Multifunctional microsphere for treating sepsis as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological materials, and particularly relates to a multifunctional microsphere for treating sepsis and a preparation method and application thereof. The multifunctional microspheres for treating sepsis, provided by the invention, are prepared by taking polydopamine nanoparticles, antibacterial peptide, eugenol and sodium alginate as main components. The multifunctional microspheres for treating sepsis, provided by the invention, have remarkable effects of pH response, inflammation resistance, immune regulation and intestinal flora regulation.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a multifunctional microsphere for treating sepsis, a preparation method and an application thereof. Background Art

[0002] Sepsis is a heterogeneous syndrome usually triggered by infection or injury, accompanied by systemic inflammatory response, which may lead to multiple organ dysfunction syndrome. Excessive inflammatory response can lead to inflammatory cell pyroptosis and massive cell necrosis, which further aggravate the inflammatory response and lead to immune system exhaustion. The activation of a large number of cytokines is the body's defense response against infection, but these inappropriate responses can lead to the exhaustion of immune cells such as T lymphocytes and change their functions. Excessive cytokine production and secretion can cause extensive tissue damage and multiple organ dysfunction and even death. Uncontrolled inflammatory response in sepsis often affects organs such as the intestine, affecting the composition of the intestinal microbiome, leading to internal environmental disorders, severe dysbiosis, and ultimately the development of multiple organ failure. Inflammation, immunity and intestinal flora are important participants in the process of sepsis and are related to the occurrence and progression of sepsis. Sepsis is a common cause of death in intensive care units and one of the top ten most common causes of death in the world. It has a high incidence, severe condition, difficult treatment, poor prognosis and high mortality. Currently, sepsis is mostly treated with antibiotics, which not only causes patients to develop drug resistance and corresponding toxic side effects, but also has a long treatment cycle, poor prognosis, and high mortality rate.

[0003] So far, there is no targeted drug therapy for the treatment of excessive inflammatory response, immune imbalance and intestinal flora disorder in sepsis. Therefore, it is urgent to invent a drug that can treat excessive inflammatory response, immune imbalance and intestinal flora disorder in sepsis. Summary of the invention

[0004] In view of the defects of the prior art, the present invention provides a multifunctional microsphere for treating sepsis, and a preparation method and application thereof. The multifunctional microsphere MDC / EO / PDA-SA for treating sepsis provided by the present invention uses polydopamine nanoparticles as carriers, and the polydopamine nanoparticles are loaded with antimicrobial peptides and eugenol with combined anti-inflammatory effects, and the outer layer is wrapped with pH-responsive sodium alginate. Through pH response, the drug is accurately released to the intestinal part, thereby achieving the purpose of anti-inflammatory, immune imbalance regulation and intestinal flora regulation to treat sepsis. The multifunctional microspheres for treating sepsis provided by the present invention have significant pH response, anti-inflammatory, immune regulation and intestinal flora regulation effects.

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

[0006] A multifunctional microsphere for treating sepsis. The multifunctional microsphere is prepared with polydopamine nanoparticles, antimicrobial peptides, eugenol and sodium alginate as main components.

[0007] Furthermore, the antimicrobial peptide is the antimicrobial peptide MDC, and the amino acid sequence of the antimicrobial peptide MDC is shown in SEQ.ID.NO.1.

[0008] Ac-GWLKKIGKKIERVGQHTRDATIQTIGVAQQAANVAATLKG-NH2 (SEQ. ID. NO. 1).

[0009] Another object of the present invention is to provide a method for preparing the multifunctional microspheres for treating sepsis, comprising the following steps:

[0010] S1. preparing a polydopamine nanoparticle solution;

[0011] S2, preparing antimicrobial peptide preparatory solution, eugenol storage solution, sodium alginate storage solution and calcium chloride storage solution;

[0012] S3, uniformly mixing the polydopamine nanoparticle solution prepared in step S1 with the antimicrobial peptide preparatory solution and eugenol storage solution prepared in step S2, stirring, centrifuging, collecting, and freeze-drying;

[0013] S4, uniformly mixing the precipitate obtained in step S3 with the sodium alginate storage solution prepared in step S2, squeezing the mixture into the calcium chloride storage solution prepared in step S2, post-processing, and freeze-drying to obtain the product.

[0014] Furthermore, the specific operation of the post-treatment in step S4 is: hardening in a calcium chloride storage solution for 1-3 hours, and rinsing with water after collection.

[0015] Furthermore, the preparation method of the polydopamine nanoparticle solution is:

[0016] (1) preparing a dopamine hydrochloride aqueous solution;

[0017] (2) mixing ethanol, water and ammonia water, stirring to obtain a mixed solution, adding the dopamine hydrochloride aqueous solution prepared in step (1) to the mixed solution, stirring for 20-30 hours, centrifuging, washing, and freeze-drying to obtain polydopamine nanoparticles;

[0018] (3) dissolving the polydopamine nanoparticles obtained in step (2) in water to prepare a polydopamine nanoparticle solution with a concentration of 1.5-2 mg / mL.

