DNase I loaded liposome and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to DNase I loaded lipidosome and application thereof. The invention provides a bionic long-acting deoxyribonuclease I (DNase I) liposome with free DNA (cfDNA) responsiveness and a dual-targeting function, the liposome is subjected to layered fusion modification through an erythrocyte membrane and a neutrophile granulocyte membrane, a release window is accurately regulated and controlled by utilizing a 4ME-PC proportion, and a liposome drug carrier for cfDNA targeted combination and accurate delivery of an inflammation part is realized. The polypeptide can be used for cfDNA-related inflammatory diseases, and has an important application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a liposome loaded with DNase I and application thereof. BACKGROUND

[0002] Sepsis is a life-threatening disease caused by the host's response to infection disorder, which can rapidly progress to multiple organ dysfunction with extremely high mortality. Its pathological process is not only related to the pathogenic microorganism itself, but also closely related to the excessive activation of the body's immune response. As an important part of innate immunity, neutrophils can capture and eliminate pathogens through the release of neutrophil extracellular traps (NETs). NETs are composed of depolymerized chromatin fibers, accompanied by the release of a large amount of antibacterial proteins and cell-free DNA (cfDNA).

[0003] Under normal circumstances, NETs help to defend against infection; but in sepsis, the excessive accumulation of NETs and cfDNA can significantly exacerbate the inflammatory response, activate the Toll-like receptor 9 (TLR9) signaling pathway, trigger an inflammatory factor storm, promote tissue damage, and induce impaired red blood cell function, leading to sepsis-related anemia and multiple organ dysfunction. Therefore, efficient and sustained clearance of excessive NETs and cfDNA is an important treatment strategy to improve the prognosis of sepsis.

[0004] Deoxyribonuclease I (DNase I) is a kind of endonuclease that can degrade DNA and has been proven to effectively decompose NETs and cfDNA. However, free DNase I has a short half-life in the body, is easy to inactivate, has poor stability, and lacks lesion targeting ability and the ability to release on demand, which limits its application in sepsis treatment. And the existing cfDNA targeting strategies mainly rely on chemical modification or artificial ligand recognition, which usually have low recognition efficiency, high immunogenicity, and complex preparation.

[0005] Therefore, there is an urgent need for a biomimetic nanodrug delivery system that combines high safety, simple preparation, long circulation, dual targeting of inflammatory lesions, and cfDNA-responsive release function to break through the limitations of existing treatment options and improve the effectiveness and safety of sepsis treatment. SUMMARY

[0006] Therefore, the present application aims to solve the technical problem of providing a liposome loaded with DNase I and application thereof.

[0007] The present application provides a liposome loaded with DNase I, which comprises a liposome and DNase I.

[0008] The liposome is prepared from 2,3-bis[(3,7,11,15-tetramethylhexadecyl)oxy]propyl 2-(trimethylammonium)ethyl phosphate (4ME-PC), phosphatidylcholine disodium stearate (DSPC), cholesterol and 1,2-diacetyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) according to the molar ratio (0.3-1.8): (22.7-1.22): 2: 0.015.

[0009] The raw materials involved in the preparation of the liposome are screened and combined, and the ratio of the raw materials is optimized.

[0010] ME-Liposome 0: the molar ratio of DSPC, cholesterol and DSPE-PEG 2000 is 3:2:0.015;

[0011] ME-Liposome 1: the molar ratio of 4ME-PC, DSPC, cholesterol and DSPE-PEG 2000 is 0.3:2.7:2:0.015;

[0012] ME-Liposome 2: the molar ratio of 4ME-PC, DSPC, cholesterol and DSPE-PEG 2000 is 0.6:2.4:2:0.015;

[0013] ME-Liposome 3: the molar ratio of 4ME-PC, DSPC, cholesterol and DSPE-PEG 2000 is 0.9:2.1:2:0.015;

[0014] ME-Liposome 4: the molar ratio of 4ME-PC, DSPC, cholesterol and DSPE-PEG 2000 is 1.2:1.8:2:0.015;

[0015] ME-Liposome 5: the molar ratio of 4ME-PC, DSPC, cholesterol and DSPE-PEG 2000 is 1.5:1.5:2:0.015;

[0016] ME-Liposome 6: the molar ratio of 4ME-PC, DSPC, cholesterol and DSPE-PEG 2000 is 1.8:1.2:2:0.015;

[0017] The liposome ME-Liposome 0~ME-Liposome 6 encapsulates DNase I to obtain a DNase I-loaded liposome; wherein ME-Liposome 3 has optimal encapsulation efficiency (the encapsulation efficiency is preferably 60%~90%), drug loading, particle size, and potential, and other physicochemical properties.

