Phase separation mediated nano trap for removing extracellular free DNA (deoxyribonucleic acid) as well as preparation method and application of nano trap

Abstract

The invention discloses a phase separation mediated nano trap for removing extracellular free DNA (Deoxyribonucleic Acid) as well as a preparation method and application thereof, and relates to the technical field of medical nano biological materials. The nano trap takes hollow mesoporous cerium dioxide as a core, DNA degrading enzyme is loaded in the nano trap through a coordinate bond, and a cationic polymer layer is modified on the surface of the nano trap, so that the nano trap has positive zeta potential. The structure can be used as a nucleation center to efficiently capture negatively charged cfDNA; the cfDNA is mediated to generate liquid-liquid phase separation to form a stable aggregate, and then the cfDNA is subjected to in-situ degradation through a loaded enzyme. The invention further discloses a construction method of the nano trap and application of the nano trap in preparation of drugs for treating or preventing systemic inflammatory response syndromes (such as septicopyemia) mediated by abnormal cfDNA level rise. The nano trap provided by the invention has the advantages of high cfDNA removal efficiency, innovative mechanism, synergistic effects of oxidation resistance and bacteria resistance and the like, and provides an effective new way for treatment of related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanobiomaterials for medical use, in particular to a phase separation mediated nanotraps for removing extracellular free DNA and a preparation method and application thereof. BACKGROUND

[0002] As a key damage-associated molecular pattern (DAMP), extracellular free DNA (cfDNA) is a core driver in the development of various diseases. In conditions such as sepsis, severe trauma, ischemia-reperfusion injury, and certain autoimmune diseases, cfDNA is released into the circulatory system in large quantities. These cfDNA can form DNA condensates through mechanisms such as liquid-liquid phase separation (LLPS), thereby activating immune cells and triggering a severe inflammatory storm, leading to tissue and organ damage. In sepsis, a life-threatening organ dysfunction caused by infection, the pathological role of cfDNA is particularly prominent and clear. The rapid increase of cfDNA in serum has been confirmed as a key factor leading to immune disorders and inflammatory cascades in sepsis. Therefore, developing a technology that can efficiently remove cfDNA in the circulatory system has become one of the core treatment strategies for intervening in such major diseases.

[0003] Current methods for removing cfDNA are not perfect. Existing strategies (such as the use of cationic polymers) mainly rely on unstable electrostatic adsorption, and there is a risk of complex dissociation and cfDNA re-release, limiting the therapeutic effect. Traditional mesoporous nanomaterials are usually negatively charged, and there is electrostatic repulsion with cfDNA which is also negatively charged, and they do not have degradation ability, so it is difficult to achieve application without special functionalization.

[0004] Therefore, there is an urgent need in the art for a technology that can serve as a universal platform for developing new biomaterials that can stably capture and efficiently degrade pathological cfDNA, thereby providing more effective and fundamental solutions for treating a series of related major diseases including sepsis. SUMMARY

[0005] The present application provides a phase separation mediated nanotraps for removing extracellular free DNA and a preparation method and application thereof. The nanotraps efficiently remove extracellular free DNA (cfDNA) by mediating the phase separation process. The nanotraps also have a synergistic therapeutic effect of resisting oxidative stress and resisting bacteria, which can solve the problem of low cfDNA removal efficiency and single mechanism of action in the prior art.

[0006] In view of the deficiencies of the prior art, the present application provides the following technical solutions.

[0007] In a first aspect, the present application provides a phase separation mediated nanotrap for removing extracellular free DNA, which comprises:

[0008] (a) a hollow mesoporous nanoparticle core;

[0009] (b) a DNA degradation enzyme loaded inside the mesoporous nanoparticle core;

[0010] (c) a cationic layer with positive zeta potential modified on the surface of the mesoporous nanoparticle core.

[0011] Further, the mesoporous nanoparticle core is mesoporous ceria and / or mesoporous manganese dioxide.

[0012] Further, the positive zeta potential of the cationic layer is not less than +10 mV.

[0013] The present application constructs a cationic layer on the surface of the mesoporous nanoparticle core, which aims to capture negatively charged cfDNA by the positive charge thereof.

[0014] Further, the material constituting the cationic layer is a cationic polymer and / or a silane coupling agent.

[0015] Specifically, the material constituting the cationic layer is at least one of polyethyleneimine, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)trimethoxysilane.

[0016] Further, the average particle size of the mesoporous nanoparticle core is 180-200 nm, and the average particle size of the nanotrap is 240-280 nm.

[0017] It should be noted that the mesoporous nanoparticle core of the nanotrap of the present application can be directly purchased as a commercial product, and can also be prepared by referring to the methods in the existing literature journals, or can be prepared by self-development.

[0018] Alternatively, the DNA degradation enzyme is a common DNA degradation enzyme in the art. For example, the DNA degradation enzyme can be at least one of type 1 DNA enzyme, type 2 DNA enzyme, and type 3 DNA enzyme, and the protein is combined with the hollow mesoporous nanoparticle with nanoscale enzyme activity by coordination bond.

