Novel hydrogen sulfide donor, preparation method and application of novel hydrogen sulfide donor in preparation of medicine for treating inflammatory diseases

Abstract

The invention discloses a novel hydrogen sulfide donor, a preparation method and application of the novel hydrogen sulfide donor in preparation of drugs for treating inflammatory diseases, and belongs to the technical field of biological medicines. The hydrogen sulfide donor comprises hydrogen sulfide synthetase, a drug carrier loaded with the hydrogen sulfide synthetase, and a functional coating coated outside the drug carrier. The drug carrier is a metal organic framework material, a polymer nano material, a liposome material, an exosome material or a hydrogel material, and can provide protection, slow release, enrichment and other functions for biological enzyme according to the characteristics of different inflammatory diseases. The functional coating is a surfactant, a polymer, an edge active agent, a biological membrane or a lipid double layer, and is used for improving the physicochemical properties of the drug carrier, so that the drug carrier is further endowed with special properties such as in-vivo targeting, long circulation, transdermal drug delivery and the like. According to the invention, an accurate, safe and lasting hydrogen sulfide supply means is realized, and the method has a wide application prospect. And a new treatment thought and strategy are expected to be provided for treatment of inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a novel hydrogen sulfide donor, a preparation method and application thereof in preparing medicines for treating inflammatory diseases. Background Art

[0002] H2S is the third gaseous signaling molecule with biological effects discovered after nitric oxide and carbon monoxide. It can be endogenously produced and freely pass through biological membranes to directly trigger signal cascade reactions in cells, and then widely participate in various physiological processes in the body, with multiple functions such as immune regulation, barrier repair, anti-apoptosis, and anti-oxidation. H2S at physiological concentrations can reduce the production of proinflammatory cytokines and chemokines, and induce neutrophil apoptosis and macrophages to transform to an anti-inflammatory phenotype. H2S can also inhibit the adhesion of leukocytes to the vascular endothelium and the migration of leukocytes to the site of inflammation, thus playing a key role in the repair of damaged tissues. Studies have shown that inhibiting the synthesis of endogenous H2S in healthy rats can induce significant intestinal inflammation and mucosal damage; administration of H2S can significantly reduce the expression of the proinflammatory cytokine TNF-α, reduce colon inflammation, and promote mucosal healing. Related studies have publicly shown that regulating Caspase-3 activity can inhibit the apoptosis of gastric epithelial cells in rat ischemia-reperfusion or cerebral ischemia models, thereby protecting cells. Related studies have shown that in a mouse liver ischemia-reperfusion injury model, low-concentration Na2S treatment can be observed to reduce the level of lipid peroxidation in liver cells. In summary, the functions of H2S in repairing mucosal damage, anti-apoptosis, and anti-oxidation are well in line with the needs of inflammatory disease treatment, providing a new opportunity for the treatment of related diseases.

[0003] Existing H2S supply methods include the most basic inorganic sulfide salt donors, such as Na2S, NaHS, CaS, etc. These donors will degrade rapidly in solution and release H2S in large quantities and quickly. Inorganic sulfide salt donors have poor stability in solution and release H2S rapidly after direct local administration. They cannot simulate the process of slow and continuous production of H2S in the body, and local excess H2S will cause certain harm to the body. Slow-release donors, such as GYY4137, a derivative of Lawesson's reagent (dithiophosphate), can slowly release H2S during hydrolysis, thereby achieving a relatively gentle and gentle H2S supply. The slow-release donor GYY4137 can slowly release H2S during hydrolysis and is currently the most widely used H2S donor. However, its H2S release is greatly affected by pH and temperature, and the rate is uncontrollable. Under normal physiological conditions, the efficiency of H2S production is low. Allicin in natural products, such as diallyl trisulfide (DATS), is also a type of H2S donor containing polysulfide bonds, which can react with glutathione to release H2S. As H2S donors, natural products have poor water solubility, produce more byproducts after H2S release, and have complex biological activities. In the past decade, researchers have successively synthesized a variety of H2S donors with different properties and explored their biological applications. Related studies have disclosed an in situ spraying drug delivery system CAP-w-FC based on DATS nanomicelles, which achieved good therapeutic effects in a mouse ulcerative colitis model after rectal administration. Related studies have disclosed a spleen-targeted liposome ST-H2S lipo loaded with H2S donor GYY4137, and explored its multiple therapeutic effects in a DSS-induced mouse colitis model.