[0019] Furthermore, the concentration of the dopamine hydrochloride aqueous solution prepared in step (1) is 0.02-0.1 g / mL.

[0020] Furthermore, in step (2), the volume ratio of ethanol, water and aqueous ammonia is 35-45:85-95:2-4.

[0021] Furthermore, in step S2, the concentration of the antimicrobial peptide preparatory solution is 1-4 mg / mL; the concentration of the eugenol storage solution is 1-4 mg / mL; the mass concentration of the sodium alginate storage solution is 1-3%; and the mass concentration of the calcium chloride storage solution is 1-3%.

[0022] Furthermore, in step S3, the stirring time is 8-15 hours, the centrifugal speed is 12000-15000 r / min, and the centrifugal time is 10-20 minutes.

[0023] Another object of the present invention is to provide a drug for treating sepsis, wherein the drug comprises the multifunctional microspheres for treating sepsis or the multifunctional microspheres prepared by the preparation method of the multifunctional microspheres for treating sepsis.

[0024] Another object of the present invention is to provide the use of the multifunctional microspheres for treating sepsis or the multifunctional microspheres prepared by the preparation method of the multifunctional microspheres for treating sepsis in preparing drugs for treating sepsis. The drug pH responds to the intestinal site of sepsis to release the drug to have a therapeutic effect.

[0025] Design ideas and principles of the present invention:

[0026] The multifunctional microspheres MDC / EO / PDA-SA provided by the present invention mainly include active ingredients, anti-inflammatory parts, immunomodulatory and intestinal flora regulating parts, and pH response parts; the active ingredients include loaded antimicrobial peptides MDC; the anti-inflammatory parts, immunomodulatory and intestinal flora regulating parts include MDC / EO / PDA-SA; the pH response part includes sodium alginate SA. First, the present invention uses polydopamine nanoparticles PDA, antimicrobial peptides MDC, eugenol EO, and sodium alginate SA as the main components of multifunctional microspheres, and loads antimicrobial peptides MDC and eugenol EO into polydopamine nanoparticles; secondly, sodium alginate SA is used to wrap MDC / EO / PDA to form microspheres. The process of sepsis inflammation involves the interaction of multiple cells and molecules, including the activation of inflammatory cells, the release of cytokines, and mutual adhesion between cells. The present invention can be used to respond to the pH of the intestine through sodium alginate, which is not easy to swell in the low pH environment of gastric juice, and gradually swells rapidly in the environment of high pH of small intestinal fluid, and the MDC / EO / PDA contained inside is released in the intestinal part. The antimicrobial peptide MDC and eugenol EO work together to treat various conditions such as inflammation, immune disorders and intestinal flora disorders caused by sepsis.

[0027] Specifically, in the present invention, sodium alginate is used as a pH-responsive drug delivery method, which can accurately transfer active substances to the lesion site for treatment. Sodium alginate can load various drugs, RNA, DNA, etc., and can protect these drugs from the influence of the external environment. It is a non-toxic natural pH-sensitive polymer that is not easy to swell in the low pH environment of gastric juice, and gradually swells in the high pH environment of small intestinal fluid, which can reduce toxicity and prolong the effectiveness of drugs, and promote the effective delivery of drugs to pathological sites. Polydopamine nanoparticles are widely used as a multifunctional carrier with many advantages, such as near-infrared absorption, huge surface area, and good biocompatibility. They have begun to be used in biomedical treatment. The antimicrobial peptide MDC is an insect antimicrobial peptide cloned from the cDNA library of the fat body of housefly larvae. Previous studies have found that MDC can inhibit inflammation and oxidative stress and regulate intestinal flora. Eugenol EO is a natural phenolic compound that has the effects of regulating the structure and abundance of intestinal microorganisms, immunomodulation, anti-inflammatory, and antioxidant. The present invention adopts a material design method of simultaneously loading antimicrobial peptide MDC and eugenol EO on polydopamine nanoparticles, so that the combination of antimicrobial peptide and eugenol can better play the role of anti-inflammatory, immunomodulatory and flora regulation. It can be seen that the multifunctional microsphere material MDC / EO / PDA-SA constructed by the present invention has multiple functions, and can perform targeted treatment on the corresponding diseases of the intestinal site of sepsis by pH response, which has important significance and application prospects.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention provides a multifunctional microsphere MDC / EO / PDA-SA capable of treating sepsis. Studies have shown that

[0030] MDC / EO / PDA-SA multifunctional microspheres have significant pH responsiveness, making them an ideal choice for intestinal targeted drug delivery systems;

[0031] (2) The present invention conducted a study on the treatment of sepsis using the obtained MDC / EO / PDA-SA multifunctional microspheres and found that

[0032] MDC / EO / PDA-SA multifunctional microspheres have significant anti-inflammatory, immunomodulatory and intestinal flora regulatory effects;

[0033] (3) The present invention uses polydopamine nanoparticles PDA, antimicrobial peptides MDC, eugenol EO, and sodium alginate SA as main components to prepare MDC / EO / PDA-SA multifunctional microspheres with sepsis treatment effects. The preparation method of the multifunctional microspheres is simple and easy to operate, and is suitable for industrial production.

