Dual-regulation medicine composition for treating sepsis as well as preparation method and application of dual-regulation medicine composition

Abstract

The invention discloses a dual-regulation pharmaceutical composition for sepsis treatment, a preparation method and application, and belongs to the technical field of biological medicine, and the pharmaceutical composition comprises doxorubicin liposome, all-trans retinoic acid phospholipid complex nanoparticles and antibiotics. Preferably, the doxorubicin liposome is prepared from the sialic acid modified doxorubicin liposome, the sialic acid modified all-trans retinoic acid phospholipid compound nanoparticles and meropenem. Dynamic monitoring of myeloid-derived suppressor cells MDSCs and sialic acid binding immunoglobulin type lectin-E (SiglecE) positively expressed myeloid-derived suppressor cells, namely SiglecE + MDSCs cell subsets, in the middle and later periods of sepsis is used as an evaluation index of the immunosuppression state, and dual regulation and control of sepsis acute inflammation and immunosuppression are achieved. The method has remarkable advantages in the aspects of increasing the survival rate, precisely targeting immunosuppression, improving the drug delivery efficiency and the like, and a brand new solution is provided for clinical treatment of sepsis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a dual-regulatory drug combination for treating sepsis, a preparation method and application thereof. Background Art

[0002] In recent years, as researchers have continued to delve deeper into the pathological mechanisms of sepsis, its treatment strategies have continued to improve. From the 1950s to the 1980s, antibiotics were the leading treatment for sepsis, primarily acting through broad-spectrum bactericidal activity. For example, third-generation cephalosporins and carbapenems significantly reduced the early mortality rate of Gram-negative bacterial infections (from over 50% to approximately 30%). However, the limitations of these drugs quickly became apparent: simple bactericidal activity was unable to reverse the microcirculatory disturbances and multi-organ damage caused by host immune disorders, and some patients still died from secondary infections caused by immunosuppression in the late stages of sepsis.

[0003] Since the 1990s, anti-inflammatory treatment strategies have emerged. Anti-inflammatory drugs attempt to improve prognosis by blocking the cytokine storm caused by excessive inflammation. Drugs such as anti-TNF-α monoclonal antibodies and IL-1 receptor antagonists have entered clinical trials. However, trial results have shown that these drugs not only fail to reduce mortality but may even exacerbate the body's immunosuppression by excessively suppressing inflammation and increase the risk of secondary fungal or drug-resistant bacterial infections.

[0004] As problems with anti-inflammatory drugs have gradually been uncovered, immunomodulatory therapeutic strategies have begun to attract attention. Their primary goal is to balance the dynamic imbalance between inflammation and immunosuppression. For example, in a mouse model of sepsis, anti-PD-1 antibodies were able to reverse T cell exhaustion and improve survival. Immunomodulation has shown groundbreaking potential in the treatment of sepsis. Future research may further integrate multi-omics data and artificial intelligence predictive models, shifting from "population-based approaches" to "specific immune remodeling" to overcome the multiple dilemmas of drug resistance, organ dysfunction, and poor long-term prognosis. Summary of the Invention

[0005] In view of the above-mentioned deficiencies or defects in the prior art, the primary purpose of the present invention is to provide a dual-regulatory drug combination for the treatment of sepsis, and to inhibit the expression of sialic acid-binding immunoglobulin-type lectin-E (Siglec-E)-positive myeloid-derived suppressor cells (MDSCs) in the middle and late stages of sepsis. + Dynamic monitoring of MDSCs cell subsets can be used as an evaluation indicator of immunosuppression status to achieve dual regulation of acute inflammation and immunosuppression in sepsis.

[0006] A dual-regulatory drug combination for the treatment of sepsis, comprising doxorubicin liposomes, all-trans retinoic acid phospholipid complex nanoparticles, and antibiotics.

[0007] Preferably, the doxorubicin liposomes are modified with sialic acid.

[0008] Preferably, the all-trans retinoic acid phospholipid complex nanoparticles are modified with sialic acid.

[0009] Preferably, the antibiotic is selected from meropenem, imipenem, panipenem, ertapenem, cefepime, ceftaroline fosamil, and monocyclic β-lactam antibiotics.

[0010] More preferably, a dual-modulatory drug combination for the treatment of sepsis consists of sialic acid-modified doxorubicin liposomes (DOX-SAL), sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles (ATRA-SN), and meropenem (Mp). The sialic acid modifier used is a sialic acid-cholesterol derivative (SA-CH), prepared using the method described in patent CN113024400A.

[0011] In the drug combination of the present invention, the membrane material of the DOX-SAL is composed of phospholipids, cholesterol and SA-CH in a molar ratio of 50-55:40-45:1-5, preferably 55:40:5, and the modification density of SA-CH is 1 mol%-10 mol%, preferably 5 mol%.

