Multifunctional double-drug co-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses multifunctional double-drug co-loaded nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of medicines. A Janus nano material composed of mesoporous silica and mesoporous polydopamine is respectively loaded with salvianolic acid B and polymyxin B under the condition of pH 4.5 to obtain the double-drug co-loaded nano-particles, the double-drug co-loaded nano-particles have good biocompatibility and synergistic treatment effect, and the double-drug co-loaded nano-particles can reduce active oxygen accumulation and inhibit oxidative stress and inflammatory response, so that the double-drug co-loaded nano-particles can be used for preparing the anti-inflammatory drug for treating the salvianolic acid B and the polymyxin B. The endothelial injury caused by sepsis is effectively relieved. The salvianolic acid B and the polymyxin B are loaded at the same time, the salvianolic acid B protects endothelial cells by removing active oxygen and reducing inflammatory reaction, the polymyxin B kills gram-negative bacteria by destroying bacterial cell membranes and reduces infection sources and endotoxin release, and through the synergistic effect of the salvianolic acid B and the polymyxin B, the blood vessel leakage can be reduced, and the blood vessel leakage can be reduced. And the integrity of the endothelial barrier can be enhanced, so that the aim of treating sepsis endothelial injury is fulfilled.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to multifunctional dual-drug co-loaded nanoparticles and a preparation method and application thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection. Its core pathological mechanisms include oxidative stress induced by bacterial endotoxins, inflammatory storms caused by neutrophil infiltration, and endothelial barrier dysfunction, leading to multiple organ failure and high mortality, with approximately 11 million deaths per year worldwide. Current treatments mainly rely on antibiotics and anti-inflammatory drugs, but there are the following problems: (1) Single-drug limitations: Antibiotics can only kill bacteria, but cannot neutralize endotoxins or repair endothelial damage; although anti-inflammatory drugs can inhibit inflammatory factors, they lack antibacterial activity. Chinese patent application CN202310846319.5 (publication date: 2023-10-17) discloses a nanoparticle loaded with ulinastatin and tannic acid, which enhances stability through metal coordination, but only relies on the simple combination of tannic acid antioxidant and ulinastatin anti-inflammatory, without integrating antibacterial components, and cannot block the source of infection; (2) Low drug delivery efficiency: the half-life of free drugs is short, such as the half-life of ulinastatin in vivo is less than 1 hour, and it is difficult to target and enrich in inflammatory sites, leading to systemic toxicity.

[0003] Polymyxin B (PMB, CAS No.: 1404-26-8) is a cationic polypeptide antibiotic that binds to lipid A in the outer membrane lipopolysaccharide of Gram-negative bacteria and destroys the integrity of the bacterial cell membrane to achieve rapid sterilization. However, after PMB sterilization, bacterial lysis occurs, a large amount of LPS is released into the blood, and the release of endotoxins exacerbates inflammation, further amplifies oxidative stress and inflammatory responses, and aggravates endothelial damage. At the same time, free PMB is prone to accumulation in the kidneys, and its dose-dependent toxicity limits its long-term use. Chinese patent application CN103735508A (publication date: 2014-04-23) uses nanocarriers (such as liposomes) to encapsulate PMB to reduce toxicity, but does not address the need for synergistic endotoxin release and endothelial repair.

[0004] Salvianolic acid B (SalB, CAS No.: 115939-25-8) is a water-soluble phenolic acid compound extracted from Salvia miltiorrhiza, which has significant antioxidant and anti-inflammatory activities. However, SalB alone has defects, such as the inability to control the source of infection, the lack of neutralization effect on endotoxins, and the inability to block the pathological cycle caused by persistent bacterial infection. Oral absorption is poor, and the half-life of free SalB in the blood is short (<1 hour), making it difficult to maintain an effective concentration and having low bioavailability.

[0005] With the rapid development of nanotechnology, nanomaterials have become a new type of drug delivery system. However, their design still has the limitations of single carrier and poor drug release synergy. Summary of the Invention

[0006] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing multifunctional dual-drug-loaded nanoparticles.