[0034] (4) The present invention establishes a sepsis treatment drug delivery system, achieves the treatment of sepsis, and provides a new treatment method for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The electron microscopic comparison of polydopamine nanoparticles, MDC / EO / PDA, sodium alginate microspheres, and MDC / EO / PDA-SA multifunctional microspheres;

[0036] Figure 2 This is the pH response release behavior diagram of the multifunctional microspheres;

[0037] Figure 3 It is a comparison chart of the cell viability results of HK2 in different groups;

[0038] Figure 4 It is a comparison chart of the cell viability results of different groups on LO2;

[0039] Figure 5 This is a comparison chart of the in vitro anti-inflammatory results of different groups;

[0040] Figure 6 This is a comparison chart of the survival rate, body temperature and weight results of different groups;

[0041] Figure 7 It is a comparison chart of the liver and kidney function results in vivo in different groups;

[0042] Figure 8 This is a comparison chart of the anti-inflammatory results of different groups in vivo;

[0043] Fig. 9 This is a comparison chart of the antioxidant results of different groups in vivo;

[0044] Fig.10 It is a bar chart comparing the in vivo immune regulation results of different groups;

[0045] Fig.11 The flow cytometric scatter plots show the in vivo immune regulation results of different groups;

[0046] Fig.12 This is a comparison chart of community composition analysis results for different groups;

[0047] Fig.13 This is a comparison chart of the Alpha diversity shannon-2 index results of different groups;

[0048] Fig.14 This is a comparison chart of the Alpha diversity dominance index results for different groups. DETAILED DESCRIPTION

[0049] The present invention is further explained below in conjunction with specific examples, but it should be noted that the following examples are only used to explain the present invention, and cannot be used to limit the present invention, and all technical solutions that are the same or similar to the present invention are within the protection scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0050] 1. Reagents and biological materials

[0051] Mouse mononuclear macrophage Raw 264.7 cells, human renal cortical proximal tubule epithelial cells HK2, and human normal hepatocyte LO2 cell lines were purchased from the China Center for Type Culture Collection, Wuhan University;

[0052] The antimicrobial peptide MDC was commissioned to be synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd.;

[0053] Dopamine hydrochloride was purchased from MedChemexpress Biotechnology Company, USA;

[0054] Unless otherwise specified, the culture medium was the commonly used culture medium in the laboratory, DMEM and DMEM / F12 medium.

[0055] Example 1 Preparation of polydopamine (PDA) nanoparticle solution

[0056] (1) adding 0.5 g of dopamine hydrochloride into 10 mL of deionized water to prepare an aqueous solution of dopamine hydrochloride with a concentration of 0.05 g / mL;

[0057] (2) Mix 40 mL of ethanol, 90 mL of deionized water, and 3 mL of aqueous ammonia, and stir at 30 °C for 30 min to obtain a mixed solution;

[0058] (3) adding the dopamine hydrochloride aqueous solution prepared in step (1) to the mixed solution stirred in step (2), stirring under magnetic force at 30° C. for 24 hours, rotating at 600 r / min, centrifuging at 15,000 r / min for 15 minutes, washing with deionized water, repeating three times, and then freeze-drying to obtain polydopamine nanoparticles; dissolving the polydopamine nanoparticles in deionized water to obtain a polydopamine nanoparticle solution with a concentration of 2 mg / mL.

[0059] Example 2 Preparation of solution

[0060] (1) The preparation of the antimicrobial peptide (MDC) preparatory solution includes the following steps:

[0061] 30 mg of antimicrobial peptide MDC powder was added into 30 mL of sterile PBS solution to prepare an antimicrobial peptide (MDC) stock solution with a concentration of 1 mg / mL for later use.

[0062] (2) The preparation of eugenol (EO) storage solution comprises the following steps:

[0063] 28.11 μL of eugenol was added to 30 mL of sterile PBS solution to prepare a 1 mg / mL eugenol (EO) stock solution for later use.

[0064] (3) The preparation of sodium alginate (SA) storage solution includes the following steps:

[0065] 1.5 g of sodium alginate powder was added into 100 mL of sterile PBS solution to prepare a sodium alginate (SA) stock solution with a mass concentration of 1.5% for later use.

[0066] (4) The preparation of calcium chloride (CaCl2) storage solution includes the following steps:

[0067] Add 2 g of calcium chloride powder into 100 mL of sterile PBS solution to prepare a calcium chloride (CaCl2) stock solution with a mass concentration of 2% for later use.