[0012] In the pharmaceutical combination of the present invention, the composition of ATRA-SN is all-trans retinoic acid (ATRA), egg yolk phosphatidylglycerol (EPG) and SA-CH, with a mass ratio of 1-5:11-14:5-6, preferably 1:13.0:5.6, and the modification density of SA-CH is 10%-50%, preferably 28%-30%.

[0013] The method for preparing a dual-regulatory drug combination for treating sepsis according to the present invention comprises:

[0014] Preparation of sialic acid-modified doxorubicin liposomes (DOX-SAL):

[0015] Phospholipids, cholesterol and sialic acid modifiers were weighed, dissolved in anhydrous ethanol, and stirred in a constant temperature water bath at 60°C-65°C until transparent; after the ethanol evaporated, citric acid-sodium citrate buffer preheated to 60°C-65°C was injected and stirred to form a preliminary liposome product, which was extruded through a polycarbonate membrane to prepare blank liposomes; the pH of the external aqueous phase was adjusted with sodium phosphate buffer, doxorubicin solution was added, the drug was incubated at 60°C-65°C, and the mixture was terminated in an ice water bath to prepare DOX-SAL.

[0016] Preparation of sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles (ATRA-SN):

[0017] ATRA, EPG and sialic acid modifier were weighed, anhydrous ethanol was added, and the mixture was dissolved in a water bath at 60-65°C and the ethanol was evaporated until it became viscous. Preheated glucose injection solution was injected, stirred for hydration, and passed through a microporous filter membrane. ATRA-SN was obtained under light-protected conditions.

[0018] When preparing the drug combination, the prepared DOX-SAL and ATRA-SN are combined with antibiotics to form the drug combination system.

[0019] A dual-regulatory drug combination for the treatment of sepsis is used to dual-regulate acute inflammation and immunosuppression in sepsis. The drug combination is administered as follows:

[0020] One hour after cecal ligation and puncture (CLP) model establishment, meropenem (10 mg·kg -1 ), 3 consecutive days;

[0021] 3 h after model establishment, DOX-SAL (0.5 mg·kg -1 ), 1 time in total;

[0022] On the 3rd, 5th and 7th days after model establishment, ATRA-SN (8 mg·kg -1 ), 1 time each.

[0023] The application of the drug combination improves the immunosuppressive state by regulating the number of MDSCs in a sepsis mouse model.

[0024] The application of the drug combination can regulate Siglec-E in the sepsis mouse model. + The number of MDSCs can improve the immunosuppressive state.

[0025] A method for evaluating the immunosuppressive state of sepsis by dynamically monitoring the expression of MDSCs and Siglec-E in a mouse model of sepsis after administration of a drug combination. + The changes in the number of MDSCs are used as an evaluation indicator of the immunosuppressive state in the middle and late stages of sepsis to assess the improvement of the immunosuppressive state.

[0026] A method for evaluating the immunosuppression state of sepsis, further comprising detecting MDSCs and Siglec-E in a sepsis mouse model + The proportion of MDSCs.

[0027] A method for evaluating the immunosuppressive state of sepsis, further comprising regulating MDSCs and Siglec-E +Improve the survival rate of sepsis mouse model by increasing the number of MDSCs.

[0028] A method for evaluating the immunosuppression state of sepsis, further comprising detecting Gr-1 in bone marrow and peripheral blood + CD11b + Siglec-E + The proportion of cells.

[0029] A preparation comprising the above drug combination is in the form of liposomes or nanoparticles and is used for intravenous injection or intraperitoneal injection.

[0030] Beneficial effects of the present invention:

[0031] Through the triple innovation of "monitoring indicators-drug carriers-treatment strategies", this invention has achieved a breakthrough in sepsis treatment from "group-based programs" to "specific immune remodeling". It has significant advantages in improving survival rates, precisely targeting immunosuppression, and improving drug delivery efficiency, providing a new solution for the clinical treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the survival rate of septic mice in the present invention, each value is expressed as mean ± standard deviation, n = 6;

[0033] Figure 2 The mouse bone marrow MDSCs (Gr-1 + CD11b + ) Flow cytometry analysis detection scatter plot;

[0034] Figure 3 The mouse bone marrow MDSCs (Gr-1 + CD11b + ) ratio of flow cytometry statistical results; p>0.05, ** p<0.01, **** p<0.0001;

[0035] Figure 4 The mouse blood MDSCs (Gr-1 + CD11b + ) Flow cytometry analysis detection scatter plot;

[0036] Figure 5 The mouse blood MDSCs (Gr-1 + CD11b + ) Ratio flow cytometry statistical results; p>0.05, ** p<0.01, **** p<0.0001;