[0007] Another object of the present invention is to provide multifunctional dual-drug-loaded nanoparticles.

[0008] Another object of the present invention is to provide the application of the above-mentioned multifunctional dual-drug-loaded nanoparticles.

[0009] The object of the present invention is achieved by the following technical solutions:

[0010] A method for preparing multifunctional dual-drug-loaded nanoparticles, comprising the following steps:

[0011] S1. Take mesoporous silica spheres and disperse them evenly in an organic solution. Respectively add cetyltrimethylammonium bromide (CTAB), dopamine (DA), and stir until clear and transparent; continue to add mesitylene (TMB) under stirring, and stir to form an emulsion; add tris(hydroxymethyl)aminomethane (Tris), continue to stir, centrifuge, and wash to obtain Janus nanoparticles composed of mesoporous silica and mesoporous polydopamine, named MSN@mPDA;

[0012] S2. Dissolve salvianolic acid B (SalB) in phosphate buffer solution (PBS) to obtain a salvianolic acid B solution; add the MSN@mPDA obtained in step S1 to the salvianolic acid B solution, stir in the dark, centrifuge, collect the particles, and wash to obtain MSN@mPDA particles loaded with salvianolic acid B, named MPS;

[0013] S3. Dissolve polymyxin B (PMB) in acetic acid buffer solution containing an emulsifier to obtain a polymyxin B solution; add the MPS obtained in step S3 to the polymyxin B solution, oscillate in the dark, centrifuge, collect the particles, and wash to obtain MSN@mPDA particles loaded with salvianolic acid B and polymyxin B, named MPPS.

[0014] Further, the mesoporous silica spheres described in step S1 are prepared by the following steps:

[0015] (1) Add cetyltrimethylammonium bromide (CTAB) and ultrapure water to a two-necked flask, stir until clear, add triethanolamine (TEA), and stir to obtain a mixed solution A; preheat an oil bath, and assemble the flask and condenser;

[0016] (2) Measure tetraethyl orthosilicate (TEOS) and cyclohexane and mix them evenly to obtain a mixed solution B;

[0017] (3) Slowly drop the mixture B into the mixture A to form a two-phase stratified solution and react; after cooling to room temperature, centrifuge to obtain the precipitate, wash it to obtain mesoporous silica spheres containing the template; disperse the obtained mesoporous silica spheres containing the template in a sodium chloride methanol solution, ultrasonicate, and wash to obtain mesoporous silica spheres.

[0018] Furthermore, the mixing ratio of cetyltrimethylammonium bromide, ultrapure water, triethanolamine, tetraethyl orthosilicate (TEOS), and cyclohexane is 2 - 4 g: 50 - 60 mL: 0.1 - 0.2 g: 3 - 5 mL: 10 - 20 mL; preferably 3 g: 55 mL: 0.112 g: 4 mL: 16 mL.

[0019] Furthermore, the preheating temperature of the oil bath is 60 ± 2 °C.

[0020] Furthermore, the reaction time is 48 ± 2 h.

[0021] Furthermore, the sodium chloride methanol solution is a sodium chloride methanol solution with a concentration of 1 wt%.

[0022] Furthermore, the organic solution in step S1 is an ethanol solution; preferably an ethanol solution obtained by mixing ultrapure water and absolute ethanol in equal proportions.

[0023] Furthermore, in step S1, the mesoporous silica spheres, organic solution, cetyltrimethylammonium bromide, The mixing ratio of dopamine, m - xylene, tris(hydroxymethyl)aminomethane is 4 - 6 mg: 8 - 12 mL: 8 - 12 mg: 150 - 250 mg: 50 - 150 mg: 0.5 - 2 mL: 8 - 12 mg; preferably 5 mg: 10 mL: 10 mg: 200 mg: 100 mg: 1 mL: 10 mg.

[0024] Furthermore, the stirring state in step S1 refers to a state with a rotation speed of 500 ± 50 rpm.

[0025] Furthermore, the stirring time in step S1 is 30 ± 2 min.

[0026] Furthermore, the continuous stirring time in step S1 is 12 ± 2 h.