[0004] Although significant progress has been made in the development of exogenous H2S donors, these donors have not been able to simulate the production of H2S in the body well, and cannot release H2S stably and uniformly, which has a great impact on the biological function of H2S. In addition, excessive local concentration in a short period of time will produce toxic side effects. Therefore, developing new H2S supply strategies and achieving controllable and stable release of H2S in the body are crucial to improving the treatment effect of H2S-mediated inflammatory diseases. Summary of the invention

[0005] In view of the problem that current H2S donors cannot well simulate the production of H2S in the body and cannot release H2S stably and uniformly, the purpose of the present invention is to design and provide a new hydrogen sulfide donor, a preparation method and its use in the preparation of drugs for treating inflammatory diseases.

[0006] The present invention uses a drug carrier to encapsulate and protect the modified cystathionine gamma lyase (CGL-E339V) to ensure the stable progress of the enzymatic reaction. At the same time, a functional coating with functions such as targeting and long circulation is coated on the surface of the hydrogen sulfide donor, giving different hydrogen sulfide donors new performance. When the particles are enriched in the lesion, the H2S enzymatic reaction is carried out, which well simulates the function of stable release of endogenous H2S. This innovation realizes a precise, safe and long-lasting hydrogen sulfide supply method and has broad application prospects. It is expected to provide new treatment ideas and strategies for the treatment of inflammatory diseases.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a novel hydrogen sulfide donor, which comprises hydrogen sulfide synthase, a drug carrier loaded with hydrogen sulfide synthase, and a functional coating coated on the outside of the drug carrier. The drug carrier is a metal organic framework material, a polymer nanomaterial, a liposome material, an exosome material or a hydrogel material, which can provide protection, sustained release, enrichment and other functions for biological enzymes according to the characteristics of different inflammatory diseases. The functional coating is a surfactant, a polymer, a marginal active agent, a biological membrane or a lipid bilayer, which is used to improve the physical and chemical properties of the drug carrier, thereby further giving it special properties such as in vivo targeting and long circulation.

[0009] Preferably, the metal organic framework material is a UiO series material, a ZIFs series material, a PCN series material or a MIL series material; more preferably, the PCN series material is a PCN-333 nanoparticle;

[0010] Preferably, the polymer nanomaterial is a polymer nanoparticle, a polymer micelle or a polymer nanofiber; more preferably, the polymer nanomaterial is polylactic acid-co-glycolic acid (PLGA);

[0011] Preferably, the liposome material is dipalmitoylphosphatidylcholine liposome, distearoylphosphatidylcholine liposome or dioleoylphosphatidylcholine liposome; more preferably, the liposome material is dioleoylphosphatidylcholine liposome;

[0012] Preferably, the exosome material is mesenchymal stem cell MSC, hematopoietic stem cell exosome or M2 macrophage exosome; more preferably, the exosome material is M2 macrophage exosome;

[0013] Preferably, the hydrogel material is sodium alginate-chitosan hydrogel, polyethylene glycol-polylactic acid-glycolic acid, or gelatin-oxidized sodium alginate hydrogel; more preferably, the hydrogel material is sodium alginate-chitosan hydrogel;

[0014] The novel hydrogen sulfide donor, the functional coating is a surfactant, a polymer, an edge active agent, a biological membrane or a lipid bilayer.

[0015] Preferably, the surfactant is polysorbate-20 (Tween 20);

[0016] Preferably, the polymer is hyaluronic acid (HA);

[0017] Preferably, the marginal active agent is sorbitan oleate (Span 80);

[0018] In a second aspect, the present invention provides a method for preparing a novel hydrogen sulfide donor as described in any one of the above, comprising the following steps:

[0019] (1) expressing an enzyme capable of catalyzing the production of hydrogen sulfide from sulfur-containing amino acids;

[0020] The specific preparation process includes:

[0021] The amino acid sequence of the enzyme CGL-E339V capable of catalyzing sulfur-containing amino acids to produce hydrogen sulfide was synthesized, and a PCR reaction was performed to obtain the full-length sequence of the CGL gene. After adding a His tag at the rear end, double restriction digestion with the expression vector was performed, and the target gene fragment and plasmid vector were recovered, transformed into competent bacteria, and recombinant bacteria were obtained after cultivation;

[0022] The recombinant plasmid is transformed into competent bacteria by chemical transformation, incubated, and non-resistant LB liquid medium is added for shaking culture, and then transferred to an LB plate containing antibiotics, and positive colonies are subjected to PCR, restriction digestion and sequencing identification, and the target protein is purified to obtain a prokaryotic expression system, and the modified cystathionine gamma lyase with an amino acid sequence as shown in SEQ ID NO.1 is expressed;

[0023] The amino acid sequence of SEQ ID NO.1 is:

