Clostridium butyricum targeted delivery system based on GelMA microspheres as well as preparation method and application of clostridium butyricum targeted delivery system

Abstract

The invention discloses a clostridium butyricum targeted delivery system based on GelMA microspheres and a preparation method and application thereof, and relates to the technical field of biological medicines.The clostridium butyricum targeted delivery system based on GelMA microspheres comprises a methacrylated gelatin microsphere carrier, a GelMA microsphere carrier, a GelMA microsphere carrier, a GelMA microsphere carrier, a GelMA microsphere carrier, a GelMA microsphere carrier and a GelMA microsphere carrier, the clostridium butyricum is entrapped in the GelMA microspheres; wherein the GelMA microspheres form a porous structure through photo-crosslinking, can be adhered to the damaged mucous membrane part of the intestinal tract, and can delay the release of butyric acid. According to the GelMA microsphere delivery system for targeting the damaged intestinal mucosa, the C.butyricum can be wrapped, the activity of bacteria is guaranteed, the C.butyricum is adhered to the damaged part of the intestinal mucosa for 6-12 hours, and meanwhile volatilization of a metabolite butyric acid is delayed. The problems that in the UC micro-ecological therapy, probiotics are difficult to resist the gastrointestinal tract environment to reach the inflammation part, the probiotics are difficult to colonize, and probiotics metabolites are too fast to lose are effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to a targeted delivery system of Clostridium butyricum based on GelMA microspheres, and its preparation method and application. Background Art

[0002] Ulcerative colitis (UC) is a chronic non-specific intestinal inflammatory disease caused by multiple factors such as genetics, immunity, microorganisms, and environment, and belongs to the main type of inflammatory bowel disease (IBD). UC patients face problems such as low endoscopic remission rate, repeated symptoms, high hospitalization rate, and the risk of colectomy (10%-15%), and heavy medical burden. And existing treatment means (such as aminosalicylates, glucocorticoids, biological agents, etc.) have problems such as unstable curative effect, long-term medication dependence, and side effects, and are listed as refractory diseases by the World Health Organization, and there is an urgent need for safe and efficient new treatment strategies.

[0003] In recent years, the role of intestinal flora and its metabolites (such as short-chain fatty acids, SCFAs) in the treatment of UC has attracted much attention. Clostridium butyricum, as an anaerobic resident bacterium in the intestine, can play key roles such as repairing intestinal barrier function, regulating immune response, and remodeling intestinal flora by producing butyric acid. However, Clostridium butyricum faces problems such as poor targeting, difficult colonization, and rapid release of metabolites in clinical applications.

[0004] To solve the above problems, researchers have developed a variety of probiotic targeted delivery systems. Although targeted delivery systems (such as hydrogel microcapsules, adhesive carriers) show potential in the treatment of IBD, for example, poly-γ-glutamic acid microgels, hyaluronic acid hydrogels, etc. can protect probiotics and release in response to the inflammatory environment, but the specific delivery system for Clostridium butyricum still has the following deficiencies:

[0005] The existing carriers have limited adhesion ability to the specific mucosal damaged parts of UC and are difficult to accurately enrich in the distal colon lesion area. And there is no exclusive sustained-release mechanism designed for the volatility of butyric acid, the metabolite release cycle is short, and the treatment window is narrow. Some synthetic materials have risks of immunogenicity or toxicity of degradation products, which limit long-term application.

[0006] Therefore, we propose a targeted delivery system of Clostridium butyricum based on GelMA microspheres, and its preparation method and application, in order to solve the problems raised above. Summary of the Invention

[0007] The purpose of the present invention is to provide a targeted delivery system of Clostridium butyricum based on GelMA microspheres, and its preparation method and application, so as to solve the problems in the current market proposed in the above background art.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] The Clostridium butyricum targeted delivery system based on GelMA microspheres includes:

[0010] Methacrylated gelatin microsphere carriers;

[0011] Clostridium butyricum encapsulated in the GelMA microspheres;

[0012] Among them, the GelMA microspheres form a porous structure through photocrosslinking, can adhere to the damaged intestinal mucosa site, and delay the release of butyric acid.

[0013] Preferably, the pore size of the GelMA microspheres is 10 - 100 μm.

[0014] Preferably, the loading amount of Clostridium butyricum is 1×10 6 CFU / mL.

[0015] Preferably, the GelMA microspheres can adhere to the intestinal inflammation site for 6 - 12 hours.