[0027] Furthermore, the phosphate buffer solution in step S2 refers to a phosphate buffer solution with a pH of 4.5 ± 0.2.

[0028] Furthermore, the concentration of the salvianolic acid B solution in step S2 is 2 ± 1 mg / mL.

[0029] Further, the ratio of MSN@mPD to salvianolic acid B solution described in step S2 is 4 - 6 mg : 8 - 12 mL; preferably 5 mg : 10 mL.

[0030] Further, the light - avoiding stirring described in step S2 means stirring at 4°C in the dark for 6 ± 1 h.

[0031] Further, the conditions for centrifugation in step S2 are 12000 ± 1000 rpm and 10 ± 2 min.

[0032] Further, the phosphate buffer solution with pH 4.5 ± 0.2 is used for washing in step S2.

[0033] Further, the emulsifier described in step S3 includes at least one of Tween 80, Tween 20, and Pluronic F68.

[0034] Further, the addition amount of the emulsifier described in step S3 is calculated based on its concentration of 0.1 ± 0.05 wt%.

[0035] Further, the acetic acid buffer solution described in step S3 is an acetic acid buffer solution with pH 4.5 ± 0.2.

[0036] Further, the concentration of the polymyxin B solution described in step S3 is 1.5 ± 0.5 mg / mL.

[0037] Further, the light - avoiding oscillation described in step S3 means oscillating in the dark for 12 ± 2 h.

[0038] Further, the conditions for centrifugation in step S3 are 12000 ± 2000 rpm and 10 ± 2 min.

[0039] Further, the phosphate buffer solution with pH 7.4 is used for washing in step S3.

[0040] A multifunctional dual - drug - loaded nanoparticle is obtained by the above - mentioned preparation method.

[0041] The application of the above - mentioned multifunctional dual - drug - loaded nanoparticle in the preparation of a multifunctional drug.

[0042] Further, the functions include antibacterial, antioxidant, and / or anti - inflammatory.

[0043] The application of the above - mentioned multifunctional dual - drug - loaded nanoparticle in the preparation of a drug for treating sepsis.

[0044] The present invention is based on Janus - structured mesoporous nanoparticles (MSN@mPDA) and solves the defects of the prior art through the following designs: (1) Zoned loading and pH - responsive release: The MSN side has hydrophilic pores loaded with SalB, achieving slow release through hydrogen bonding and electrostatic interaction. The mPDA side has hydrophobic aromatic rings loaded with PMB, achieving rapid release triggered by pH, synchronously sterilizing and inhibiting endotoxin. The method provided by the present invention can simultaneously achieve source control and downstream protection, rapidly kill pathogenic bacteria and neutralize endotoxin, block inflammatory signal transduction; continuously scavenge ROS, inhibit inflammasome activation, and repair endothelial barrier function.

[0045] The present invention has the following advantages and effects compared with the prior art:

[0046] (1) Dual - drug zoned loading of Janus structure: Mesoporous silica (hydrophilic phase) is used to load SalB, and mesoporous polydopamine (hydrophobic phase) is used to load PMB. Through the difference in material physical and chemical properties, dual - drug independent loading and controlled release are achieved. At pH 4.5, the silanol groups on the surface of MSN are partially protonated, and combine with the carboxylate groups of SalB through electrostatic interaction, enhancing the drug - loading efficiency. At the same time, the phenolic hydroxyl groups of SalB form hydrogen bonds with the silanol groups on the surface of MSN, further enhancing the loading stability. The hydrophobic fatty acid chain of PMB combines with the aromatic ring skeleton of mPDA through hydrophobic interaction, and at the same time, the amino group of PMB forms electrostatic attraction with the residual amino groups on the surface of mPDA, enhancing the loading stability.

[0047] (2) pH - responsive sequential release: The low pH of the inflammatory microenvironment triggers a decrease in the degree of protonation of the amino groups of mPDA, weakens the hydrophobic interaction, and promotes the rapid release of PMB for sterilization. At the same time, MSN slowly releases SalB to continuously inhibit oxidative damage, forming a "first antibacterial then repair" synergistic sequence.