[0018] The concentration of the DNase I in the liposome is 500 μg / mL~2000 μg / mL.

[0019] The application provides a double-membrane layered modified liposome, which is prepared by sequentially mixing the liposome, red blood cell membranes, and neutrophil membranes.

[0020] The mass ratio of the liposome to the red blood cell membranes in the double-membrane layered modified liposome is 1:1.

[0021] The mass ratio of the product obtained by mixing the liposome and the red blood cell membranes to the neutrophil membranes in the double-membrane layered modified liposome is 1:1.

[0022] The application provides a preparation method of the double-membrane layered modified liposome, which comprises the following steps:

[0023] Step 1: mixing 4ME-PC, DSPC, cholesterol, and DSPE-PEG2000 to form a DNase I-loaded liposome precursor;

[0024] Step 2: mixing the DNase I-loaded liposome precursor and DNase I to obtain a DNase I-loaded liposome;

[0025] Step 3: sequentially mixing the DNase I-loaded liposome and red blood cell membranes and neutrophil membranes to obtain the double-membrane layered modified liposome.

[0026] The components in the bilayer modified liposomes of this invention are the optimal combination obtained through screening. These optimal components interact and work together to affect the performance of the final bilayer modified liposomes. In the bilayer modified liposomes of this invention, DSPC (phospholipid distearate-sn-glycerol-3-phosphocholine) provides a stable bilayer membrane framework, enhancing structural rigidity; cholesterol regulates membrane fluidity and reduces drug leakage; DSPE-PEG2000 (1,2-diacetyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) provides steric hindrance protection, prolonging cycle time; 4ME-PC (2,3-bis[(3,7,11,15-tetramethylhexadecyl)oxy]propyl 2-(trimethylamino)phosphate ethyl ester) enhances membrane compactness and regulates the release window through hydrophobic chain methylation; further optimization of the component addition ratio, specifically the ratio of 4ME-PC to cholesterol, makes DNase... The release window of DNase I and the stability of the liposome membrane are precisely regulated, allowing the drug release window to be within 12–24 hours. Furthermore, by loading appropriate concentrations of DNase I, and by modifying the first layer of the erythrocyte membrane to endow the liposomes with immune escape and long-circulation capabilities, and by modifying the second layer of the neutrophil membrane to achieve active targeting of the inflammatory microenvironment and responsive release of cfDNA.

[0027] The dual-membrane cfDNA-responsive long-acting DNase I liposome of this invention can be used for the treatment of sepsis via intravenous injection, and is particularly suitable for pathological states such as sepsis, post-traumatic sepsis, and severe pneumonia complicated with sepsis accompanied by high levels of NETs and cfDNA. It can also be extended to other cfDNA-related inflammatory diseases, including acute respiratory distress syndrome (ARDS), systemic lupus erythematosus (SLE), and post-traumatic systemic inflammatory response syndrome (SIRS).