[0016] The preparation method of the Clostridium butyricum targeted delivery system based on GelMA microspheres includes the following steps:

[0017] S1. Prepare GelMA: React gelatin with methacrylic anhydride at 50°C for 1 - 3 hours, dialyze and then freeze-dry;

[0018] S2. Prepare the GelMA precursor solution: Dissolve GelMA in phosphate buffer solution (PBS) and add a photoinitiator;

[0019] S3. Load Clostridium butyricum: Mix Clostridium butyricum cultured to the logarithmic growth phase with the GelMA precursor solution;

[0020] S4. Microsphere forming: Use microfluidic technology to form emulsion droplets, and cure them by ultraviolet photocrosslinking to obtain GelMA microspheres encapsulating Clostridium butyricum.

[0021] Preferably, the photoinitiator is phenyl-2,4,6-trimethylbenzoylphosphinic acid ester (LAP).

[0022] Preferably, in step S3, the culture conditions of Clostridium butyricum are an anaerobic environment, incubated at 37°C for 14 - 16 hours.

[0023] The application of the Clostridium butyricum targeted delivery system in the preparation of drugs for treating ulcerative colitis. Preferably, the drug is used by oral administration, enema or endoscopic delivery, and the drug can improve the intestinal barrier function, reduce the level of inflammatory factors, and regulate the balance of intestinal flora.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] The present invention develops a GelMA microsphere delivery system targeting damaged intestinal mucosa, which can encapsulate C. butyricum and ensure the viability of the bacteria, adhere to the damaged site of the intestinal mucosa for 6 - 12 hours, and at the same time delay the volatilization of the metabolite butyric acid. It effectively solves the problems in the UC microecological therapy that probiotics are difficult to resist the gastrointestinal environment and reach the inflammatory site, difficult to colonize, and the metabolites of probiotics are lost too quickly.

[0026] In the present invention, the GelMA microspheres can specifically adhere to the damaged site of the intestinal mucosa, precisely deliver Clostridium butyricum to the UC lesion area, avoid the loss in environments such as gastric acid and bile in traditional oral preparations, and improve the targeting of Clostridium butyricum. Moreover, the GelMA microspheres provide a good living environment for Clostridium butyricum, extend its residence time in the intestine, promote its colonization and growth in the intestine, thereby enhancing the therapeutic effect of Clostridium butyricum. At the same time, the GelMA microspheres can control the release rate of the metabolite butyric acid of Clostridium butyricum, enabling it to be slowly released in the intestine, maintaining the effective concentration in the intestine, and avoiding the decline in therapeutic effect caused by the too-fast volatilization of butyric acid.

[0027] In the present invention, Clostridium butyricum can produce butyric acid, which can promote the growth of intestinal epithelial cells and repair the intestinal barrier function, thereby reducing intestinal permeability, reducing the release of inflammatory factors, and improving the intestinal health of UC patients. Moreover, Clostridium butyricum can regulate the balance of the intestinal flora and inhibit the production of inflammatory factors, thereby reducing the inflammatory response of UC patients and alleviating UC symptoms.

[0028] The above summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present invention will be readily apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the targeted delivery system of the present invention;

[0030] Figure 2 It is a schematic diagram for the preparation of butyricum - GelMA microspheres in the present invention;

[0031] Figure 3 It is a diagram showing that C. butyricum alleviates intestinal inflammation in the present invention; wherein, (A) is a schematic diagram of the experiment; (B) is the abundance of C. butyricum in the colon; (C) is the improvement of weight loss; (D - E) are the improvements in colon shortening; (F - G) are the improvements in colon pathology; (H - J) are the decreases in serum inflammatory factor levels.

[0032] Figure 4This is the figure showing the improvement of intestinal barrier function by C. butyricum in the present invention; among them, the intestinal barrier proteins ZO-1, Occludin, Claudin1, and MUC2 were significantly increased after C. butyricum treatment.

[0033] Figure 5 This is the figure showing the improvement of colitis by C. butyricum through SCFAs in the present invention; among them, by utilizing the characteristics that SCFAs will volatilize under heating conditions and cannot pass through a 100KD molecular filter, combined with targeted SCFAs metabolomics, it was verified that the main anti-inflammatory component in the metabolites of C. butyricum is SCFAs. After intervening in colitis mice with C. butyricum culture medium with heating (CBCM) and without heating (DCB), there was no curative effect after heating (SCFAs volatilized); (A) Weight loss / (B-C) Colon length; The small molecule substances in the C. butyricum culture solution are the main anti-inflammatory components / (D) Weight loss / (E) Colon length / (F) Targeted metabolomics analysis of SCFAs in the C. butyricum culture solution.