[0037] Figure 6 The mouse bone marrow Siglec-E + MDSCs (Gr-1 + CD11b + Siglec-E + ) Flow detection histogram;

[0038] Figure 7 The mouse bone marrow Siglec-E + Statistical results of MDSCs ratio flow cytometry analysis; p>0.05, *** p<0.001;

[0039] Figure 8 Siglec-E in mouse blood + MDSCs (Gr-1 + CD11b + Siglec-E + ) Flow detection histogram;

[0040] Figure 9 Siglec-E in mouse blood + Statistical results of MDSCs ratio flow cytometry analysis; p>0.05, *** p<0.001. DETAILED DESCRIPTION

[0041] To better understand the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents and materials used in the following examples are all commercially available unless otherwise specified.

[0042] Example 1: Preparation of DOX liposomes

[0043] 1. Solution Preparation

[0044] NaOH solution: Accurately weigh 0.25 g of NaOH and place it in a 10 mL volumetric flask. Dissolve it with distilled water and dilute to the mark. Mix well and filter through a 0.22 μm microporous filter to obtain a concentration of 25 mg mL. -1 of NaOH solution.

[0045] Hydration medium: Accurately weigh 4.20 g of citric acid, dissolve it in distilled water, and adjust the pH to 4.0 with NaOH solution. Then, dilute it to 100.0 mL with distilled water, mix well, and filter it through a 0.22 μm microporous membrane to obtain a citric acid-sodium citrate buffer solution with a pH of 4.0 and a concentration of 200 mM.

[0046] External aqueous phase pH adjuster: Accurately weigh 0.38 g of sodium phosphate, dissolve it in distilled water and dilute it to 2.0 mL, mix well, and filter it through a 0.22 μm microporous membrane to obtain a sodium phosphate buffer solution with a concentration of 500 mM.

[0047] 2. Preparation of Blank Liposomes

[0048] Based on preliminary laboratory research, a relatively mature formulation has been established, in which a phospholipid-to-cholesterol ratio of 55:45 (n / n) is selected in the liposome membrane to produce blank liposomes with good performance. The SA-CH modification density is 5 mol%. The membrane compositions of the two liposomes are shown in Table 1.

[0049] Table 1 Prescriptions of different liposome DOX

[0050]

[0051] In Table 1, DOX-CL represents standard doxorubicin liposomes, and DOX-SAL represents sialic acid-modified doxorubicin liposomes. The sialic acid used is a sialic acid-cholesterol derivative (SA-CH), prepared using the method described in patent CN113024400A. HSPC represents hydrogenated soy lecithin, and CH represents cholesterol.

[0052] After accurately weighing the membrane material according to the prescription in Table 1, add anhydrous ethanol at a ratio of 10% of the final volume, and continue stirring in a constant temperature water bath at 65°C until it is completely dissolved to form a transparent solution. Then extend the stirring time to allow the ethanol to evaporate completely. Preheat the citric acid-sodium citrate buffer (200mM, pH 4.0) to 65°C and inject it into the lipid solution at a rate of 5mL / min. Maintain constant temperature and stirring for 20min to form the initial liposome product. Transfer to the extrusion device and drive it through 400nm, 200nm, 100nm and 80nm pore size polycarbonate membranes in sequence under nitrogen pressure for cyclic extrusion to finally obtain a blank liposome suspension with uniform particle size.

[0053] 3. Preparation of Gradient Liposomes and Loading of DOX

[0054] DOX liposomes were prepared using the pH gradient method. 1.0 mL of blank liposome suspension was taken, 0.3 mL of sodium phosphate buffer (500 mM) was added to adjust the pH of the external aqueous phase, and the pH gradient liposomes were obtained after thorough mixing. -1 The DOX solution was mixed with the blank liposomes at a drug-lipid ratio of 1:10 (w / w), and 0.2 mL of sterile water for injection was added. The mixture was stirred in a 60°C constant temperature water bath for 20 min to complete the active drug loading. The mixture was immediately transferred to an ice water bath for 2 min to terminate the drug encapsulation process. The final concentration was 1.0 mg mL -1 DOX liposomes.

[0055] Example 2: Preparation of ATRA phospholipid complex nanoparticles

[0056] Based on preliminary laboratory research, the drug-lipid ratio of ATRA to EPG was determined to be 1:5 (n / n) to prepare ordinary ATRA phospholipid complex nanoparticles and ATRA phospholipid complex nanoparticles with a SA-CH modification density of approximately 30%. According to the prescription in Table 2, the components of each phospholipid complex nanoparticle were accurately weighed and transferred to a 10 mL vial. 700 μL of anhydrous ethanol was added and stirred in a 60 ° C water bath for 30 minutes to completely dissolve. The system was then opened and the ethanol was evaporated until the membrane material became yellow, transparent and viscous with no solid precipitation. A 5 mL syringe was used to inject the membrane at a rate of 10 mL min. -1 Preheat 5% glucose injection to the same temperature and inject at a constant rate. Stir and hydrate for 10 minutes. Finally, filter through 0.8μm, 0.45μm, and 0.22μm microporous membranes to remove free ATRA and sterilize. The entire preparation process should be performed in the dark.