[0068] Example 3 Loading of active ingredients

[0069] The polydopamine nanoparticle solution prepared in Example 1 is mixed with the antimicrobial peptide preparatory solution and eugenol storage solution prepared in Example 2, stirred, centrifuged, washed, and freeze-dried to prepare MDC / EO / PDA, comprising the following steps:

[0070] 5 mL of the polydopamine nanoparticle solution prepared in Example 1, 10 mL of the antimicrobial peptide preparatory solution prepared in Example 2 and 10 mL of the eugenol stock solution were mixed evenly, and magnetic stirring was performed for 12 h at a speed of 600 r / min. To remove free antimicrobial peptides and eugenol, ultracentrifugation was performed at 15000 rpm at 4°C for 15 min, and the washing operation was repeated three times. MDC / EO / PDA was obtained after freeze-drying.

[0071] Example 4 Synthesis of MDC / EO / PDA-SA Multifunctional Microspheres

[0072] 1 mg of MDC / EO / PDA prepared in Example 3 was mixed evenly with 1 mL of sodium alginate storage solution prepared in Example 2, and the mixture was transferred to a 1 mL sterile syringe and squeezed into 20 mL of calcium chloride storage solution prepared in Example 2. The prepared hydrogel microspheres were hardened in the calcium chloride storage solution for 2 h, collected, rinsed 3 times with distilled water to wash away the residual calcium chloride on the surface, and freeze-dried to obtain MDC / EO / PDA-SA multifunctional microspheres.

[0073] 1 mL of the sodium alginate (SA) storage solution prepared in Example 2 was taken into a 1 mL sterile syringe and squeezed into 20 mL of the calcium chloride storage solution prepared in Example 2. The prepared hydrogel microspheres were hardened in the calcium chloride storage solution for 2 h, collected, rinsed with distilled water 3 times to wash away the residual calcium chloride on the surface, and freeze-dried to obtain sodium alginate microspheres.

[0074] The polydopamine nanoparticles prepared in Example 1 and the MDC / EO / PDA prepared in Example 3 were photographed by transmission electron microscopy, and the sodium alginate microspheres and the constructed MDC / EO / PDA-SA multifunctional microspheres were photographed by scanning electron microscopy. The electron microscope comparison of polydopamine nanoparticles, MDC / EO / PDA, sodium alginate microspheres, and MDC / EO / PDA-SA multifunctional microspheres is shown in FIG. Figure 1 As shown. Among them, Figure 1 A is a transmission electron microscope image of polydopamine nanoparticles prepared in Example 1, Figure 1 B is the transmission electron microscope image of MDC / EO / PDA prepared in Example 3, Figure 1 C is the scanning electron microscope image of sodium alginate microspheres. Figure 1 D is the scanning electron microscope image of MDC / EO / PDA-SA multifunctional microspheres. Figure 1 The transmission electron microscopy results of A show that the polydopamine nanoparticles are obviously spherical and have a relatively smooth surface. Figure 1 The transmission electron microscopy results of B show that after loading the active ingredients, the MDC / EO / PDA particles have increased in size and still maintain a good spherical shape. Figure 1 The scanning electron microscopy results of C show that SA presents a relatively rounded spherical state and the surface of SA is relatively smooth; Figure 1 D It can be seen that after the SA encapsulated the drug, the surface of the microspheres showed a certain degree of roughness, covered with wrinkles and a small amount of cracks, indicating that the construction was successful, which was MDC / EO / PDA-SA.

[0075] Comparative Example 1 Synthesis of MDC / PDA-SA

[0076] 5 mL of the polydopamine nanoparticle solution prepared in Example 1 and 10 mL of the antimicrobial peptide preparatory solution prepared in Example 2 were mixed evenly, and magnetic stirring was performed for 12 h at a speed of 600 r / min. The solution was centrifuged at 15000 rpm for 15 min, and washed with deionized water. The mixture was repeated three times, and then collected and freeze-dried to obtain MDC / PDA.

[0077] 1 mg of MDC / PDA was mixed evenly with 1 mL of the sodium alginate storage solution prepared in Example 2, and the mixture was transferred to a 1 mL sterile syringe and squeezed into 20 mL of the calcium chloride storage solution prepared in Example 2. The prepared hydrogel microspheres were hardened in the calcium chloride storage solution for 2 h, collected, rinsed 3 times with distilled water to wash away the residual calcium chloride on the surface, and freeze-dried to obtain MDC / PDA-SA.

[0078] Comparative Example 2 Synthesis of EO / PDA-SA

[0079] 5 mL of the polydopamine nanoparticle solution prepared in Example 1 and 10 mL of the eugenol stock solution prepared in Example 2 were mixed evenly, and magnetic stirring was performed for 12 h at a speed of 600 r / min. The solution was centrifuged at 15000 rpm for 15 min, and washed with deionized water. The mixture was repeated three times, and then collected and freeze-dried to obtain EO / PDA.

[0080] 1 mg of EO / PDA was mixed evenly with 1 mL of the sodium alginate storage solution prepared in Example 2, and the mixture was transferred to a 1 mL sterile syringe and squeezed into 20 mL of the calcium chloride storage solution prepared in Example 2. The prepared hydrogel microspheres were hardened in the calcium chloride storage solution for 2 h, collected, rinsed with distilled water 3 times to wash away the residual calcium chloride on the surface, and freeze-dried to obtain EO / PDA-SA.

