response of the whole body.

[0067] Compared with the prior art, the DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system prepared in the present application has good biocompatibility, targeting, sustained release and multi-target action mechanism, which is specifically manifested as follows: the HAMA hydrogel itself has good biocompatibility, and the introduction of DNA enzyme and rhubarb effective ingredients does not significantly affect its biocompatibility, so that it will not cause serious immune response when used in vivo.

[0068] The hydrogel can be applied locally at the site of inflammation, which has a certain degree of targeting. At the same time, the slow release of rhubarb active ingredients and DNA enzymes in the hydrogel can continuously exert anti-inflammatory and immunomodulatory effects and reduce the frequency of administration.

[0069] In addition, through the synergistic effects of the anti-inflammatory and immunomodulatory active ingredients of rhubarb, the degradation of its own DNA by DNase, and the physical barrier effect of hydrogel, the systemic inflammatory response in the pathogenic process of SLE can be treated more effectively.

Claims

1. A DNA enzyme modified photopolymerization traditional Chinese medicine hydrogel system, characterized in that: The hydrogel system is prepared by loading the effective ingredients in the rhubarb traditional Chinese medicine into the DNA enzyme modified ultraviolet light polymerized HAMA hydrogel.

2. The DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to claim 1, characterized in that: The active ingredient in the rhubarb Chinese medicine is rhein, and its general structural formula is as follows:

3. A DNA enzyme modified photopolymerization traditional Chinese medicine hydrogel system according to claim 2, characterized in that: The preparation method of the hydrogel system is as follows: S1: Preparation of HAMA hydrogel; S2: Loading DNA enzyme and rhein onto HAMA hydrogel by chemical coupling; S3: UV curing of hydrogel system.

4. The DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to claim 3, characterized in that: The specific steps of S1 are: dissolving hyaluronic acid in a phosphate buffer solution, adjusting the pH value of the HA solution after complete dissolution, then slowly adding methacrylic anhydride, stirring the reaction, and centrifuging to remove unreacted MA after the reaction is completed to obtain a HAMA hydrogel.

5. The DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to claim 4, characterized in that: The hyaluronic acid in S1 is dissolved in phosphate buffer solution at a concentration of 2% (w / v).

6. The DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to claim 5, characterized in that: The centrifugation conditions in S1 are: 3500 g, 3 min.

7. The DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to claim 6, characterized in that: The specific steps of S2 are as follows: dissolving the HAMA solution prepared in S1 in a PBS solution, adding NHS and EDC and stirring after the solution is completely dissolved, and then adding DNA enzyme and rhein, stirring and reacting overnight; The pH was adjusted and dialyzed in pure water with a molecular weight cutoff of >1 kDa. After the dialysis was completed, the hydrogel was pre-frozen at 20°C overnight and freeze-dried to obtain a HAMA hydrogel loaded with DNA enzyme and rhein.

8. The DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to claim 7, characterized in that: The specific steps of S3 are as follows: dissolve the HAMA hydrogel loaded with DNA enzyme and rhein obtained in S2 in a PBS solution, add HMPP high-efficiency photosensitive initiator, vortex and shake evenly, and irradiate under ultraviolet light to obtain a DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system.

9. The DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to claim 8, characterized in that: The wavelength of the ultraviolet light in S3 is 284 nm, and the irradiation time is 30 s.

10. Use of the DNA enzyme-modified photopolymerized traditional Chinese medicine hydrogel system according to any one of claims 1 to 8 in the preparation of a drug for treating systemic inflammatory response in the pathogenic process of SLE.

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