[0057] Table 2 Formulations of different ATRA phospholipid complex nanoparticles

[0058]

[0059] Example 3: Pharmacodynamics Experimental Protocol

[0060] Thirty C57BL / 6 mice were randomly divided into five groups (n=6): sham group (Sham group), control group (Control group), meropenem combined with sialic acid-modified doxorubicin liposomes treatment group (Mp+DOX-SAL group), meropenem combined with sialic acid-modified doxorubicin liposomes combined with ordinary all-trans retinoic acid phospholipid complex nanoparticles treatment group (Mp+DOX-SAL+ATRA-N group), and meropenem combined with sialic acid-modified doxorubicin liposomes combined with sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles treatment group (Mp+DOX-SAL+ATRA-SN group). Meropenem (Mp) drug solution was injected intraperitoneally 1 hour after cecal ligation and puncture (CLP) model establishment for 3 consecutive days. DOX drug solution or liposomes were injected into the tail vein once 3 hours after model establishment for a total of one administration. In addition, ATRA phospholipid complex nanoparticles were injected into the tail vein once on the 3rd, 5th and 7th days after model establishment. The DOX dosage was 0.5 mg kg -1 The dosage of Mp was 10 mg kg -1 The ATRA dosage was 8 mg kg -1 Throughout the experiment, the survival rate and body weight of the mice were recorded and monitored continuously for 10 days.

[0061] Establishment of sepsis mouse model:

[0062] A mouse sepsis model was established by cecal ligation and puncture surgery. The specific procedures are as follows:

[0063] (1) Preoperative anesthesia: 5% chloral hydrate (dosage: 0.1 mL kg -1 ) were injected intraperitoneally into the mice to ensure that the animals were pain-free and non-reflexive throughout the operation, proving that anesthesia was successful.

[0064] (2) Intraoperative operation: Fix the mouse in supine position and remove the hair from the abdomen. Disinfect the abdominal surgical site with alcohol. Make a longitudinal incision of about 1-2 cm in the midline of the abdomen, pull out the cecum, and ligate the end of the cecum with 4-0 sterile absorbable surgical sutures. The ligature length is 75% of the total length of the cecum. Use a 21G sterile needle to puncture the midpoint between the ligature point and the end of the cecum along the mesentery to the anti-mesentery direction. Gently press the ligated intestinal tube and squeeze out an appropriate amount of intestinal contents, and return the intestinal tube to the abdominal cavity. Use 4-0 sterile absorbable surgical sutures to suture the abdomen layer by layer (continuous suture for the inner abdomen and interrupted suture for the outer abdomen).

[0065] (3) Postoperative care: The abdominal surgical area was disinfected again with alcohol. After the mouse fully woke up, 0.5 mL of sterile saline was injected subcutaneously for fluid replacement. The mouse was then placed in a prone position on a warming pad for 1 hour to wait for recovery.

[0066] Survival analysis:

[0067] The results of the previous study confirmed that each combination group has the possibility of curing septic mice, but its survival rate is low and most deaths occur on the 5th day after the CLP model. Therefore, in the pharmacodynamics experiment of the present invention, ATRA phospholipid complex nanoparticles were introduced as an immunomodulator to address the problem of death caused by immunosuppression in the middle and late stages of sepsis. The survival of septic mice in each group was monitored to investigate the effect of ATRA phospholipid complex nanoparticles as an immunomodulator on the survival of septic mice in the middle and late stages. The results are shown in Figure 2. Figure 1 , Table 3.

[0068] Table 3 Comparison of survival rates among groups

[0069]

[0070] The experimental results show that, when comparing survival rates among treatment groups, the Mp+DOX-SAL+ATRA-SN group > the Mp+DOX-SAL+ATRA-N group > the Mp+DOX-SAL group. The Mp+DOX-SAL+ATRA-SN group showed the best therapeutic effect, with a survival rate of 83.3%, indicating that ATRA-SN effectively alleviated mortality in the middle and late stages of the CLP model.

[0071] Example 4: Immunomodulatory Effects of ATRA Phospholipid Complex Nanoparticles

[0072] In order to explore the regulatory effect of ATRA phospholipid complex nanoparticles as an immunomodulator on immunosuppression in the process of sepsis, the number of myeloid-derived suppressor cells (MDSCs) in septic mice at different stages of the disease was detected by flow cytometry to determine whether the immunosuppressive state was improved.