[0028] The application of DNase I in the treatment of sepsis is limited by its short half-life, poor in vivo stability, lack of lesion targeting, and inability to release on demand. While existing liposomes can improve pharmacokinetic characteristics, they still struggle to simultaneously meet the requirements of long circulation, inflammatory targeting, and cfDNA-responsive release. This invention provides a biomimetic long-acting DNase I liposome with cfDNA responsiveness and dual targeting functions. It employs a layered modification strategy: first, the liposome surface is coated with a red blood cell membrane to retain the CD47-SIRPα signal and prolong the circulation period; then, it is further fused with a neutrophil membrane, fully exposing adhesion molecules such as LFA-1, Mac-1, and CXCR2 on its surface, thereby enhancing inflammatory targeting. Layered modification avoids problems such as spatial interference and uneven distribution of membrane proteins caused by mixed modifications, resulting in stable and uniform dual functions for the internal liposomes. Furthermore, this invention incorporates cfDNA-responsive design. In the inflammatory microenvironment of sepsis, cfDNA / NETs accumulate in large quantities. The vector of this invention can respond to the high-level cfDNA environment and trigger the precise release of DNase I, thereby achieving targeted action at the lesion site, effectively clearing excess cfDNA / NETs, ​​and alleviating cytokine storm and organ dysfunction.

[0029] This invention provides the use of at least one of the following (I) to (II) in the preparation of a medicament for treating and / or preventing sepsis:

[0030] I) The liposomes described in this invention;

[0031] II) The double-membrane layered modified liposomes described in this invention.

[0032] In the applications described in this invention, sepsis is classified by severity as including sepsis, severe sepsis, and / or septic shock; sepsis is classified by source of infection as including community-acquired sepsis and / or hospital-acquired sepsis; and sepsis is classified by infecting pathogen as including bacterial sepsis, fungal sepsis, viral sepsis, and / or sepsis caused by other pathogens.

[0033] The mouse model of sepsis of the present invention is considered the "gold standard" for sepsis research because it can well reflect the complexity of human sepsis, including multibacterial peritonitis, bacterial translocation into the bloodstream (bacteremia), septic shock, and multiple organ dysfunction.

[0034] This invention provides a medicament for treating and / or preventing sepsis, the raw materials of which include at least one of the following i) to ii):

[0035] i) The liposomes described in this invention;

[0036] ii) The double-membrane layered modified liposomes of the present invention.

[0037] Furthermore, the medicament described in this invention also includes pharmaceutically acceptable excipients.

[0038] The pharmaceutically acceptable excipients include at least one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, disintegrants, stabilizers, plasticizers, penetration enhancers and / or sustained-release agents.

[0039] The pharmaceutically acceptable excipients include at least one or a combination of two or more of solvents, solubilizers, cosolvents, emulsifiers, disintegrants, stabilizers, plasticizers, penetration enhancers and / or sustained-release agents.

[0040] The solvent includes water or a buffer solution; the solubilizer includes a surfactant and / or cyclodextrin.

[0041] The co-solvents include organic acids and their sodium salts, amide compounds, inorganic salts and / or cyclodextrins and their derivatives;

[0042] The emulsifiers include surfactants, polysaccharides, phospholipids, proteins, and / or polyoxyethylene fatty alcohol ethers;

[0043] The disintegrants include starches, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and microcrystalline cellulose.

[0044] The stabilizers include antioxidants, chelating agents, cyclodextrins and their derivatives and / or surfactants;

[0045] The plasticizers include polyols, organic esters, and / or natural polymer derivatives;

[0046] The penetration enhancer includes alcohols, terpenes, and / or fatty acid esters;

[0047] The sustained-release agent includes hydrophilic gel, ethyl cellulose, polyethylene, polyvinyl chloride, ethylene-vinyl acetate copolymer and / or polymethacrylate.

[0048] This invention provides a combination of medicines for treating and / or preventing sepsis, comprising the medicines described herein and other medicines for treating sepsis.

[0049] Other medications used to treat sepsis include antibiotics, norepinephrine, and / or glucocorticoids.

[0050] This invention provides a liposome that mimics the growth-effect deoxyribonuclease I (DNase I) with cell-free DNA (cfDNA) responsiveness and dual targeting function. The liposome is modified by layered fusion of erythrocyte membrane and neutrophil membrane and the release window is precisely controlled by the 4ME-PC ratio to achieve targeted binding of cfDNA and precise delivery to the inflammatory site. It is a liposomal drug carrier that can be used in cfDNA-related inflammatory diseases and has important application prospects. Attached Figure Description

[0051] Figure 1 Schematic diagram of DNase I / R&N-Lipo structure;

[0052] Figure 2 Transmission electron microscopy images of DNase I / Lipo and DNase I / R&N-Lipo are shown.