[0024] MQEKDASSQGFLPHFQHFATQAIHVGQDPEQWTSRAVVPPISLSTTFKQGAPGQHSGFEYSRSGNPTRNCLEKAVAALDGAKYCLAFASGLAATVTITHLLKAGDQIICMDDVYGGTNRYFRQVASEFGLKISFVDCSKIKLLEAAITPETKLVWIETPTNPTQKVIDIEGCAHIVHKHGDIILVVDNTFMSPYFQRPLALGA DISMYSATKYMNGHSDVVMGLVSVNCESLHNRLRFLQNSLGAVPSPIDCYLCNRGLKTLHVRMEKHFKNGMAVAQFLESNPWVEKVIYPGLPSHPQHELVKRQCTGCTGMVTFYIKGTLQHAEIFLKNLKLFTLAVSLGGFESLAELPAIMTHASVLKNDRDVLGISDTLIRLSVGLEDEEDLLEDLDQALKAAHPPSGIHS.

[0025] According to different drug carriers, hydrogen sulfide synthase is loaded into the drug carrier by any one of the following methods (2)-(6), and a functional coating is coated on the outside of the drug carrier;

[0026] (2) After obtaining hydrogen sulfide synthase, when the drug carrier is a metal organic framework material, weigh the metal organic framework material, disperse it in water, treat it with a cell disruptor, slowly drip the hydrogen sulfide synthase, perform ultrasound in an ice bath, adjust the power of the cell disruptor, slowly drip the functional coating, perform ultrasound, centrifuge, take the precipitate and disperse it in ultrapure water to obtain a hydrogen sulfide donor;

[0027] (3) When the drug carrier is a liposome material, lipids, functional coatings, cholesterol and other materials are weighed and dissolved in an organic solvent, and a film is formed by rotary evaporation. An aqueous solution of hydrogen sulfide synthase is added, and ultrasonic hydration is performed to form a liposome suspension. The suspension is centrifuged and the precipitate is dispersed in ultrapure water to obtain a hydrogen sulfide donor;

[0028] (4) When the drug carrier is a polymer nanomaterial, an aqueous solution of hydrogen sulfide synthase is added and mixed with the polymer nanomaterial solution, and a nanoparticle suspension is obtained after a second ultrasonic emulsification treatment, which is collected by centrifugation and subjected to surface functionalization modification to obtain a hydrogen sulfide donor;

[0029] (5) When the drug carrier is an exosome material, the cells are cultured, the exosomes are extracted, the exosomes are mixed with an aqueous solution of hydrogen sulfide synthase, electroporation is performed to allow the exosomes to encapsulate the engineered enzyme, and the exosomes are collected by centrifugation to obtain a hydrogen sulfide donor;

[0030] (6) When the drug carrier is a hydrogel material, the aqueous solutions of hydrogen sulfide synthase or all the above hydrogen sulfide donors are mixed, and an initiator is added to induce cross-linking to obtain a hydrogen sulfide donor.

[0031] The preparation method, in step (2), the volume ratio of the drug carrier to deionized water is 1 to 5:1; the mass volume ratio of the drug carrier to the modified cystathionine gamma lyase is 1 mL: 0.3 to 0.5 mg; the functional coating is slowly added dropwise to a final concentration of 2 mg / mL. The power of the cell disruptor is 55 W; the power of the cell disruptor is adjusted to 45 W;

[0032] The conditions of the ice bath ultrasound are: power 25-40W, time 30min;

[0033] The ultrasonic conditions are: power 25-40W, time 15min.

[0034] In the preparation method, the volume ratio of the drug carrier to deionized water in step (3) is 2 to 8:1;

[0035] The ultrasonic conditions are: power 25-40W, time 15min.

[0036] In the preparation method, the ultrasonic conditions in step (4) are: power 25-40W, time 15min.

[0037] In the preparation method, the electroporation conditions in step (5) are: 250-350V, 350-550μF, 100Ω, and 1mm.

[0038] In a third aspect, the present invention provides a drug for treating inflammatory diseases, comprising any one of the novel hydrogen sulfide donors and a medically acceptable excipient.

[0039] The drug for treating inflammatory diseases is administered orally, by rectal perfusion, intravenous injection or local administration.

[0040] In a fourth aspect, the present invention provides use of a novel hydrogen sulfide donor as described in any one of the items in the preparation of a drug for treating inflammatory diseases.

[0041] In the application, the inflammatory disease is inflammatory bowel disease, psoriasis or atherosclerosis.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention uses engineered cystathionine gamma lyase (CGL-E339V) as the core for catalyzing the production of H2S. Compared with before the modification, the H2S production performance of the modified CGL enzyme is enhanced by 25 times, and it has a more excellent substrate affinity.