[0034] Figure 6 This is the figure showing the main metabolite of anti-inflammatory and barrier repair by C. butyricum in the present invention; among them, acetic acid, propionic acid, and butyric acid were used to treat colitis mice respectively, and the improvement of weight loss, (B) colon length, and (C) relative mRNA expression of cytokines IL-6 and IL-1β were comprehensively considered. The effect of the protein level (D) and mRNA level (M) of the tight junction marker ZO-1 in colitis mice after butyrate administration was the best.

[0035] Figure 7 This is the figure showing the detection of bacterial activity of GelMA microspheres and the adhesion test at the intestinal mucosal damage site in the present invention; among them, (A) Dilution coating was performed on viable C. butyricum bacteria cultured in vitro for 12 h vs. C. butyricum encapsulated by GelMA microspheres for 12 h, and the results showed that GelMA microspheres did not affect bacterial activity. (B) The intestinal mucosa of normal mice was intact, and it was basically undetectable 6 hours after oral administration of fluorescently labeled GelMA microspheres. However, in DSS-induced colitis mice, there was mucosal damage in the whole digestive tract of the mice, and fluorescence could still be detected within 6-12 hours after oral administration, indicating that the GelMA microspheres have an adhesion property to the mucosal damage site. Detailed implementation mode

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0037] Example 1: For the culture and identification of C. butyricum

[0038] In a laminar flow hood, dissolve C. butyricum tablets (import drug registration number: S20140080, Miyarisan Pharmaceutical Co., Ltd., Japan) in sterile cold PBS solution.

[0039] Use the streak inoculation method to inoculate the C. butyricum bacterial solution on MRS solid medium, put it into a sealed bag containing an anaerobic pack, and transfer it to an incubator at 37 °C for 24 h. When single colonies grow on the solid medium, take out the solid medium, pick out single colonies with an inoculation loop and culture them in MRS liquid medium, and quickly put them into a sealed bag containing an anaerobic pack that produces inert gas. Then place the whole sealed bag in an incubator at 37 °C for 14 - 16 h. If gas is generated, the medium becomes turbid, and white precipitate appears at the bottom of the culture tube, then according to the instructions of the QIAGEN kit for DNA extraction, extract bacterial DNA in sequence, and then perform PCR amplification with C. butyricum 16S rRNA specific primers, as shown in the attached Figure 3 - Attached Figure 7 figure.

[0040] After the amplification is complete, take out the product for gel electrophoresis; if there are no extra bands in the electrophoresis result, send the sample to Shanghai Sangon for bacterial identification by 16S rRNA sequencing.

[0041] After the attached figure shows that the identification result is C. butyricum, start to measure the growth curve of C. butyricum. Use Graphpad software to draw the exponential growth curve, and select the best viability time of C. butyricum live bacteria according to the results; then streak the bacteria at the best viability time on a solid medium plate and count them at an optical density (OD) of 600.

[0042] According to the optimal viability time of C. butyricum, the live C. butyricum bacterial solution cultured for the corresponding time was quickly centrifuged at a low speed in a laminar flow hood at 4 °C at a rotation speed of 2000 rpm / min for 5 min. The supernatant was poured out and filtered through a 0.22 μm filter for later use. The precipitate after centrifugation was washed 3 times with sterile cold PBS, and finally adjusted to a concentration of 1×106 colony forming units (CFU) / mL. It was prepared immediately before each experiment and used immediately.

[0043] Example 2. Establishment of an enteritis animal model

[0044] C57BL / 6 mice raised under SPF conditions were used to induce an acute ulcerative colitis model by administering 2.5% DSS (dextran sulfate sodium). Wild-type acute enteritis mouse model: C57BL / 6 mice at 6-8 weeks of age were adaptively raised and then given 2.5% DSS solution for free drinking for 7 consecutive days to induce acute colitis.

[0045] Enteritis indicators: During the experiment, the body weight changes, fecal characteristics, and rectal bleeding of the mice were monitored to evaluate the disease activity index. At the end of the experiment, the colon length, gross changes in the colon, colon pathology, serum inflammatory factor ELISA (TNF-α, IL-6, IL-1β), and HE staining of paraffin-embedded colon tissue were examined. As shown in Appendix Figure 3 Appendix Figure 4 shown.

[0046] Barrier indicators: FITC-dextran intestinal permeability detection, IF, WB, and PCR were used to detect the expression of tight junction proteins (Zo-1, Occludin, Claudin, and MUC2), and the ultrastructure of tight junctions was observed by electron microscopy. As shown in Appendix Figure 4 shown.

[0047] Analysis of intestinal flora and its metabolites: The changes in intestinal flora were analyzed by 16S rRNA gene sequencing, and the levels of SCFAs in fecal and serum samples were determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). As shown in Appendix Figure 5 Appendix Figure 6 shown.