[0053] Figure 3 The stability of DNase I / Lipo and DNase I / R&N-Lipo is shown, where A is the particle size distribution of DNase I / Lipo and DNase I / R&N-Lipo over 7 days; B is the particle size of DNase I / Lipo and DNase I / R&N-Lipo over 7 days; and C is the change in polydispersity index (PDI) of DNase I / Lipo and DNase I / R&N-Lipo over 7 days.

[0054] Figure 4 The release rate of DNase I / R&N-Lipo in response to cfDNA is shown;

[0055] Figure 5The cytotoxicity of DNase I, DNase I / Lipo, and DNase I / R&N-Lipo is shown in the following figures: A represents cell viability of BMDMs treated with different concentrations of DNase I, DNase I / Lipo, and DNase I / R&N-Lipo for 24 hours; B represents cell viability of BMDMs treated with different concentrations of DNase I, DNase I / Lipo, and DNase I / R&N-Lipo for 48 hours; C represents cell viability of BMDMs treated with different concentrations of DNase I, DNase I / Lipo, and DNase I / R&N-Lipo for 72 hours; D represents neutrophil viability of BMDMs treated with different concentrations of DNase I, DNase I / Lipo, and DNase I / R&N-Lipo for 6 hours; E represents cell viability of human umbilical vein endothelial cells (HUVECs) treated with different concentrations of DNase I, DNase I / Lipo, and DNase I / R&N-Lipo for 24 hours; and F represents cell viability of BMDMs treated with different concentrations of DNase I, DNase I / Lipo, and DNase I / R&N-Lipo for 24 hours. Cell viability of human umbilical vein endothelial cells (HUVECs) after treatment with I / Lipo and DNase I / R&N-Lipo for 48 hours;

[0056] Figure 6 Biosafety evaluation of DNase I / R&N-Lipo;

[0057] Figure 7 The effect of DNase I / R&N-Lipo on the half-life of DNase I and the accumulation of free DNase I at the site of inflammation in septic mice was shown; where A represents the effect on the half-life of DNase I and B represents the effect on the accumulation of free DNase I at the site of inflammation.

[0058] Figure 8 The therapeutic effects of DNase I / R&N-Lipo are shown; where A represents the plasma cfDNA content of mice in each group and B represents the survival curve of mice in each group. Detailed Implementation

[0059] This invention provides DNase I-loaded liposomes and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0060] Compared with the prior art, the DNase I / R&N-Lipo provided by the present invention has the following significant advantages:

[0061] 1. Improve drug stability: By introducing highly methylated phospholipid 4ME-PC, the stability of the liposome membrane is significantly enhanced, effectively reducing the burst release phenomenon and achieving stable release effects of sustained and controlled release.

[0062] 2. Long-lasting circulation: Red blood cell membrane modification endows liposomes with immune escape ability, and prolongs blood circulation time through CD47–SIRPα signal “self-recognition”, significantly improving drug utilization.

[0063] 3. Dual targeting of inflammatory lesions: The inflammatory chemokine receptors on the neutrophil membrane can actively recognize and accumulate in the inflammatory microenvironment. Combined with the long-circulating effect of RBCm, the two achieve a dual effect of "accumulation + persistence".

[0064] 4. cfDNA responsive release: Relying on the natural binding properties of TLR9-cfDNA to achieve on-demand release, reducing side effects in non-lesion sites.

[0065] 5. Superior Modification Method: Existing technologies employ mixed modification of erythrocyte and neutrophil membranes, resulting in random distribution of membrane proteins and mutual functional interference. This invention utilizes layered modification, maintaining the functional integrity of both erythrocyte and neutrophil membranes and avoiding impaired protein activity; it achieves a stable superposition of long-circulation capacity and inflammation-targeting capacity, rather than random distribution; it improves in vivo circulation time and drug accumulation efficiency at inflammatory sites, resulting in more consistent efficacy; combined with cfDNA response release, it better aligns with the pathological characteristics of sepsis, demonstrating superior efficacy compared to mixed modification schemes.