[0073] Mouse sample collection:

[0074] (1) Collection of peripheral blood samples.

[0075] (2) Extraction of bone marrow cells: Take the tibia of the mouse leg and cut halfway through the joint cavity with surgical scissors. Use a 5 mL syringe to draw an appropriate amount of PBS solution and insert it into the joint cavity to flush the bone marrow out of the tibia. After repeated pipetting, mix the cells and pass them through a 40 μm cell sieve. Centrifuge the filtrate and resuspend it to obtain a bone marrow cell suspension.

[0076] Detection of the number of MDSCs:

[0077] 0.5 mg / mL anti-mouse Gr-1 monoclonal antibody and 0.2 mg / mL anti-mouse CD11b monoclonal antibody were added to the above bone marrow cells and peripheral blood cell suspensions to label MDSCs, and flow cytometry was performed to obtain the proportion of MDSCs. The results are shown in Figure 2-Figure 5 .

[0078] like Figure 2-Figure 3As shown, the percentage of MDSCs in the bone marrow on day 5 of each treatment group was slightly different, all ranging from 45% to 50%. Comparing the percentage of MDSCs in the bone marrow on day 7 of each treatment group, the Mp+DOX-SAL group > the Mp+DOX-SAL+ATRA-N group > the Mp+DOX-SAL+ATRA-SN group. The percentage of MDSCs in the bone marrow of the Mp+DOX-SAL+ATRA-N group and the Mp+DOX-SAL+ATRA-SN group were similar, both ranging from 25% to 30%, with no significant difference (p>0.05). The two ATRA phospholipid complex nanoparticles had similar regulatory effects on MDSCs. Comparing the percentage of MDSCs in the bone marrow of each treatment group on day 14, the Mp+DOX-SAL group > the Mp+DOX-SAL+ATRA-N group > the Mp+DOX-SAL+ATRA-SN group were significantly higher. The Mp+DOX-SAL+ATRA-SN group had the lowest percentage of bone marrow MDSCs, at 10.7%, demonstrating that ATRA-SN has the strongest inhibitory effect on bone marrow MDSCs. The Mp+DOX-SAL+ATRA-N group showed a decrease in the percentage of bone marrow MDSCs from 27.4% on day 7 to 22.7% on day 14, demonstrating that ATRA-N has a limited inhibitory effect on bone marrow MDSCs.

[0079] like Figure 4-Figure 5 As shown, the number of MDSCs in peripheral blood on day 5 varied slightly among the treatment groups, all ranging from 3% to 4%. Comparing the number of MDSCs in peripheral blood on day 7 among the treatment groups, the Mp+DOX-SAL group ranked higher than the Mp+DOX-SAL+ATRA-N group and higher than the Mp+DOX-SAL+ATRA-SN group, with the Mp+DOX-SAL+ATRA-SN group showing the strongest inhibitory effect on MDSCs. Comparing the number of MDSCs in peripheral blood on day 14 among the treatment groups, the Mp+DOX-SAL group ranked higher than the Mp+DOX-SAL+ATRA-N group and higher than the Mp+DOX-SAL+ATRA-SN group. The Mp+DOX-SAL+ATRA-SN group had the lowest proportion of MDSCs in peripheral blood, at 1.52%, demonstrating that ATRA-SN has the strongest inhibitory effect on MDSCs in peripheral blood.

[0080] Siglec-E + Detection of the number of MDSCs:

[0081] Related literature in this field discloses that MDSCs from the blood of lung cancer patients and tumor-bearing mice express a large number of inhibitory Siglec-E receptors and are highly sialylated. Therefore, it is speculated whether MDSCs expanded during sepsis will also be highly sialylated and express Siglec-E. +receptors, so by detecting Siglec-E + The proportion of MDSCs in the blood was used to verify this hypothesis.

[0082] 0.5 mg / mL anti-mouse Gr-1 monoclonal antibody, 0.2 mg / mL anti-mouse CD11b monoclonal antibody, and 0.2 mg / mL anti-mouse Siglec-E monoclonal antibody were added to spleen single cell suspension and peripheral blood single cell suspension, respectively, to label MDSCs, and Siglec-E was obtained by flow cytometry. + The number of MDSCs in MDSCs is as follows: Figure 6-Figure 9 .

[0083] like Figure 6-Figure 7 As shown, the Sigiec-E in the bone marrow of each treatment group on day 5 + The difference in the number of MDSCs was small, fluctuating around 45%. + Compared with the proportion of MDSCs, the Sigiec-E of bone marrow + The difference in the number of MDSCs was small, and all were above 93%, which indicated that almost all MDSCs in the bone marrow expressed Sigiec-E receptors on the 7th day of sepsis. + The number and proportion of MDSCs were compared: Mp+DOX-SAL group > Mp+DOX-SAL+ATRA-N group > Mp+DOX-SAL+ATRA-SN group. + The number of MDSCs accounted for nearly 42%, which proved that ATRA-N had a significant effect on the immune response of Sigiec-E. + MDSCs had almost no inhibitory effect. + The number of MDSCs accounted for the least 19.8%, proving that ATRA-SN has a significant effect on the + MDSCs have a significant inhibitory effect.