[0081] Experimental Example 1: Evaluation of in vitro pH-responsive release behavior of multifunctional microspheres

[0082] MDC / EO / PDA-SA was immersed in 10 mL of artificial simulated gastric fluid (SGF) medium and 10 mL of artificial simulated intestinal fluid (SIF) medium, respectively. The release system temperature was maintained at 37°C. 100 μL of the leaching solution was taken at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and 100 μL of the original solution was immediately added to the two systems to replace the lost volume. The drug content was determined and the cumulative release percentage was calculated.

[0083] The experimental results are as follows Figure 2 As shown by Figure 2It can be seen that the pH-responsive release curve of the drug dependent on sodium alginate in the low pH environment of the stomach and the high pH environment of the intestine obtained by using the MDC / EO / PDA-SA of Example 4 of the present invention shows that the release rates of MDC and EO in the MDC / EO / PDA-SA in the low pH environment of SGF in the first 2 hours are very low, only 19.96% and 14.42%, respectively, at which time most of the MDC / EO / PDA-SA has passed through the stomach. On the contrary, MDC / EO / PDA-SA releases drugs rapidly in the high pH environment of SIF, and nearly 72.95% of MDC and 64.03% of EO are rapidly released in 24 hours, which is mainly due to the rapid swelling of sodium alginate in the alkaline environment of the intestine. This shows that MDC / EO / PDA-SA can achieve pH-responsive release behavior, effectively protect the loaded drug from being destroyed by gastric acid, and achieve pH-responsive release in the intestine, thereby improving the bioavailability of the drug.

[0084] Experimental Example 2: Evaluation of Multifunctional Microspheres on Cell Viability

[0085] Viability test: Cell Counting Kit-8 (CCK-8, APExBIO, USA) was used to detect the effect of multifunctional microspheres on cell viability. The specific steps are as follows:

[0086] The experiment was divided into a normal group (Normal group), an experimental group (the experimental groups were set up into 3 groups: MDC / PDA-SA, EO / PDA-SA and MDC / EO / PDA-SA) and a blank group (used as a blank control, without adding cells, but adding culture medium to offset the increase in OD value caused by the culture medium).

[0087] Human renal cortical proximal tubule epithelial cells HK2 and human normal liver cells LO2 were routinely cultured in the laboratory. HK2 and LO2 cells in the logarithmic growth phase were collected. When the cells grew to 70-80% of the culture bottle, the culture medium was discarded, and sterile PBS was added and gently shaken. This was repeated 3 times. The PBS was discarded, and trypsin cell digestion solution (0.25%) was added for digestion for 2-5 minutes. The culture medium was added to terminate the digestion. The cell suspension was placed in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the culture medium was added for cell counting to make the cell density 5×10 3cells / well, 100 μL per well was inoculated in a 96-well culture plate, cultured overnight in a 5% CO2 incubator, the culture medium was removed, and PBS was washed three times. 100 μL of culture medium (normal culture medium) was added to the normal group and the blank group, and 100 μL of MDC / PDA-SA, EO / PDA-SA and MDC / EO / PDA-SA were added to the experimental group respectively. After continuing to culture for 24 hours, 10 μL of CCK-8 was added to each well of cells under light-proof conditions, and incubated in an incubator for 60 minutes. According to the manufacturer's instructions, CCK-8 reagent was used to detect the effect of multifunctional microspheres on the activity of HK2 and other cells, and the absorbance value (OD value) of each well was measured at a wavelength of 450 nm using SynergyH1 microplate reader (Bio Tek, USA). Among them, the cell viability calculation formula is as follows:

[0088]

[0089] The experimental results are as follows Figure 3 , Figure 4 As shown by Figure 3 , Figure 4 It can be seen that the cell viability of the MDC / EO / PDA-SA multifunctional microspheres obtained in Example 4 of the present invention is relatively high, which indicates that the MDC / EO / PDA-SA multifunctional microspheres obtained in Example 4 of the present invention have good biocompatibility.

[0090] Experimental Example 3: Evaluation of the anti-inflammatory effect of multifunctional microspheres in vitro

[0091] Mouse mononuclear macrophage Raw 264.7 cells were routinely cultured in the laboratory. Raw 264.7 cells in the logarithmic growth phase were collected. When the cells grew to 70-80% of the culture flask, the culture medium was discarded, sterile PBS was added and gently shaken. This was repeated 3 times. PBS was discarded, culture medium was added, and the culture medium was pipetted with a pipette to blow the bottom of the culture flask to suspend the cells. The cells were counted and the culture medium was added to make the cell density 5×10 5 cells / well, 3 mL per well was inoculated into a 6-well culture plate and cultured overnight in a 5% CO2 incubator.