[0044] 2. We selected IBD, psoriasis, and atherosclerosis as representative disease models to verify the in vivo efficacy of the particles. Our strategy can not only inhibit inflammation and immune response and promote barrier repair, but also greatly avoid adverse reactions to normal tissues while fully exerting the therapeutic effect. It is expected to provide new therapeutic ideas and strategies for the treatment of inflammatory diseases.

[0045] 3. The present invention is based on a bionic strategy, with the core being to simulate the enzyme catalytic pathway for producing H2S in vivo, to construct an efficient and controllable H2S biosynthesis system, to endow H2S with the function of stable and uniform release from the source, to achieve a specific, efficient and safe new therapy for inflammatory diseases, and to provide new ideas and scientific basis for further promoting H2S-mediated biological applications and disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The performance of CGL-E339V is characterized, where a is the change of enzyme H2S production performance before and after the transformation, and b is the enzyme kinetic curve;

[0047] Figure 2 It is the characterization of CPH synthesis, where a is the change of hydrated particle size and b is the change of Zeta potential;

[0048] Figure 3 is the CPH hydrogen sulfide release curve;

[0049] Figure 4 It is the CPT synthesis characterization, where a is the change of hydrated particle size and b is the change of Zeta potential;

[0050] Figure 5 It is the CDS synthesis characterization, where a is the change of hydrated particle size and b is the change of Zeta potential;

[0051] Figure 6 is the therapeutic effect of CPH on IBD, where a is the colon white light image and b is the disease activity index;

[0052] Figure 7 is the therapeutic effect of CE on IBD, where a is the colon white light image and b is the disease activity index;

[0053] Figure 8 is the therapeutic effect of CG on IBD, where a is the colon white light image and b is the disease activity index;

[0054] Fig. 9It is the treatment effect of CPT for psoriasis;

[0055] Fig.10 For the treatment effect of CDS psoriasis;

[0056] Fig.11 is the efficacy of CE on atherosclerosis, where a is the plaque area and b is the necrotic area. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and the accompanying drawings.

[0058] Example 1: Synthesis of engineered CGL enzymes

[0059] Hydrogen sulfide synthase is an enzyme in the body that has the ability to catalyze sulfur-containing amino acids to produce hydrogen sulfide, including but not limited to cystathionine β-synthase (CBS), cystathionine γ-lyase (CGL), 3-mercaptopyruvate sulfotransferase (3-MST) and their corresponding engineered forms such as cystathionine γ-lyase with mutation 339 (CGL-E339V). In order to achieve efficient hydrogen sulfide production performance, we compared the hydrogen sulfide production efficiency of the engineered enzymes before and after the modification. The results are as follows: Figure 1 As shown in a, preferably, the present invention selects cystathionine gamma lyase (CGL-E339V) with the highest hydrogen sulfide production efficiency as the hydrogen sulfide synthase. Specifically, we optimized the cystathionine gamma lyase CGL sequence, mutated site 339, obtained the CGL-E339V gene fragment, and subcloned the gene fragment into the expression vector pET28a through a one-step cloning kit (Nanjing Novozyme Biotechnology Co., Ltd.) to obtain the recombinant plasmid pET28a-CGL-E339V. After Escherichia coli BL21 was made competent, the obtained recombinant plasmid was transferred thereto by heat shock method and the correctly identified single colony was transferred to LB+Amp liquid culture medium, and further PCR, enzyme digestion and sequencing identification were performed, and the obtained positive clone strain was named CGL-E339V-BL21 recombinant bacteria. After culture amplification, the magnetic bead method was used to purify the target protein, and the results are shown in Figure 1 As shown in Figure b, we successfully constructed a prokaryotic expression system capable of mass production of CGL-E339V. The CGL-E339V enzyme produced by the engineered bacteria has excellent enzyme kinetics.

[0060] Example 2: Preparation and surface functionalization of a novel hydrogen sulfide donor using a metal organic framework as a delivery carrier