[0066] 6. Multiple pathological blockades: The continuously released DNase I can effectively clear NETs / cfDNA, block the TLR9-mediated inflammatory amplification circuit, and inhibit cytokine storm.

[0067] 7. Improved treatment efficacy: In animal model validation, it effectively improved multi-organ damage, improved multi-organ dysfunction, prolonged survival time, and had good safety.

[0068] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0069] Example 1: Preparation of liposome core and screening of 4ME-PC ratio (ME-Liposome)

[0070] The molar ratios of 4ME-PC, DSPC, cholesterol, and DSPE-PEG2000 are shown in Table 1. The core structure of the liposome was prepared by a thin-film hydration method: each component was dissolved in a chloroform / methanol (9:1, v / v) mixed solution, the solvent was removed by rotary evaporation to form a uniform lipid film; hydration was carried out with PBS buffer (pH 7.4) and dispersion was carried out by an ultrasonic probe to obtain a uniform ME-Liposome suspension.

[0071] The particle size, polydispersity index (PDI), and zeta potential of each group of ME-Liposomes were determined using dynamic light scattering. With increasing 4ME-PC content, the particle size of the methyl liposomes increased (Table 2). When the 4ME-PC percentage was 29.91%, the particle size of ME-Liposome 5 exceeded 200 nm. Liposomes with a particle size less than 400 nm were suitable for intravenous injection, and liposomes with a particle size of 5–200 nm were beneficial for drug loading. Based on this, ME-Liposome 0, ME-Liposome 1, ME-Liposome 2, ME-Liposome 3, ME-Liposome 4, and ME-Liposome 5 were selected for drug loading experiments to evaluate the drug loading rate.

[0072] Table 1. Molecular composition and proportions of methyl liposomes (ME-liposomes)

[0073]

[0074] ME-Liposome 0 a) Represents basic liposomes; b) NA means not applicable.

[0075] Table 2. Physicochemical properties of methyl liposomes (ME-liposome)

[0076]

[0077] Example 2: DNase I loading (DNase I / Lipo)

[0078] ME-Liposome 0, ME-Liposome 1, ME-Liposome 2, ME-Liposome 3, ME-Liposome 4, and ME-Liposome 5 were incubated with DNase I solution (containing 500, 1000, and 2000 μg / mL of DNase I, respectively) at 37°C for 5 minutes to load DNase I into the liposomes, thus obtaining DNase I / Lipo.

[0079] Liposomes loaded with DNase I were placed in a dialysis bag, and the release curve was measured under PBS conditions at 37°C. The optimal 4ME-PC ratio with a release window of 12-24 hours was screened.

[0080] Unencapsulated DNase I was removed by dialysis, and the encapsulation efficiency and drug loading were determined (Tables 3 and 4).

[0081] Encapsulation efficiency (%) = DNase I / (mass of DNase I in Lipo / dosage) × 100%

[0082] Drug loading (%) = DNase I / Mass of DNase I in Lipo / Total mass of DNase I in Lipo × 100%

[0083] The results showed that with increasing 4ME-PC content, the encapsulation efficiency and drug loading of DNase I by methyl liposomes increased; with increasing DNase I concentration, the drug loading of DNase I by methyl liposomes increased, and the liposome encapsulation efficiency and drug loading were optimal when the DNase I content was 1000 μg. ME-Liposome 3 (4ME-PC / DSPC / cholesterol / DSPE-PEG 2000, 17.95 / 41.87 / 39.88 / 0.30, molar percentage) exhibited optimal physicochemical properties such as encapsulation efficiency (preferably 60%~90%), drug loading, particle size, and potential.