[0048] (3) Endotoxin - ROS closed - loop block: PMB reduces the release of bacteria - derived LPS, and SalB inhibits the generation of ROS induced by LPS. The dual - drug linkage cuts off the vicious cycle of "infection - inflammation - endothelial damage". Description of the Drawings

[0049] Figure 1 is the transmission electron microscope (TEM) image of MSN@mPDA;

[0050] Figure 2 is the transmission electron microscope (TEM) image of MPPS;

[0051] Figure 3 is the drug release curve of MPPS;

[0052] Figure 4 is the graph of the effect of MPPS on the viability of human umbilical vein endothelial cells (HUVEC);

[0053] Figure 5 It is the diagram of the reactive oxygen species scavenging level of MPPS on human umbilical vein endothelial cells (HUVECs);

[0054] Figure 6 It is the diagram of the mitochondrial oxidative stress repair of MPPS on human umbilical vein endothelial cells (HUVECs);

[0055] Figure 7 It is the diagram of the inflammatory cytokine scavenging of MPPS on human umbilical vein endothelial cells (HUVECs);

[0056] Figure 8 It is the diagram of the bactericidal ability of MPPS against Escherichia coli.

[0057] Figure 9 It is the schematic diagram of the technical principle of the present invention. Detailed implementation manners

[0058] For a better understanding of the present invention, the present invention will be further described below in conjunction with embodiments. However, the scope of protection required by the present invention is not limited to the scope shown in the embodiments.

[0059] In the present invention, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0060] In the present invention, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following items (pieces)" or its similar expressions refer to any combination of these items, including any combination of single item (piece) or plural items (pieces). For example, "at least one of a, b, or c", or, "at least one of a, b, and c" can all represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0061] It should be understood that in various embodiments of the present invention, the magnitude of the serial numbers of the above processes does not mean the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present invention.

[0062] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms of "a", "the", and "said" used in the embodiments of the present invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0063] In the description of the embodiments of the present invention, the weight of the relevant components mentioned not only refers to the specific content of each component, but also represents the proportional relationship of the weights between the components. Therefore, as long as the content of the relevant components in the description of the embodiments of the present invention is scaled up or down proportionally, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the mass described in the description of the embodiments of the present invention may be mass units well-known in the chemical field such as μg, mg, g, kg, etc.

[0064] Example 1

[0065] Synthesis of MPPS:

[0066] (1) Add 3 g of cetyltrimethylammonium bromide (CTAB) and 55 mL of ultrapure water into a 100 mL two-necked flask, and stir until clear. Add 0.112 g of triethanolamine (TEA) into the flask and stir. Preheat the oil bath to 60 °C, and assemble the flask and the condenser. Measure 4 mL of tetraethyl orthosilicate (TEOS) and 16 mL of cyclohexane, mix them evenly to obtain a mixed solution, reduce the stirring speed to 150 rpm, and slowly add the mixed solution into the flask with a dropper to form a two-phase layered solution, and react for 48 h. After cooling to room temperature, centrifuge to obtain the precipitate, and centrifuge and wash it 3 times with absolute ethanol at 10,000 rpm to obtain mesoporous silica spheres containing the template. Disperse the obtained mesoporous silica spheres containing the template in a sodium chloride methanol (1 wt%) solution, sonicate for 2 h, and repeat 3 times to ensure complete removal of the template. Then centrifuge and wash it three times with absolute ethanol at 10,000 rpm to obtain mesoporous silica spheres (MSN).

[0067] (2) Take 5 mg of MSN, disperse it in 5 mL of ultrapure water and 5 mL of absolute ethanol, and sonicate and mix evenly. Add 10 mg of cetyltrimethylammonium bromide (CTAB) and 200 mg 100 mg of dopamine (DA) respectively, and stir until clear and transparent. Adjust the rotation speed to 500 rpm, add 1 mL of m-xylene (TMB), and stir for 30 min to form an emulsion. Add 10 mg of tris(hydroxymethyl)aminomethane (Tris), and stir for 12 h. Centrifuge and wash with absolute ethanol. The washing method is the same as that of MSN to obtain MSN@mPDA.