[0061] Metal-organic framework materials (MOF) are ideal bioenzyme immobilization carriers due to their special properties such as high porosity and large surface area. Many common MOFs including UiO series, ZIFs series, PCN series, MIL series, etc. can be used as excellent carriers for immobilized enzymes. Preferably, we use PCN series metal-organic framework PCN-333 with good biocompatibility to immobilize the engineered enzyme CGL-E339V. Next, according to different assembly methods, the enzyme immobilization scheme can be divided into encapsulation (enzyme and MOF are synthesized together to form a composite material), pore penetration (introducing the enzyme into the pores of the pre-synthesized MOF) and surface binding (anchoring the enzyme to the pre-synthesized MOF surface through covalent or non-covalent interactions). Since PCN-333 has a pore size that fits the size of the engineered enzyme, preferably, we use pore penetration to immobilize the engineered enzyme. Finally, depending on the specific application scenario, surface functionalization modification of MOF by chemical or physical means can give it additional properties such as targeting, long circulation, and response. Common modification materials such as polymers, small molecules, biological membranes, lipid bilayers, etc. are widely used. Preferably, we use hyaluronic acid HA with the ability to target CD44 receptors at inflammatory sites to perform surface functionalization modification on nanoparticles to complete the synthesis of the new hydrogen sulfide donor CPH. Specifically, take 1mL of nanoparticles, centrifuge and ultrasonically disperse the precipitate in deionized water, then transfer the solution to a 5mL chicken heart bottle, adjust the power of the cell disruptor to 55W, slowly add 0.3mg of the above CGL-E339V solution, then ultrasonicate at 25W for 30min in an ice bath, adjust the power to 45W after the ultrasonication, slowly add HA solution to make the final concentration of the system 2mg / mL, then ultrasonicate at 25W for 15min. Centrifuge after the ultrasonication to obtain the new hydrogen sulfide donor CPH. The results are as follows Figure 2 As shown, the changes in particle size and potential before and after hydration prove the successful synthesis of the new hydrogen sulfide donor CPH.

[0062] Example 3: CPH hydrogen sulfide production performance evaluation

[0063] In order to evaluate the hydrogen sulfide production performance of CPH, we compared it with traditional hydrogen sulfide donors such as NaHS and GYY4137, and directly measured the hydrogen sulfide release using the DTNB method. It was proved that our method has an efficient, stable, and smooth hydrogen sulfide release curve, which meets the treatment needs of inflammatory diseases. Figure 3 As shown in the data, the hydrogen sulfide release rate of traditional hydrogen sulfide donors GYY4137 and NaHS is unstable, which can easily cause local tissue hydrogen sulfide overload in a short period of time and thus cause certain harm. However, the new hydrogen sulfide donor CPH can release hydrogen sulfide slowly and stably, and has excellent clinical application prospects.

[0064] Example 4: Preparation and surface functionalization of a novel hydrogen sulfide donor using polymer nanomaterials as carriers

[0065] Polymer nanomaterials are widely used in the delivery of various drugs due to their excellent biocompatibility and degradability. Various polymer nanomaterials including polymer nanoparticles, polymer micelles, polymer nanofibers, etc. can be used for enzyme immobilization, which can achieve multiple functions such as enzyme activity protection, stability improvement, and controlled release. Preferably, we use polylactic acid-glycolic acid copolymer PLGA with good biocompatibility to encapsulate and protect CGL-E339V and synthesize CP nanoparticles. Further, according to different specific application scenarios, the surface functionalization modification of polymer nanomaterials using various materials such as biomolecules, polymers, chemical groups, lipid bilayers, etc. can give them new properties. In order to improve the skin permeability of the material in psoriasis, we use non-ionic surfactant Tween 20 to further modify the surface functionalization of nanoparticles and complete the synthesis of hydrogen sulfide donor CPT. Specifically, 150 mg of PLGA polymer was dissolved in 1.5 mL of dichloromethane to form a PLGA solution, 10 mg of CGL enzyme was dissolved in 10 mL of phosphate buffer (PBS, pH 7.4) to form a CGL enzyme solution, and a protective sugar such as 5% (w / v) trehalose was added thereto to protect the activity of the enzyme during the emulsification process. The CGL enzyme solution treated with the protective agent was slowly added dropwise to the PLGA solution, and ultrasonic emulsification was performed using an ultrasonic processor, and the first emulsion was formed by ultrasonic treatment in an ice bath for 60 s under slight stirring. Colostrum (W / O) was slowly added dropwise to 20 mL of phosphate buffered saline (PBS) containing 0.5% (w / v) polyethylene glycol (PEG), stirred slightly, and ultrasonicated for 60 s in an ice bath to form a secondary emulsion, which was then centrifuged and washed. The washed CP nanoparticle precipitate was resuspended in 10 mL of deionized water containing 1% (v / v) Tween 20, stirred for 1 hour to allow Tween 20 to adsorb on the surface of the nanoparticles, and then stirred at room temperature for 3 hours, and centrifuged and washed to obtain CPT nanoparticles. The results are shown in Figure 4 As shown, the changes in the hydrated particle size and potential before and after the hydration prove the successful synthesis of the new hydrogen sulfide donor CPT. The evaluation of CPT hydrogen sulfide production performance is the same as that in Example 3, so it will not be repeated here.