[0092] The experiment was divided into a normal group (Normal group), an LPS group (Model group), an experimental group (the experimental groups were set to 3 groups: MDC / PDA-SA, EO / PDA-SA and MDC / EO / PDA-SA), and a blank group (used as a blank control, without adding cells, and then adding culture medium to offset the increase in OD value caused by the culture medium). The working concentration of LPS was set to 1 μg / mL. The normal group and the blank group were added with culture medium (without LPS, normal culture medium), the LPS group (Model group) and the experimental group were added with LPS and placed in the incubator for 24 hours, then the culture medium was discarded, and the cells were washed three times with PBS. Then, 1 mL of culture medium (normal culture medium) was added to the normal group and the blank group, 1 mL of culture medium without drugs was added to the LPS group (Model group), and 1 mL of MDC / PDA-SA, EO / PDA-SA and MDC / EO / PDA-SA were added to the experimental groups respectively; after incubation for 24 hours, the cell supernatant was collected, and TNF-α, IL-6 and IL-1β were detected using the method described in the instructions of the ELISA kit (purchased from ELISA Biotechnology Co., Ltd.).

[0093] The experimental results are as follows Figure 5 As shown by Figure 5 It can be seen that the contents of TNF-α, IL-6 and IL-1β in the Model group were significantly higher than those in the normal group (Normal group). The contents of inflammatory factors TNF-α, IL-6 and IL-1β in the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were significantly lower than those in the Model group. Among the three experimental groups of MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, the contents of inflammatory factors TNF-α, IL-6 and IL-1β in the MDC / EO / PDA-SA group were the lowest, which shows that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention have a certain anti-inflammatory effect.

[0094] Experimental Example 4: Evaluation of Multifunctional Microspheres on Survival Rate, Body Temperature and Weight

[0095] SPF male C57 (18–22 g) mice were purchased from the Guangdong Medical Experimental Animal Center and bred at the Medical Experimental Animal Center of Guangdong Pharmaceutical University (Experimental Animal Production License No.: SCXK(Guangdong)2022-0002), (Experimental Unit Use License SYXK(Guangdong)2022-0125). Animal experiments strictly abided by the regulations on experimental animal management.

[0096] Mice were grouped and CLP modeling was performed: 6 mice were randomly selected as the Normal group without treatment, and the other mice were routinely disinfected and depilated in the abdominal surgical area after anesthesia. Among them, 6 mice were randomly selected as the Sham group, and only the abdomen was opened and sutured, without CLP modeling; the remaining mice were made an incision of about 1-1.5 cm in length on the abdominal wall with a scalpel under sterile conditions, and a puncture was made at the ligation site to form a cecal leak, and a small amount of intestinal contents was gently squeezed out. The processed cecum was placed in situ in the abdominal cavity, and the peritoneum and skin were sutured layer by layer. After modeling, the mice were randomly divided into: Model group, MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, with 6 mice in each group. Two hours after successful modeling, the Normal, Sham and Model groups were gavaged with 200 μL of saline solution; the MDC / PDA-SA, EO / PDA-SA and MDC / EO / PDA-SA groups were gavaged with 0.2 g of MDC / PDA-SA + 200 μL of saline solution, EO / PDA-SA + 200 μL of saline solution and MDC / EO / PDA-SA + 200 μL of saline solution, respectively. The drugs were administered for 7 consecutive days, and the weight, temperature and survival of the mice were recorded at fixed times.

[0097] The experimental results are as follows Figure 6 As shown by Figure 6 The survival rate results show that the Model group died on the 4th day, and the survival rates of the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were higher than those of the Model group. Among the three experimental groups of MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, the survival rate in the MDC / EO / PDA-SA group was the highest. The results of body temperature and weight showed that the body temperature of each group began to rise on the first day, accompanied by a sharp drop in body weight; the body temperature and weight of the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were restored. Among the three experimental groups of MDC / PDA-SA group, EO / PD A-SA group and MDC / EO / PDA-SA group, the body temperature and weight in the MDC / EO / PDA-SA group were restored the best. This shows that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention can improve the survival rate of septic mice and improve the survival status of mice.

[0098] Test Example 5: In vivo liver and kidney function evaluation

[0099] Mice were grouped and CLP modeled according to the method of Experiment 4 above: Normal group, Sham group, Model group, MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, with 6 mice in each group. Drugs were administered 2 hours after successful modeling. The Normal group, Sham group and Model group were gavaged with 200 μL of normal saline; the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were gavaged with 0.2 g of MDC / PDA-SA + 200 μL of normal saline, EO / PDA-SA + 200 μL of normal saline and MDC / EO / PDA-SA + 200 μL of normal saline, respectively. After 24 hours, peripheral blood and intestinal tissues of mice were collected. The peripheral blood of the collected mice was taken for the detection of blood biochemical liver and kidney function indicators according to the methods described in the instructions for liver and kidney function indicators BUN, CRE, ALT, AST. The experimental results are as follows Figure 7 As shown by Figure 7 It can be seen that the content of BUN, CRE, ALT and AST in the Model group was significantly higher than that in the Sham group, and the content of BUN, CRE, ALT and AST in the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group was significantly lower than that in the Model group. Among the three experimental groups of MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, the content of BUN, CRE, ALT and AST in the MDC / EO / PDA-SA group was the lowest. This shows that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention have an improving effect on the liver and kidney function of mice.