[0068] (3) Dissolve SalB in phosphate buffer (PBS) at pH 4.5 with a concentration of 2 mg / mL. Add 50 mg of MSN@mPDA to 10 mL of the SalB solution, stir in the dark at 4 °C for 6 hours, centrifuge at 12000 rpm for 10 min, collect the particles, wash them twice with PBS at pH 4.5 to remove the surface-adsorbed drugs, and obtain MPS. Disperse the MPS particles in the PMB solution (1.5 mg / mL, acetic acid buffer at pH 4.5 containing 0.1% Tween 80), and oscillate in the dark for 12 h. Centrifuge at 12000 rpm for 10 min, collect the particles, and wash them three times with PBS at pH 7.4 to obtain MPPS.

[0069] Example 2

[0070] In this example, the MPPS prepared in Example 1 was used for characterization and drug release evaluation:

[0071] (1) Morphological characterization evaluation of MSN@mPDA and MPPS

[0072] The morphological characteristics of MSN@mPDA and MPPS were observed by transmission electron (TEM) microscopy.

[0073] The results are as Figure 1 and Figure 2 shown. It can be seen from the TEM results that the obtained Janus nanoparticles have an asymmetric double-sphere structure, with a uniform structure and good dispersion. Figure 2 It can be seen that the pore sizes of the two parts become smaller after MPPS is loaded with drugs.

[0074] (2) MPPS drug release curve

[0075] Disperse 10 mg of MPPS in 5 mL of the release medium and load it into a dialysis bag (MWCO 3.5 kDa). Immerse the dialysis bag in 50 mL of PBS at the corresponding pH, stir at a constant temperature, and sample 1 mL at regular intervals. At the same time, add an equal volume of fresh medium, and detect the drug concentration with a UV spectrophotometer.

[0076] The results are as Figure 3 shown. Under the condition of pH 4.5, the release rate of PMB is accelerated and the release amount increases; while the release of SalB is relatively slow.

[0077] Example 3

[0078] In this example, the MPPS prepared in Example 1 was used for related cell experiment tests:

[0079] (1) Cytotoxicity test

[0080] The toxic effects of different concentrations of MPPS on human umbilical vein endothelial cells (HUVECs) were detected by a CCK-8 kit. The HUVEC cell suspension was inoculated into a 96-well cell culture plate, and 1 mL of the cell suspension with a density of 1×10 5 cells / mL was added to each well. After culturing in an incubator with 5% carbon dioxide at 37 °C for 24 h, different concentrations of MPPS were added and incubated for 12 h. The OD 450 wavelength was measured according to the operation steps of the CCK-8 kit instructions, and the cell viability was calculated.

[0081] The effect of MPPS on cell viability is shown in Figure 4 the figure. The results showed that MPPS had low toxicity to cells, and the cell viability was above 90%.

[0082] (2) Reactive oxygen species scavenging ability test

[0083] The intracellular reactive oxygen species content was characterized by a DCFH-DA fluorescent probe using a flow cytometer. The HUVEC cell suspension was inoculated into a 6-well cell culture plate, and 1 mL of the cell suspension with a density of 5×10 5 cells / mL was added to each well. After culturing in an incubator with 5% carbon dioxide at 37 °C for 24 h, 100 μg / mL LPS was added to each well to stimulate the cells for 2 h. Different concentrations of MPPS were added according to the experimental grouping and treated for 2 h. After staining with the DCFH-DA probe according to the operation steps of the reagent instructions, the effect of reactive oxygen species scavenging was analyzed by a flow cytometer.

[0084] The results are shown in Figure 5 the figure. The reactive oxygen species level in the treatment group decreased significantly compared with that in the control group, indicating that MPPS had a good effect on scavenging reactive oxygen species.