[0066] Example 5: Preparation and surface functionalization of a novel hydrogen sulfide donor using liposomes as carriers

[0067] Liposome materials have excellent biocompatibility and biodegradability as drug carriers and are ideal drug carriers. Currently, a variety of liposome materials such as dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine liposomes are widely used in clinical practice. By selecting appropriate liposome components and surface modifications, the biodistribution, stability and efficacy of drugs can be significantly improved, while reducing side effects. Preferably, we use dioleoylphosphatidylcholine (DOPC) liposomes with a stable lipid structure to encapsulate and protect CGL-E339V and synthesize CD nanoparticles. Furthermore, in order to improve the skin permeability of the material in psoriasis, preferably, we use the marginal active agent Span 80 for surface treatment. Span 80 can enhance the elasticity of liposomes so that they can penetrate the skin more easily, thereby obtaining hydrogen sulfide donor CDS. The specific steps are as follows: 3 mg DOPC, 0.5 mg Span 80, 2 mg cholesterol and 1 mg PE-PEG are dissolved in an appropriate amount of chloroform and placed in a chicken heart bottle, and the solvent is removed by rotary evaporation to form a film, and then the film is dried in a vacuum dryer for 1 hour, and then 5 mg CGL-E339V PBS solution is added, shaken and ultrasonically treated to form a liposome suspension, and a new hydrogen sulfide donor CDS is obtained. The results are as follows. Figure 5 As shown, the changes in the hydrated particle size and potential before and after the hydration prove the successful synthesis of the novel hydrogen sulfide donor CDS. The evaluation of CDS hydrogen sulfide production performance is the same as that in Example 3, so it will not be repeated here.

[0068] Example 7: Preparation of a novel hydrogen sulfide donor using exosomes as carriers

[0069] For the treatment of inflammatory diseases, cellular exosomes are good drug carriers. Various cellular exosomes, including mesenchymal stem cells MSC, hematopoietic stem cell exosomes and M2 macrophage exosomes, have strong anti-inflammatory effects. Preferably, we use M2 macrophage exosomes as drug carriers. M2 macrophage exosomes can produce a variety of anti-inflammatory factors to significantly reduce inflammation. Depending on the assembly method, the engineered enzyme can be loaded by different methods including direct incubation, dialysis, electroporation, etc. Preferably, in order to improve the encapsulation efficiency of the engineered enzyme, we use electroporation to prepare the new hydrogen sulfide donor CE. The specific steps are: when culturing RAW264.7 macrophages, use DMEM high-glucose medium containing IL-4 (20ng / mL) for culture. Wait for the macrophages to grow all over the bottom of the culture bottle, collect the supernatant, and centrifuge to remove cells, apoptotic bodies and cell debris. The supernatant was then centrifuged (2500g, 15min) with an ultrafiltration tube to concentrate M2exo, and then filtered (diameter 450nm) to remove exosomes above 450nm. Finally, the filtrate was centrifuged and filtered to obtain M2exo, which was resuspended in PBS and added to the electroporation dish together with the engineered enzyme solution, and electroporated under the conditions of 250V, 350μF, 100Ω, and 1mm. The resulting solution was then incubated at 37°C for 30min to ensure membrane fusion of M2exo, thereby encapsulating CGL-E339V inside. Finally, an ultracentrifuge was used to centrifuge to obtain the hydrogen sulfide donor CE. The CE hydrogen sulfide production performance evaluation is the same as that in Example 3, so it will not be repeated here.

[0070] Example 8: Synthesis of a novel orally administrable hydrogen sulfide donor

[0071] In order to give the above hydrogen sulfide donor gastric performance and improve patient compliance, hydrogel is a good drug carrier, including sodium alginate-chitosan, polyethylene glycol-polylactic acid-glycolic acid, gelatin-oxidized sodium alginate hydrogel, etc. Preferably, we use sodium alginate-chitosan hydrogel as a drug carrier to directly coat CGL-E339V enzyme or all hydrogen sulfide donors mentioned in Examples 3, 5, and 7. The specific steps are to dissolve sodium alginate and chitosan powder in deionized water, stir well to form a uniform mixed solution, add hydrogen sulfide synthase / donor solution to the mixed solution, and continue stirring to prevent the formation of uneven gel particles, and add a cross-linking agent to the mixed solution to induce cross-linking between sodium alginate and chitosan. Finally, the gel is poured into a mold to form a desired shape to obtain a hydrogen sulfide donor CG. The hydrogen sulfide production performance evaluation is the same as that in Example 3, so it is not repeated here.