[0100] Experimental Example 6: Evaluation of the anti-inflammatory effect of multifunctional microspheres in vivo

[0101] Inflammatory factors were detected in the peripheral blood of mice collected in the above-mentioned Experimental Example 5 according to the method described in the instructions of the ELISA kit (purchased from ELISA Biotechnology Co., Ltd.).

[0102] The experimental results are as follows Figure 8 As shown by Figure 8It can be seen that the contents of CRP, PCT, TNF-α, IL-6 and IL-1β in the Model group were significantly higher than those in the Sham group, and the contents of inflammatory factors CRP, PCT, TNF-α, IL-6 and IL-1β in the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were significantly lower than those in the Model group. Among the three experimental groups of MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, the contents of inflammatory factors CRP, PCT, TNF-α, IL-6 and IL-1β in the MDC / EO / PDA-SA group were the lowest, which shows that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention can reduce inflammation, have good anti-inflammatory and systemic infection relief effects.

[0103] Experimental Example 7: Evaluation of Antioxidant Damage in Vivo

[0104] The intestinal tissue oxidative damage was detected according to the method described in the instructions of the SOD, MDA, MPO, and CAT kits (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.). The intestinal tissue collected in the above-mentioned test example 5 was taken out, and the tissue weight (g): volume (mL) was accurately weighed and added to 9 volumes of pre-cooled saline. The tissue was minced with autoclaved surgical scissors and homogenized as completely as possible. The supernatant after centrifugation at 3000rpm for 10min was prepared for total protein concentration determination according to the BCA assay kit (Beyotime, Shanghai, China).

[0105] The experimental results are as follows Fig. 9 As shown by Fig. 9It can be seen that the SOD and CAT contents in the Model group were significantly lower than those in the Sham group, and the SOD and CAT contents in the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were significantly higher than those in the Model group. Among the three experimental groups, the MDC / PDA-SA group, the EO / PDA-SA group and the MDC / EO / PDA-SA group, the SOD and CAT contents in the MDC / EO / PDA-SA group were the highest, which indicates that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention significantly promoted the activity of CAT and SOD antioxidant enzymes. The MDA and MPO contents of the Model group were significantly higher than those of the Sham group. The MDA and MPO contents of the MDC / PDA-SA group, the EO / PDA-SA group and the MDC / EO / PDA-SA group were significantly lower than those of the Model group. Among the three experimental groups of the MDC / PDA-SA group, the EO / PDA-SA group and the MDC / EO / PDA-SA group, the MDA and MPO contents of the MDC / EO / PDA-SA group were the lowest, indicating that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention can inhibit the activity of MPO and the level of MDA. In summary, it can be seen that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention have a good antioxidant effect.

[0106] Test Example 8: Immunomodulation Evaluation

[0107] According to the method of the above-mentioned experimental example 4, mice were grouped and CLP modeling was performed: Normal group, Sham group, Model group, MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, with 6 mice in each group. The drug was administered 2 hours after successful modeling, and the Normal group, Sham group and Model group were gavaged with 200 μL of normal saline; the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were gavaged with 0.2 g of MDC / PDA-SA + 200 μL of normal saline, EO / PDA-SA + 200 μL of normal saline and MDC / EO / PDA-SA + 200 μL of normal saline, respectively. After 24 hours, the spleens of mice were collected for flow cytometry detection.

[0108] The experimental results are as follows Fig.10 , Fig.11 As shown by Fig.10 Comparison of in vivo immune regulation results in different groups (average of the experimental results of 3 mice in each group), and Fig.11 The flow cytometry scatter plots of the in vivo immune regulation results of different groups (taking the experimental results of one mouse in each group as an example) show that the CD4 + and CD4 + / CD8 +The content of CD4 + and CD4 + / CD8 + The content of CD4 in the MDC / EO / PDA-SA group was significantly higher than that in the Model group. + and CD4 + / CD8 + The content is the highest, which shows that the MDC / EO / PDA-SA multifunctional microspheres prepared by the present invention can regulate the immune function of mice.

[0109] Test Example 9: Evaluation of intestinal flora regulation

[0110] According to the method of the above-mentioned test example 4, mice were grouped and CLP modeling was performed: Normal group, Sham group, Model group, MDC / PDA-SA group (MDC.PDA.SA), EO / PDA-SA group (EO.PDA.SA) and MDC / EO / PDA-SA group (MDC.EO.PDA.SA), with 6 mice in each group. The drug was administered 2 hours after successful modeling, and the Normal group, Sham group and Model group were gavaged with 200 μL of normal saline; the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were gavaged with 0.2g of MDC / PDA-SA+200μL normal saline, EO / PDA-SA+200μL normal saline and MDC / EO / PDA-SA+200μL normal saline, respectively. After 24 hours, the feces of the mice were collected and sent for testing.