[0048] Example 3. Preparation of C. butyricum-GelMA microspheres

[0049] Synthesis of GelMA: Weigh 5 g of type A porcine skin gelatin and dissolve it in 50 mL of phosphate buffer solution (DPBS) (10% W / V) at 50 °C with stirring until completely dissolved. Subsequently, gradually add about 4 ml of appropriate amount of methacrylic anhydride (MA), and continuously stir at 50 °C for acylation reaction for 1-3 hours.

[0050] Then, 100 ml of diluent DPBS (warmed to 40 °C) was added to terminate the excessive methacrylic acid reaction. The resulting mixture was dialyzed in warm distilled water at 40 °C using a 12 - 14 kDa cut-off dialysis tube for 1 week to remove unreacted reagents and by-products, and finally freeze-dried for one week to obtain porous foam-like GelMA, which was collected and stored long-term in a -80 °C refrigerator. As shown in the appendix Figure 1 as follows

[0051] Loading of C.butyricum: C.butyricum was cultured to the logarithmic growth phase, collected and washed to remove the culture medium components. C.butyricum was mixed with GelMA microspheres and adsorbed under suitable conditions so that C.butyricum adhered firmly to the surface or inside of the microspheres (C.butyricum was incorporated into 1.0 g of GelMA precursor solution, and 0.0125 g of phenyl lithium-2,4,6-trimethylbenzoylphosphinate initiator and the continuous phase were respectively injected into the inlet of the microfluidic device from a syringe, and the flow rates of the dispersed phase and the continuous phase were controlled by a micro-injection pump. The resulting emulsion droplets were collected in a cryogenic bath at about -30 °C, so that stable ice crystals were formed inside the emulsion droplets). As shown in the appendix Figure 2 as follows

[0052] Through a photocrosslinking step or a chemical crosslinking agent, the stable binding of C.butyricum to the microspheres was ensured. Then, morphological analysis was carried out, and the surface morphology of the microspheres and the distribution of C.butyricum were observed using a scanning electron microscope (SEM). Nanoparticle tracking analysis (NTA) was used to determine the pore size of the microspheres. In a simulated intestinal inflammatory environment, the release rate and survival rate of C.butyricum-GelMA microspheres were analyzed by timed sampling. As shown in the appendix Figure 7 as follows

[0053] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples

[0054] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A Clostridium butyricum targeted delivery system based on GelMA microspheres, characterized in that, Comprising: Methacrylated gelatin microsphere carriers; Clostridium butyricum encapsulated in the GelMA microspheres; Wherein, the GelMA microspheres form a porous structure through photocrosslinking, can adhere to the damaged intestinal mucosa site, and delay the release of butyric acid.

2. The Clostridium butyricum targeted delivery system based on GelMA microspheres according to claim 1, wherein: The loading amount of Clostridium butyricum is 1×10 6 CFU / mL.

3. The Clostridium butyricum targeted delivery system based on GelMA microspheres according to claim 1, characterized in that: The GelMA microspheres can adhere to the intestinal inflammation site for 6 - 12 hours.

4. Preparation method of Clostridium butyricum targeted delivery system based on GelMA microspheres, characterized in that, Including the following steps: S1. Prepare GelMA: React gelatin with methacrylic anhydride at 50°C for 1 - 3 hours, dialyze and then lyophilize; S2. Prepare a GelMA precursor solution: Dissolve GelMA in phosphate buffer and add a photoinitiator; S3. Load Clostridium butyricum: Mix Clostridium butyricum cultured to the logarithmic growth phase with the GelMA precursor solution; S4. Microsphere forming: Use microfluidic technology to form emulsion droplets, and cure by ultraviolet photocrosslinking to obtain GelMA microspheres encapsulating Clostridium butyricum.

5. The preparation method of the Clostridium butyricum targeted delivery system based on GelMA microspheres according to claim 4, wherein: The photoinitiator is phenyl-2,4,6-trimethylbenzoylphosphinic acid ester.

6. The preparation method of the Clostridium butyricum targeted delivery system based on GelMA microspheres according to claim 4, wherein: In step S3, the culture conditions of Clostridium butyricum are an anaerobic environment, incubated at 37°C for 14 - 16 hours.

7. Use of the Clostridium butyricum targeted delivery system according to claims 1 - 3 in the preparation of a drug for treating ulcerative colitis.

8. Use of the Clostridium butyricum targeted delivery system according to claim 7 in the preparation of a drug for treating ulcerative colitis, characterized in that: The drug is used by oral, enema or endoscopic delivery methods, and the drug can improve intestinal barrier function, reduce the levels of inflammatory factors, and regulate the balance of intestinal flora.

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