[0084] Table 3. Encapsulation efficiency of DNase I methyl liposomes

[0085]

[0086] Table 4. Drug loading capacity of DNase I methyl liposomes

[0087]

[0088] Example 3: Double-film layered modification and TLR9 outward orientation retention (DNase I / R&N-Lipo)

[0089] (I) Isolation and purification of erythrocyte membrane (RBCm) and neutrophil membrane (NEm)

[0090] 1. RBC extraction: EDTA-anticoagulated whole blood from healthy C57BL / 6J mice was centrifuged at 600g for 5 min to remove the plasma and leukocyte layer, and washed three times with PBS. The red blood cell suspension was placed in hypotonic buffer and incubated on ice for 30 min to allow the cells to swell and rupture. The membrane precipitate was collected by centrifugation at 13,000g for 5 min. The hypotonic treatment was repeated 5-7 times until a light pink precipitate was obtained. The precipitate was resuspended in PBS and the protein was quantified. The precipitate was stored at -80℃.

[0091] 2. NEm extraction: Neutrophils derived from mouse bone marrow were isolated, purified by Percoll density gradient centrifugation, and then subjected to hypotonic treatment to obtain cell membranes. Protein denaturation was avoided at 4°C to ensure the native conformation of the TLR9 receptor.

[0092] (II) Double-film layered modification and TLR9 outward orientation retention (DNase I / R&N-Lipo)

[0093] 1. DNase I-loaded liposomes were mixed with RBCm at a mass ratio of 1:1 and extruded sequentially through 400nm, 200nm, and 100nm filter membranes to obtain homogeneous RBCm-Lipo.

[0094] 2. RBCm-Lipo and NEm were mixed in a 1:1 ratio and extruded sequentially through 400nm, 200nm, and 100nm filter membranes to obtain homogeneous RBCm–NEm double-membrane liposomes (DNase I / R&N-Lipo). Figure 1 );

[0095] 3. Membrane protein orientation detection: TLR9 was labeled with immunofluorescence to confirm that it maintains outward activity in the outer NEm layer.

[0096] Transmission electron microscopy revealed that DNase I / R&N-Lipo exhibited a typical bilayer phospholipid structure. Figure 2 DNase I / Lipo and DNase I / R&N-Lipo were characterized and identified, and the results are shown in Table 5. The particle size of DNase I / R&N-Lipo increased to 120-140 nm. Zeta potential and WB detection of membrane proteins showed that both membranes were successfully coated. The changes in particle size and PDI of DNase I / R&N-Lipo after storage at 4℃ for 7 days were monitored, showing that the formulation has good stability and is conducive to storage. Figure 3 ).

[0097] Table 5. Characterization and identification of DNase I / Lipo and DNase I / R&N-Lipo

[0098]

[0099] Example 4 Performance Evaluation

[0100] (I) cfDNA responsiveness experiment

[0101] The outer membrane of NEm retains the outward conformation of TLR9, which can specifically recognize and bind to cfDNA. When cfDNA binds to TLR9 on the membrane surface, it can promote the increase of liposome membrane permeability through local perturbation of the membrane-liposome interface structure, accelerate the release of DNaseI, and achieve cfDNA concentration-dependent responsive drug release.

[0102] The drug release rate was detected by co-incubating DNase I / R&N-Lipo with known concentrations of cfDNA sequences (CpG ODN 1826, PBS + 0, 1, 5, 10 μg / mL cfDNA). The control group did not contain cfDNA (PBS + 0). Results showed that in the presence of cfDNA, TLR9–cfDNA binding induced membrane relaxation, significantly accelerating the drug release rate (p<0.01), while release remained slow in the absence of cfDNA. Furthermore, 10 μg / mL cfDNA increased the DNase I release over 2 hours to 2.8 times that of the control group, demonstrating that cfDNA can act as a biosignal molecule triggering drug release. Figure 4 ).

[0103] (II) Safety Evaluation

[0104] The cytotoxicity of DNase I / R&N-Lipo was assessed using the CCK-8 assay. Different concentrations of DNase I / R&N-Lipo (0.1–1000 μg / mL) were co-incubated with BMDMs, neutrophils, and HUVEC cells, and cell viability was measured. DNase I / R&N-Lipo showed no significant cytotoxicity across a wide concentration range and exhibited good biocompatibility. Figure 5 ).