[0111] The experimental results are as follows Figure 12-14 As shown, Fig.12Middle: Microbial Community Composition, Relative abundance (Phylum), Firmicutes, Bacteroidetes, Proteobacteria, Epsilonbacteraeota, Verrucomicrobia, Pat escibacteria, Deferribacteres, Actinobacteria, Tenericutes. The Firmicutes in the Model group were significantly higher than those in the Sham group, and the Bacteroidetes were significantly lower than those in the Sham group. The amounts of Firmicutes and Bacteroidetes in the MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group were restored. Figure 13-14 Middle: The Shannon-2 index and dominance index of each group in Alpha diversity in Family show that the Alpha diversity of intestinal flora in the Model group is significantly lower than that in the Sham group. Among the three experimental groups of MDC / PDA-SA group, EO / PDA-SA group and MDC / EO / PDA-SA group, the Alpha diversity of intestinal flora in the MDC / EO / PDA-SA group is the best, indicating that MDC / EO / PDA-SA can improve the Alpha diversity of intestinal flora in sepsis and increase species richness. Therefore, Figure 12-14 It can be seen that the MDC / EO / PDA-SA multifunctional microspheres prepared in the present invention can improve the intestinal flora disorder caused by sepsis.

[0112] In summary, the MDC / EO / PDA-SA multifunctional microspheres provided by the present invention have a therapeutic effect on sepsis, can respond to the intestinal site of sepsis, bring the antimicrobial peptides MDC and eugenol EO into the lesion site for release, and at the same time, under the condition of inflammation, MDC and EO work together to alleviate and treat various conditions such as inflammation, immune disorders and intestinal flora disorders caused by sepsis.

[0113] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A multifunctional microsphere for treating sepsis, characterized in that: The multifunctional microspheres are prepared with polydopamine nanoparticles, antimicrobial peptides, eugenol and sodium alginate as main components.

2. The multifunctional microsphere for treating sepsis according to claim 1, characterized in that: The antimicrobial peptide is the antimicrobial peptide MDC, and the amino acid sequence of the antimicrobial peptide MDC is shown in SEQ.ID.NO.

1.

3. A method for preparing multifunctional microspheres for treating sepsis according to claim 1 or 2, characterized in that: The following steps are involved: S1. preparing a polydopamine nanoparticle solution; S2, preparing antimicrobial peptide preparatory solution, eugenol storage solution, sodium alginate storage solution and calcium chloride storage solution; S3, uniformly mixing the polydopamine nanoparticle solution prepared in step S1 with the antimicrobial peptide preparatory solution and eugenol storage solution prepared in step S2, stirring, centrifuging, collecting, and freeze-drying; S4, uniformly mixing the precipitate obtained in step S3 with the sodium alginate storage solution prepared in step S2, squeezing the mixture into the calcium chloride storage solution prepared in step S2, post-processing, and freeze-drying to obtain the product.

4. The method for preparing multifunctional microspheres for treating sepsis according to claim 3, characterized in that: The preparation method of the polydopamine nanoparticle solution is: (1) preparing an aqueous solution of dopamine hydrochloride; (2) mixing ethanol, water and ammonia water, stirring to obtain a mixed solution, adding the dopamine hydrochloride aqueous solution prepared in step (1) to the mixed solution, stirring for 20-30 hours, centrifuging, washing, and freeze-drying to obtain polydopamine nanoparticles; (3) dissolving the polydopamine nanoparticles obtained in step (2) in water to prepare a polydopamine nanoparticle solution with a concentration of 1.5-2 mg / mL.

5. The method for preparing multifunctional microspheres for treating sepsis according to claim 4, characterized in that: The concentration of the dopamine hydrochloride aqueous solution prepared in step (1) is 0.02-0.1 g / mL.

6. The method for preparing multifunctional microspheres for treating sepsis according to claim 4, characterized in that: In the step (2), the volume ratio of ethanol, water and aqueous ammonia is 35-45:85-95:2-4.

7. The method for preparing multifunctional microspheres for treating sepsis according to claim 3, characterized in that: The concentration of the antimicrobial peptide preparatory solution in step S2 is 1-4 mg / mL; the concentration of the eugenol storage solution is 1-4 mg / mL; the mass concentration of the sodium alginate storage solution is 1-3%; and the mass concentration of the calcium chloride storage solution is 1-3%.

8. The method for preparing multifunctional microspheres for treating sepsis according to claim 3, characterized in that: In step S3, the stirring time is 8-15 hours, the centrifugal speed is 12000-15000 r / min, and the centrifugal time is 10-20 minutes.

9. A drug for treating sepsis, characterized in that: The drug includes the multifunctional microspheres for treating sepsis according to claim 1 or 2 or the multifunctional microspheres prepared by the preparation method of the multifunctional microspheres for treating sepsis according to any one of claims 3-8.

10. Use of the multifunctional microspheres for treating sepsis according to claim 1 or 2 or the multifunctional microspheres prepared by the method for preparing the multifunctional microspheres for treating sepsis according to any one of claims 3 to 8 in preparing drugs for treating sepsis.

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