[0084] like Figure 8-Figure 9 As shown, the peripheral blood Sigiec-E of each treatment group on the 5th day + The difference in the number of MDSCs was small, fluctuating around 30%. + The percentage of MDSCs was compared with that of peripheral blood Sigiec-E +The number of MDSCs accounted for more than 92%, which indicates that almost all MDSCs in peripheral blood expressed Sigiec-E receptors on the 7th day of sepsis. + The percentage of MDSCs was compared in the Mp+DOX-SAL+ATRA-N group>Mp+DOX-SAL group>Mp+DOX-SAL+ATRA-SN group. + The number of MDSCs accounted for nearly 38%, which proved that ATRA-N had a significant effect on the growth of Sigiec-E. + MDSCs had almost no inhibitory effect. + The number of MDSCs accounted for the least 16.9%, proving that ATRA-SN has a great effect on the + MDSCs have a significant inhibitory effect.

[0085] Example 5: Analysis of the immunomodulatory effect of ATRA phospholipid complex nanoparticles

[0086] MDSCs have a strong immunosuppressive function. After the onset of sepsis, the persistent inflammatory microenvironment stimulates the abnormal activation and expansion of MDSCs. Driven by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), the differentiation of immature myeloid cells in the bone marrow is blocked and released in large quantities into the peripheral circulation and tissues. ATRA has an immunomodulatory effect that inhibits the expansion of MDSCs, promotes their differentiation into dendritic cells, and enhances antigen presentation ability. Therefore, in the pharmacodynamics experiment of the present invention, the immunosuppressive state of septic mice after treatment with ATRA phospholipid complex nanoparticles was evaluated by examining the dynamic changes in the number of MDSCs.

[0087] (1) Immunomodulatory effects of ATRA phospholipid complex nanoparticles on bone marrow and peripheral blood MDSCs

[0088] Since sham surgery has almost no effect on the health of mice, only the percentage of MDSCs in the bone marrow and peripheral blood of the Sham group on the 5th day was measured as a control. The experimental results of the changes in the number of MDSCs in the bone marrow and peripheral blood showed that the percentage of MDSCs in the Mp+DOX-SAL group not only showed a significant upward trend in the early and middle stages of the disease (compared with the control group, the number of MDSCs in the bone marrow increased from 11.6 times (51.0% / 4.4%) on the 5th day to 16.2 times (71.4% / 4.4%) on the 7th day), Figure 2-Figure 3The number of MDSCs in peripheral blood increased from 1.7 times (3.35% / 1.94%) on day 5 to 4.7 times (9.20% / 1.94%) on day 7. Figure 4-Figure 5 ), and although it decreased slightly 14 days after onset, it still maintained a high proportion (compared to the control group, the number of MDSCs in the bone marrow was maintained at 15.6 times, and the number of MDSCs in the peripheral blood was maintained at 3.5 times). Therefore, it is speculated that in mice not treated with ATRA, immunosuppressive cells continued to proliferate in the early and middle stages of sepsis, and accumulated in large quantities in the late stages of sepsis, forming a strong immunosuppressive state, and the risk of death due to immunosuppression in mice was very high. The proportion of MDSCs in the bone marrow of the Mp+DOX-SAL+ATRA-N group showed a significant downward trend in the early and middle stages of the disease (compared to the control group, the number of MDSCs in the bone marrow decreased from 10.2 times on the 5th day to 6.2 times), and decreased slightly 14 days after onset, with no significant downward trend (compared to the control group, the number of MDSCs in the bone marrow was maintained at 5.2 times). The proportion of MDSCs in the peripheral blood of the Mp+DOX-SAL+ATRA-N group showed an upward trend in the early and middle stages of the disease (compared to the control group, the number of MDSCs in the peripheral blood increased from 1.8 times on the 5th day to 2.8 times), and decreased significantly 14 days after the onset of the disease (compared to the control group, the number of MDSCs in the peripheral blood remained at 1.6 times). Therefore, it is speculated that although the expansion of immunosuppressive cells in mice treated with ATRA-N was restricted in the early and middle stages of sepsis, the number did not decrease significantly, and even showed a certain growth trend. This resulted in some immunosuppressive cells surviving and lurking until the late stage of sepsis, forming a moderately strong immunosuppressive state, and there was a certain probability that the mice would die due to immunosuppression. In the Mp+DOX-SAL+ATRA-SN group, the proportion of MDSCs in the bone marrow showed a significant downward trend in the early and middle stages of the disease, while the proportion of MDSCs in the peripheral blood increased slightly in the early and middle stages of the disease (compared to the control group, the number of MDSCs in the bone marrow decreased from 10.1 times on day 5 to 6.1 times, and the number of MDSCs in the peripheral blood increased from 1.9 times on day 5 to 2.1 times). 14 days after the onset of the disease, both groups showed a significant downward trend and were even lower than the control group (compared to the control group, the number of MDSCs in the bone marrow decreased to 2.4 times, and the number of MDSCs in the peripheral blood decreased to 0.8 times). Therefore, it is speculated that the expansion of immunosuppressive cells in mice treated with ATRA-SN was strongly controlled in the early and middle stages of sepsis, and was significantly reduced in the late stages of sepsis. This proves that ATRA-SN has a significant regulatory effect on MDSCs, almost relieving the immunosuppressive state of the mice and restoring them to a healthy level.