[0085] (3) Mitochondrial membrane potential test

[0086] The mitochondrial membrane potential was characterized by a JC-1 fluorescent probe using a fluorescence microscope. The human umbilical vein endothelial cell (HUVEC) suspension was inoculated into a 6-well cell culture plate, and 1 mL of the cell suspension with a density of 5×10 5 cells / mL was added to each well. After culturing in an incubator with 5% carbon dioxide at 37 °C for 24 h, 100 μg / mL LPS was added to each well to stimulate the cells for 2 h. Different concentrations of MPPS were added according to the experimental grouping and treated for 2 h. After staining with the JC-1 probe according to the operation steps of the reagent instructions, photographs were taken with a fluorescence microscope for observation.

[0087] The results are shown in Figure 6As shown, the strong red fluorescence in the treatment group indicates a high mitochondrial negative charge, and JC-1 enters the mitochondria and exists as a polymer. In the model group, the cells show strong green fluorescence, mitochondrial depolarization occurs, the negative charge decreases, and JC-1 exists as a monomer in the cytoplasm. This shows that MPPS can relieve mitochondrial oxidative stress in endothelial cells.

[0088] (4) Determination of cellular inflammatory factors

[0089] The content of IL-6 in endothelial cells was measured by an ELISA kit. HUVEC cell suspensions were seeded in 6-well cell culture plates, and 100 μg / mL LPS was added to each well to stimulate the cells for 4 h. Different concentrations of MPPS were added according to the experimental grouping for 20 h of treatment. The cell supernatants were collected and centrifuged at 1500 g for 15 minutes, and then aliquoted and stored at -80 °C. The expression level of the pro-inflammatory cytokine IL-6 was detected by ELISA according to the operation steps of the reagent instructions.

[0090] The results are as Figure 7 shown. The model group of cells produced more cellular inflammatory factors after LPS stimulation, while the cytokine levels in the treatment group were significantly decreased compared with the model group, indicating that MPPS has a good anti-inflammatory effect.

[0091] Example 4

[0092] In this example, the MPPS prepared in Example 1 was used for relevant bacterial experimental tests:

[0093] The Escherichia coli strain was activated in LB liquid medium, proliferated and cultured in LB liquid medium to obtain the corresponding bacterial solution, and the OD value was adjusted to 0.4. 100 μL of the bacterial solution was taken into a 96-well plate, and different concentrations of MPPS were added and incubated at 37 °C with shaking for 4 h, and the OD 600 value was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0094] The results are as Figure 8 shown. It can be seen from the OD value that the higher the concentration of MPPS, the lower the OD value, indicating that MPPS has a certain killing ability against Escherichia coli.

[0095] Example 5

[0096] In this example, an animal experiment on a sepsis mouse model was conducted on the MPPS prepared in Example 1. The specific experimental steps are as follows:

[0097] (1) Construction of a mouse sepsis model

[0098] Male C57BL / 6J mice at 6 - 8 weeks of age were housed in a standard environment in an SPF breeding room and randomly divided into 3 groups (5 mice in each group): control group, model group, and treatment group (treated with MPPS nanoparticles after inducing sepsis). 1) The mice were fasted for 12 h before surgery and their abdominal hair was removed; 2) The mice were anesthetized with 2% isoflurane using a small animal gas anesthesia machine: 800 L / min in the induction box and 400 L / min in the anesthesia tube; 3) The abdominal skin of the mice was disinfected three times with iodophor; 4) A 1 - 2 cm incision was made layer by layer along the mid - abdomen to expose the cecum, and feces were squeezed into the cecum with forceps; The cecum was ligated with a suture about 1 cm from the end of the cecum, and then a 5 mL needle was used to penetrate the cecum, and feces the size of sesame seeds were squeezed out with forceps; Then the intestine was placed back into the abdominal cavity, the incision was continuously sutured with a suture, and the abdomen was disinfected by wiping with iodophor; 5) After surgery, 0.5 mL of pre - warmed sterile normal saline at 37℃ was injected subcutaneously on the back of the mice for resuscitation. Among them, the mice in the control group were not ligated and perforated in the cecum, and other surgical steps were the same as above. 4 h after modeling, MPPS was injected intraperitoneally into the treatment group. The mice were sacrificed 24 hours after modeling.