[0105] Healthy adult male C57BL / 6 mice were randomly divided into two groups. The experimental group received DNase I / R&N-Lipo via tail vein injection at a dose of 5 mg / kg (based on DNase I), administered every other day for 14 consecutive days. The control group received an equal volume of physiological saline under the same conditions. During the administration period, the general condition of the mice was monitored daily, including weight, diet, activity level, and coat condition. After 14 days, major organs (heart, liver, spleen, lungs, and kidneys) were collected for H&E staining. Compared with the control group, mice in the DNase I / R&N-Lipo experimental group survived well and did not exhibit significant behavioral abnormalities or weight loss during the administration period. Histological examination showed no significant pathological differences in the major organs of the experimental group compared with the control group, including inflammatory infiltration or structural abnormalities. Figure 6 The results indicate that the formulation did not induce toxic reactions and has good biocompatibility and in vivo safety, providing important safety evidence for the long-term use of the formulation in subsequent clinical applications.

[0106] Example 5: Therapeutic effect of DNase I / R&N-Lipo in septic mice

[0107] Establishment of a mouse model of sepsis: C57BL / 6J mice underwent cecal ligation and puncture after anesthesia. Control mice underwent the same procedure but without cecal puncture. Three hours post-surgery, mice were injected via the tail vein with free DNase I, DNase I / Lipo, or DNase I / R&N-Lipo, equivalent to 5 mg / kg DNase I. Results showed that the circulating half-life of the DNase I / R&N-Lipo group was prolonged by approximately 4 times; the amount of liposomes accumulated at the inflammatory lesion site was 3.5 times that of the free DNase I group. Figure 7 Plasma cfDNA concentration decreased by more than 70%; survival rate was significantly improved (p<0.01). Figure 8 ).

[0108] Furthermore, the formulation of this invention, DNase I / R&N-Lipo, can significantly reduce plasma cfDNA and NETs levels, inhibit the release of inflammatory factors such as TNF-α, IL-6, and IL-1β, improve multi-organ dysfunction, and increase the 7-day survival rate to 70%.

[0109] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Liposomes loaded with DNase I, characterized in that, Including liposomes and DNase I; The liposomes comprise 2,3-bis[(3,7,11,15-tetramethylhexadecyl)oxy]propyl 2-(trimethylamino) phosphate, phospholipid distearate-sn-glycerol-3-phosphocholine, cholesterol, and 1,2-diacetyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; The molar ratio of 2,3-bis[(3,7,11,15-tetramethylhexadecyl)oxy]propyl 2-(trimethylamino) phosphate, phospholipid distearate-sn-glycerol-3-phosphocholine, cholesterol, and 1,2-diacetyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] is (0.3~1.8):(22.7~1.22):2:0.

015.

2. The liposomes according to claim 1, characterized in that, The concentration of DNase I is 500 μg / mL to 2000 μg / mL.

3. A double-membrane layered modified liposome, characterized in that, The liposomes described in claim 1 or 2 are prepared by sequentially mixing them with erythrocyte membranes and neutrophil membranes.

4. The dual-membrane layered modified liposome according to claim 3, characterized in that, The mass ratio of liposomes to erythrocyte membranes according to claim 1 or 2 is 1:

1.

5. The dual-membrane layered modified liposome according to claim 4, characterized in that, The product obtained by mixing the liposomes of claim 1 or 2 with the erythrocyte membrane has a mass ratio of 1:1 to the neutrophil membrane.

6. Use of at least one of the following (I) to (II) in the preparation of medicaments for the treatment and / or prevention of sepsis: I) The liposomes according to claim 1 or 2; II) The double-membrane layered modified liposomes according to any one of claims 3 to 5.

7. A medicine for treating and / or preventing sepsis, characterized in that, The raw materials include at least one of the following: i) to ii) i) The liposomes according to claim 1 or 2; ii) The double-membrane layered modified liposomes according to any one of claims 3 to 5.

8. The medicament according to claim 7, characterized in that, It also includes pharmaceutically acceptable excipients.

9. A combination of drugs for the treatment and / or prevention of sepsis, characterized in that, This includes the drug as described in claim 7 or 8 and other drugs used to treat sepsis.

10. The pharmaceutical combination according to claim 9, characterized in that, Other medications used to treat sepsis include antibiotics, norepinephrine, and / or glucocorticoids.

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