[0072] Example 9: Evaluation of the efficacy of the novel hydrogen sulfide donor CPH in inflammatory bowel disease

[0073] Female C57BL / 6 mice were randomly divided into groups. The experimental group mice drank 4% DSS every day for 6 days to establish an ulcerative colitis model. The control group only received an equal amount of deionized water. The ulcerative colitis mice began to be treated with drugs on the evening of the 7th day after induction. Before drug administration, the mice were fasted in advance and replaced with 50% glucose solution instead of mouse food. The experimental group mice were enema 300μL 60mg / mL CPH nanoparticles, and the Healthy group and the Control group were enema administered with the corresponding dose of normal saline. During the experiment, the weight, rectal bleeding and diarrhea of ​​each mouse were recorded to calculate the Disease Activity Index (DAI). The occult blood in the feces was detected by the blood occult blood test and given 0 to 4 points, which were defined as follows: 0 points for no blood; 2 points for positive blood occult blood; 4 points for severe bleeding. The severity of diarrhea was scored from 0 to 4 points, and the scoring criteria were: 0 points for good particle shape; 2 points for pasty and semi-formed stools; 4 points for liquid stools. On the morning of day 11, all mice were killed and dissected, and the colons were removed and photographed. Figure 6 As shown, the condition of the CPH treatment group was significantly improved compared with the control group.

[0074] Example 10: Evaluation of the efficacy of a novel hydrogen sulfide donor using exosomes as a carrier in inflammatory bowel disease

[0075] A mouse ulcerative colitis model was established. The mice in the experimental group were given 300 μL 60 mg / mL new hydrogen sulfide donor CE by enema, and the mice in the Healthy group and the Control group were given corresponding doses of normal saline by enema. During the experiment, the DAI of each mouse was recorded and calculated. After the treatment, all the mice were killed and dissected, and the colon length of each mouse was counted. The results are shown in Figure 7 As shown, the condition of the CE-treated group was significantly improved compared with the control group.

[0076] Example 11: Evaluation of the efficacy of a novel orally available hydrogen sulfide donor in inflammatory bowel disease

[0077] A mouse ulcerative colitis model was established. The mice in the experimental group were intragastrically administered with the novel hydrogen sulfide donor CG, and the mice in the Healthy group and the Control group were intragastrically administered with the corresponding dose of normal saline. The DAI of each mouse was recorded and calculated during the experiment. After the treatment, all the mice were killed and dissected, and the colon was photographed. Figure 8 As shown, the condition of the CG treatment group was significantly improved compared with the control group.

[0078] Example 12: Evaluation of the efficacy of the novel hydrogen sulfide donor CPT in psoriasis

[0079] C57BL / 6 mice were randomly divided into groups. After anesthesia, the hair in the central area of ​​the mouse's back was carefully shaved with an electric hair clipper to form an exposed area of ​​2cm×3cm, and then a mild depilatory cream was used to remove the hair on the skin surface. Imiquimod cream was applied continuously to establish the model (day 0-day 5), and the drug was administered for 5 consecutive days. During the induction and treatment, the mice in the experimental group were applied with CPT nanoparticles every day, and the model group was applied with the corresponding dose of normal saline. The skin condition of the mice was monitored by taking pictures. The results are as follows Fig. 9 As shown, the skin condition of the CPT treatment group was significantly improved compared with the control group.

[0080] Example 13: Evaluation of the efficacy of the novel hydrogen sulfide donor CDS in psoriasis

[0081] The mouse psoriasis model was constructed by the method in Example 12. The mice in the experimental group were smeared with CDS nanoparticles every day, and the mice in the model group were smeared with a corresponding dose of normal saline. The skin condition of the mice was monitored by taking photos. Fig.10 As shown, the skin condition of the CDS-treated group was significantly improved compared with the control group.

[0082] Example 14: Evaluation of the therapeutic effect of the novel hydrogen sulfide donor CE on atherosclerosis

[0083] Male Myh11-CreERT2 ROSA26 STOP-flox eYFP+ / +Apoe- / -6-week-old mice were selected and injected with 1 mg of tamoxifen per mouse per day for 10 days, with a total dose of 10 mg / mouse. Subsequently, the mice were fed a high-fat diet (20-23% milk fat, 34% sucrose, 0.2% cholesterol) for 2 weeks, and then treated and received H2S donor CE through tail vein injection. The Health group and the Control group were given corresponding doses of normal saline. After one month of treatment, the plaque area of ​​each group was counted. Results Fig.11 As shown, the vascular condition in the CE treatment group was significantly improved compared with the control group.

Claims

1. A novel hydrogen sulfide donor, characterized in that: The hydrogen sulfide donor comprises hydrogen sulfide synthase, a drug carrier loaded with the hydrogen sulfide synthase, and a functional coating coated on the outside of the drug carrier.