[0099] (2) H&E staining was used to evaluate the pathophysiological morphology of tissues and organs

[0100] Tissues of the heart, liver, spleen, lung, and kidney of mice in each group were taken and put into a pre - prepared 4% paraformaldehyde fixative solution, dehydrated step by step with ethanol of different concentrations, made transparent with xylene, embedded in paraffin, sectioned, mounted, and dried in an incubator at 45℃; Then dewaxed with xylene, stained with hematoxylin, differentiated with 1% hydrochloric acid alcohol, blued with 1% ammonia water, then stained in eosin staining solution, and then made transparent with xylene again, and sealed with resin. The morphological changes of the organ tissues of mice in each group were observed and photographed.

[0101] (3) ELISA was used to evaluate the expression of pro - inflammatory cytokines in septic mice

[0102] For all groups of mice, the mice were sacrificed 24 hours after modeling. Blood samples of the mice were collected by orbital blood collection method into EDTA - anticoagulated EP centrifuge tubes and allowed to stand and coagulate. Then, they were centrifuged at 3000 rpm for 15 min at 4℃. The supernatant was aliquoted and stored frozen at - 80℃. The expression levels of pro - inflammatory cytokines TNF - α and IL - 6 in the serum were detected by ELISA.

[0103] (4) Determination of blood routine, blood biochemical indexes, and blood bacteria load

[0104] A blood cell analyzer was used to detect the number of blood cells; A blood biochemical analyzer was used to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood. 100 μL of the collected blood was evenly spread on a culture plate and cultured overnight in an incubator at 37℃. The next day, the colony distribution and quantity were observed and photographed.

[0105] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of multifunctional nanoparticles co-loaded with two drugs, characterized in that: It includes the following steps: S1. Uniformly disperse mesoporous silica spheres in an organic solution, and respectively add cetyltrimethylammonium bromide, F-127, and dopamine, and stir until clear and transparent; continue to add mesitylene under stirring to form an emulsion; add tris(hydroxymethyl)aminomethane, continue to stir, centrifuge, and wash to obtain Janus nanoparticles composed of mesoporous silica and mesoporous polydopamine, named MSN@mPDA; S2. Dissolve salvianolic acid B in phosphate buffer solution to obtain a salvianolic acid B solution; add the MSN@mPDA obtained in step S1 to the salvianolic acid B solution, stir in the dark, centrifuge, collect the particles, and wash them to obtain MSN@mPDA particles loaded with salvianolic acid B, named MPS; S3. Dissolve polymyxin B in acetic acid buffer solution containing an emulsifier to obtain a polymyxin B solution; add the MPS obtained in step S3 to the polymyxin B solution, oscillate in the dark, centrifuge, collect the particles, and wash them to obtain MSN@mPDA particles loaded with salvianolic acid B and polymyxin B, named MPPS.

2. The preparation method of the multifunctional dual-drug co-loaded nanoparticles according to claim 1, wherein: The mesoporous silica spheres described in step S1 are prepared through the following steps: (1) Add cetyltrimethylammonium bromide and ultrapure water to a two-necked flask, stir until clear, add triethanolamine, and stir to obtain a mixed solution A; preheat an oil bath, and assemble the flask and condenser; (2) Measure and mix tetraethyl orthosilicate and cyclohexane to obtain a mixed solution B; (3) Slowly drop the mixed solution B into the mixed solution A to form a two-phase stratified solution, and react; after cooling to room temperature, centrifuge to obtain the precipitate, wash it to obtain mesoporous silica spheres containing a template; disperse the obtained mesoporous silica spheres containing a template in a sodium chloride methanol solution, ultrasonicate, and wash to obtain mesoporous silica spheres.

3. The preparation method of the multifunctional dual-drug co-loaded nanoparticles according to claim 2, wherein: The mixing ratio of the cetyltrimethylammonium bromide, ultrapure water, triethanolamine, tetraethyl orthosilicate, and cyclohexane is 2 - 4 g: 50 - 60 mL: 0.1 - 0.2 g: 3 - 5 mL: 10 - 20 mL; The preheating temperature of the oil bath is 60 ± 2 °C; The reaction time is 48 ± 2 h; The sodium chloride methanol solution is a sodium chloride methanol solution with a concentration of 1 wt%.