[0089] (2) Testing the effects of ATRA phospholipid complex nanoparticles on bone marrow and peripheral blood Siglec-E + Immunomodulatory effects of MDSCs

[0090] Cancer research has shown that MDSCs express a high number of inhibitory Siglec-E receptors and are highly sialylated, contributing to tumor immune escape and a high mortality rate. Therefore, this study examined the proportion of MDSCs positive for Siglec-E receptors during the course of septic mice and explored the potential value of surface sialic acid modification in nanoparticles.

[0091] Since sham surgery has little effect on the health of mice, only the Siglec-E in the bone marrow and peripheral blood of the Sham group was measured on the 5th day. + The number of MDSCs was used as a control. The experimental results showed that in the middle stage of sepsis, the Siglec-E in the bone marrow and peripheral blood of each treatment group + The number of MDSCs increased significantly, and the proportion was greater than 92%. This proves that on the 7th day of sepsis, almost all MDSCs expressed Siglec-E receptors on their surfaces. + MDSCs occupy an absolute dominant position, and Siglec-E receptors have become the most ideal therapeutic target for regulating MDSCs. Thinking about this special phenomenon, it may be that in the middle stage of sepsis, immunosuppressive cells begin to proliferate, and innate immune cells and adaptive immune cells begin to play their functions and regulate immunosuppressive cells. However, due to persistent inflammatory stimulation, these immunosuppressive cells begin to widely express sialic acid binding receptors and become highly sialylated in order to avoid immune surveillance, forming a molecular barrier that hinders the recognition of immune cells, thereby forming an immunosuppressive microenvironment. Combined with previous analysis of the dynamic changes in the proportion of MDSCs, ATRA-SN uses the SA-Siglec-E axis to efficiently target and regulate MDSCs, and in the late stage of sepsis, it has a significant regulatory effect on MDSCs in the bone marrow and peripheral blood.

[0092] The widespread expression of Siglec-E receptors opens a "gateway" to immunomodulatory therapy. During specific stages of disease progression, specific cell subsets widely express specific binding receptors. Nanoparticle drug carriers, acting as "aircraft," leverage sialic acid modification to efficiently reach targeted sites. Building on the foundation of immunology, they leverage the unique advantages of pharmacology to achieve the ultimate goal of targeted therapy.

[0093] In this experiment, the number of MDSCs in the bone marrow and peripheral blood was selected for detection. The development of MDSCs begins from hematopoietic stem cells in the bone marrow. Under physiological conditions, hematopoietic stem cells gradually generate normal myeloid cells such as granulocytes and monocytes through the myeloid differentiation pathway. However, under pathological conditions such as chronic inflammation, tumors or infections, abnormal signals in the bone marrow microenvironment (such as high levels of GM-CSF, IL-6, VEGF, PGE2, etc.) interfere with the normal differentiation process of myeloid progenitor cells, causing them to stagnate in the immature stage and proliferate in large numbers to form MDSCs populations. As the pathological process continues, MDSCs accumulate in large quantities in the bone marrow and migrate through the blood circulation to peripheral tissues (such as tumor microenvironment or inflammatory sites), participating in immune escape and disease progression through mechanisms such as depletion of arginine, release of immunosuppressive cytokines (such as TGF-β, IL-10) and direct inhibition of T cell activation.

[0094] ATRA phospholipid complex nanoparticles are administered intravenously and first reach the peripheral blood, where they regulate already-migrated immunosuppressive cells, improving the immunosuppressive environment there and preventing them from circulating to various tissues and organs throughout the body. Simultaneously, the bone marrow contains a rich network of blood vessels, particularly in the red bone marrow region, which supplies blood to the bone marrow via microcirculation. Once the drug enters these vessels, it reaches the bone marrow, where it regulates immunosuppressive cells, improving the immunosuppressive state of the bone marrow microenvironment and preventing them from re-migrating into the bloodstream. This positively regulates the immune environment in the circulation, thereby achieving a benign blood-bone marrow-blood immune cycle.