2. A novel hydrogen sulfide donor as claimed in claim 1, characterized in that: The hydrogen sulfide synthase is a modified cystathionine gamma lyase.

3. A novel hydrogen sulfide donor as claimed in claim 2, characterized in that: The modified cystathionine gamma lyase is obtained by mutating the E at position 339 of the cystathionine gamma lyase to V. The amino acid sequence of the modified cystathionine gamma lyase is shown in SEQ ID NO.

1.

4. A novel hydrogen sulfide donor as claimed in claim 1, characterized in that: The drug carrier is a metal organic framework material, a polymer nanomaterial, a liposome material, an exosome material or a hydrogel material; Preferably, the metal organic framework material is a UiO series material, a ZIFs series material, a PCN series material or a MIL series material; more preferably, the PCN series material is a PCN-333 nanoparticle; Preferably, the polymer nanomaterial is a polymer nanoparticle, a polymer micelle or a polymer nanofiber; more preferably, the polymer nanomaterial is a polylactic acid-glycolic acid copolymer; Preferably, the liposome material is dipalmitoylphosphatidylcholine liposome, distearoylphosphatidylcholine liposome or dioleoylphosphatidylcholine liposome; more preferably, the liposome material is dioleoylphosphatidylcholine liposome; Preferably, the exosome material is mesenchymal stem cell MSC, hematopoietic stem cell exosome or M2 macrophage exosome; more preferably, the exosome material is M2 macrophage exosome; Preferably, the hydrogel material is sodium alginate-chitosan hydrogel, polyethylene glycol-polylactic acid-glycolic acid or gelatin-oxidized sodium alginate hydrogel; more preferably, the exosome material is M2 macrophage exosomes.

5. A novel hydrogen sulfide donor as claimed in claim 1, characterized in that: The functional coating is a surfactant, a polymer, an edge active agent, a biological membrane or a lipid bilayer; Preferably, the surfactant is polysorbate-20; Preferably, the polymer is hyaluronic acid; Preferably, the marginal active agent is sorbitan oleate.

6. A method for preparing the novel hydrogen sulfide donor according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) expressing and obtaining hydrogen sulfide synthase capable of catalyzing the production of hydrogen sulfide from sulfur-containing amino acids, loading the hydrogen sulfide synthase in the drug carrier using any of the following methods (2) to (6) according to different drug carriers, and coating the drug carrier with a functional coating; (2) When the drug carrier is a metal organic framework material, the metal organic framework material is weighed, dispersed in water, treated with a cell disruptor, and then slowly dripped with hydrogen sulfide synthase, ultrasonicated in an ice bath, the power of the cell disruptor is adjusted, the functional coating is slowly dripped, ultrasonicated, centrifuged, and the precipitate is dispersed in ultrapure water to obtain a hydrogen sulfide donor; (3) When the drug carrier is a liposome material, lipids, functional coatings, cholesterol and other materials are weighed and dissolved in an organic solvent, and a film is formed by rotary evaporation, and an aqueous solution of hydrogen sulfide synthase is added, and ultrasonic hydration is performed to form a liposome suspension to obtain a hydrogen sulfide donor; (4) When the drug carrier is a polymer nanomaterial, an aqueous solution of hydrogen sulfide synthase is added and mixed with the polymer nanomaterial solution, and a nanoparticle suspension is obtained after a second ultrasonic emulsification treatment, which is collected by centrifugation and subjected to surface functionalization modification to obtain a hydrogen sulfide donor; (5) When the drug carrier is an exosome material, the cells are cultured, the exosomes are extracted, the exosomes are mixed with an aqueous solution of hydrogen sulfide synthase, and the hydrogen sulfide synthase is encapsulated by electroporation to obtain a hydrogen sulfide donor; (6) When the drug carrier is a hydrogel material, the aqueous solutions of hydrogen sulfide synthase or all the above hydrogen sulfide donors are mixed, and an initiator is added to induce cross-linking to obtain a hydrogen sulfide donor.

7. A drug for treating inflammatory diseases, characterized in that: The method comprises the novel hydrogen sulfide donor as described in any one of claims 1 to 5 and a pharmaceutically acceptable auxiliary material.

8. A drug for treating inflammatory diseases according to claim 7, characterized in that: The drug can be administered orally, by rectal instillation, intravenous injection or topical administration.

9. Use of a novel hydrogen sulfide donor as claimed in any one of claims 1 to 5 in the preparation of a drug for treating inflammatory diseases.

10. The use according to claim 9, characterized in that The inflammatory disease is inflammatory bowel disease, psoriasis or atherosclerosis.

Images