4. The preparation method of the multifunctional dual-drug co-loaded nanoparticles according to any one of claims 1 - 3, wherein: The organic solution described in step S1 is an ethanol solution; The mesoporous silica spheres, organic solution, cetyltrimethylammonium bromide, F-127, dopamine, mesitylene, and tris(hydroxymethyl)aminomethane are in a ratio of 4 - 6 mg: 8 - 12 mL: 8 - 12 mg: 150 - 250 mg: 50 - 150 mg: 0.5 - 2 mL: 8 - 12 mg; The phosphate buffer solution described in step S2 refers to a phosphate buffer solution with a pH of 4.5 ± 0.2; The concentration of the salvianolic acid B solution described in step S2 is 2 ± 1 mg / mL; The mixing ratio of the MSN@mPD and the salvianolic acid B solution described in step S2 is 4 - 6 mg: 8 - 12 mL; The emulsifier described in step S3 includes at least one of Tween 80, Tween 20, and Pluronic F68; The addition amount of the emulsifier described in step S3 is calculated based on its concentration of 0.1 ± 0.05 wt%; The concentration of the polymyxin B solution described in step S3 is 1.5 ± 0.5 mg / mL.

5. The preparation method of the multifunctional dual-drug co-loaded nanoparticles according to any one of claims 1 - 3, wherein: The organic solution described in step S1 is an ethanol solution obtained by mixing ultrapure water and absolute ethanol in an equal ratio; The mesoporous silica spheres, organic solution, cetyltrimethylammonium bromide, F-127, dopamine, mesitylene, and tris(hydroxymethyl)aminomethane in step S1 are in a ratio of 5 mg: 10 mL: 10 mg: 200 mg: 100 mg: 1 mL: 10 mg The phosphate buffer solution described in step S2 refers to a phosphate buffer solution with a pH of 4.5; The concentration of the salvianolic acid B solution described in step S2 is 2 mg / mL; The ratio of MSN@mPD to the salvianolic acid B solution described in step S2 is 5 mg:10 mL; The emulsifier described in step S3 is Tween 80; The addition amount of the emulsifier described in step S3 is calculated based on its concentration of 0.1 wt%; The concentration of the polymyxin B solution described in step S3 is 1.5 mg / mL.

6. The method for preparing the multifunctional dual-drug co-loaded nanoparticles according to any one of claims 1-3, characterized in that: The stirring state described in step S1 refers to a state with a rotation speed of 500 ± 50 rpm; The stirring time described in step S1 is 30 ± 2 min; The continuous stirring time described in step S1 is 12 ± 2 h; The light-shielded stirring described in step S2 refers to light-shielded stirring at 4°C for 6 ± 1 h; The centrifugation conditions described in step S2 are 12000 ± 1000 rpm and 10 ± 2 min; The washing described in step S2 uses a phosphate buffer solution with a pH of 4.5 ± 0.

2.

7. The method for preparing the multifunctional dual-drug co-loaded nanoparticles according to any one of claims 1-3, characterized in that: The acetate buffer solution described in step S3 refers to an acetate buffer solution with a pH of 4.5 ± 0.2; The light-shielded oscillation described in step S3 refers to light-shielded oscillation for 12 ± 2 h; The centrifugation conditions described in step S3 are 12000 ± 2000 rpm and 10 ± 2 min; The washing described in step S3 uses a phosphate buffer solution with a pH of 7.

4.

8. A multifunctional dual-drug-loaded nanoparticle, characterized in that: Obtained by the preparation method described in any one of claims 1-7.

9. Use of the multifunctional dual-drug-loaded nanoparticles described in claim 8 in the preparation of multifunctional drugs, characterized in that: The functions described include antibacterial, antioxidant and / or anti-inflammatory.

10. The application of the multifunctional dual-drug co-loaded nanoparticles described in claim 8 in the preparation of a drug for treating sepsis.

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