Claims

1. A dual-regulatory drug combination for the treatment of sepsis, characterized in that: Including doxorubicin liposomes, all-trans retinoic acid phospholipid complex nanoparticles and antibiotics.

2. The dual-regulatory drug combination for treating sepsis according to claim 1, characterized in that: Doxorubicin liposomes are modified with sialic acid; all-trans retinoic acid phospholipid complex nanoparticles are modified with sialic acid; and the antibiotics are selected from meropenem, imipenem, panipenem, ertapenem, cefepime, ceftaroline fosamil, and monocyclic β-lactam antibiotics.

3. The dual-regulatory drug combination for treating sepsis according to claim 2, characterized in that: It is composed of sialic acid-modified doxorubicin liposomes, sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles and meropenem.

4. The dual-regulatory drug combination for treating sepsis according to claim 3, characterized in that: The membrane material composition of the sialic acid-modified doxorubicin liposomes is phospholipids, cholesterol and sialic acid modifiers in a molar ratio of 50-55:40-45:1-5; the composition of the sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles is all-trans retinoic acid, egg yolk phosphatidylglycerol and sialic acid modifiers in a mass ratio of 1-5:11-14:5-6.

5. A method for preparing the dual-regulatory drug combination for treating sepsis according to claim 4, characterized in that: The following steps are involved: Preparation of sialic acid-modified doxorubicin liposomes: Phospholipids, cholesterol, and sialic acid modifiers were weighed, dissolved in anhydrous ethanol, and stirred in a constant temperature water bath at 60°C-65°C until transparent; after ethanol evaporation, citric acid-sodium citrate buffer preheated to 60°C-65°C was injected and stirred to form a preliminary liposome product, which was extruded through a polycarbonate membrane to prepare blank liposomes; the pH of the external aqueous phase was adjusted with sodium phosphate buffer, and the doxorubicin solution was added. The drug loading was incubated at 60°C-65°C and terminated in an ice water bath; Preparation of sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles: weigh all-trans retinoic acid, egg yolk phosphatidylglycerol and sialic acid modifier, add anhydrous ethanol, dissolve in a 60-65°C water bath and evaporate the ethanol until it becomes viscous, inject preheated glucose injection, stir to hydrate, pass through a microporous filter membrane, and prepare under light-proof conditions; When preparing the drug combination, the prepared sialic acid-modified doxorubicin liposomes and sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles are combined with meropenem to form the drug combination system.

6. A use of the dual-regulatory drug combination for treating sepsis according to any one of claims 1 to 4, characterized in that: Used for dual regulation of acute inflammation and immunosuppression in sepsis; the drug combination is administered as follows: One hour after model establishment, meropenem 10 mg kg was injected intraperitoneally. -1 , 3 consecutive days; Three hours after model establishment, 0.5 mg kg of sialic acid-modified doxorubicin liposomes were injected into the tail vein. -1 , 1 time in total; On the 3rd, 5th and 7th days after model establishment, 8 mg kg of sialic acid-modified all-trans retinoic acid phospholipid complex nanoparticles were injected into the tail vein. -1 , 1 time each; The drug combination improves the immunosuppressive state by regulating the number of MDSCs and by regulating Siglec-E + The number of MDSCs can improve the immunosuppressive state.

7. A method for evaluating the immunosuppression state of sepsis, characterized in that: After administration of the drug combination according to any one of claims 1 to 4, the expression of MDSCs and Siglec-E in the sepsis mouse model was dynamically monitored. + The changes in the number of MDSCs are used as an evaluation indicator of the immunosuppressive state in the middle and late stages of sepsis to assess the improvement of the immunosuppressive state.

8. A method for evaluating the immunosuppression state of sepsis, characterized in that: After administering the drug combination according to any one of claims 1 to 4, detecting MDSCs and Siglec-E in the sepsis mouse model + The proportion of MDSCs; or detection of Gr-1 in bone marrow and peripheral blood + CD11b + Siglec-E + The proportion of cells.

9. A method for evaluating the immunosuppression state of sepsis, characterized in that: After administering the drug combination according to any one of claims 1 to 4, the effect of the drug combination on the expression of MDSCs and Siglec-E is regulated. + Improve the survival rate of sepsis mouse model by increasing the number of MDSCs.

10. A preparation comprising the pharmaceutical combination according to any one of claims 1 to 4, characterized in that: The preparation is in the form of liposomes or nanoparticles and is used for intravenous or intraperitoneal injection.

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