[0019] The application provides the above-mentioned nanotrap, wherein the nanotrap interacts with the negatively charged cfDNA through the positive zeta potential, participates in and mediates the liquid-liquid phase separation process, forms a coacervate to capture the cfDNA, limits the cfDNA in the coacervate, has a small influence on the external environment, has a more stable binding state, can continuously play a cleaning role, and finally realizes in-situ degradation of the cfDNA; and the DNA degradation enzyme is combined with the hollow mesoporous nanoparticle with nanoscale enzyme activity through coordination bonds.

[0020] The nanotrap provided by the application provides a brand-new cfDNA cleaning mechanism, has high cleaning efficiency, and has the advantages of synergistic effects of antioxidation and antibiosis, and the like, thereby providing an effective new way for treatment of related diseases.

[0021] In a second aspect, the application provides a preparation method of the above-mentioned nanotrap for cleaning extracellular free DNA through phase separation mediation, comprising the following steps:

[0022] A first dispersion liquid containing the mesoporous nanoparticle core is prepared, the DNA degradation enzyme is added, and a reaction is performed; the precipitate is collected by centrifugation to obtain a loaded enzyme intermediate;

[0023] A second dispersion liquid containing the loaded enzyme intermediate is prepared, a modifier is added, ultrasonic treatment is performed, and a reaction is performed; the precipitate is collected by centrifugation to obtain the nanotrap; and the modifier contains a material constituting the cationic layer.

[0024] In the loaded enzyme intermediate, the DNA degradation enzyme is combined with the interior of the mesoporous nanoparticle core through coordination bond action. The material constituting the cationic layer is modified on the surface of the mesoporous nanoparticle core through electrostatic adsorption, so that the originally negatively charged loaded enzyme intermediate is converted into a positively charged one.

[0025] Further, the mass ratio of the mesoporous nanoparticle core to the DNA degradation enzyme is 1:(1-10).

[0026] Further, the mass ratio of the mesoporous nanoparticle core to the DNA degradation enzyme is 1:10.

[0027] The application finds through experimental research that when the mesoporous nanoparticle core and the DNA degradation enzyme are adjusted to the above-mentioned mass ratio, the effective loading amount of the DNA degradation enzyme is relatively optimal, the encapsulation efficiency is increased from 46% to 78%, and relatively good encapsulation effect is achieved.

[0028] Further, the mass ratio of the loaded enzyme intermediate to the material constituting the cationic layer in the modifier is 1:(1-8).

[0029] Further, the mass ratio of the loaded enzyme intermediate to the material constituting the cationic layer in the modifier is 1:8.

[0030] Optionally, the solvent of the first dispersion and the second dispersion can be selected from the commonly used and pharmaceutically acceptable agents in the art; including but not limited to PBS, physiological saline, deionized water, etc.

[0031] Optionally, the concentration of the mesoporous nanoparticle core in the first dispersion is 1-10 mg / mL, and specifically 1 mg / mL can be selected.

[0032] Optionally, the DNA degradation enzyme can also be configured into a solution in advance before being added. The solvent can be the same as that of the first dispersion and the second dispersion. Further optionally, the concentration of the DNA degradation enzyme solution is 1-10 mg / mL, and specifically 10 mg / mL can be selected.

[0033] Optionally, the concentration of the enzyme intermediate loaded in the second dispersion is 1-10 mg / mL, and specifically 1 mg / mL can be selected.

[0034] Optionally, the solvent in the modifier also adopts the commonly used and pharmaceutically acceptable agents in the art; including but not limited to PBS, an ethanol aqueous solution or anhydrous ethanol, physiological saline, deionized water, etc.

[0035] Further optionally, the concentration of the material constituting the cationic layer in the modifier is 1-4 mg / mL, and specifically 4 mg / mL can be selected.

[0036] In a third aspect of the present application, the application of the above-mentioned phase separation-mediated nanotrap for removing extracellular free DNA is provided for preparing a medicine for preventing or treating systemic inflammatory response syndrome mediated by abnormally high cfDNA level.

[0037] In a fourth aspect of the present application, a composition is also provided, which comprises the above-mentioned phase separation-mediated nanotrap for removing extracellular free DNA, and is used for preventing or treating systemic inflammatory response syndrome mediated by abnormally high cfDNA level.

[0038] Further, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0039] It should be noted that the "systemic inflammatory response syndrome mediated by abnormally high cfDNA level" claimed in the present application refers to an uncontrolled systemic excessive inflammatory response driven by abnormally high cfDNA level in blood or other body fluids. For example, such conditions include but are not limited to sepsis, skin infection, etc.

[0040] The inventive concept of the present application is that: by utilizing the structural characteristics and nano-enzyme activity of the core hollow mesoporous nanoparticles, the DNA degradation enzyme is first stably loaded through coordination bond, giving it the core function of degrading cfDNA; then, the surface is modified with a cationic polymer (such as PEI), so that the nanotrap surface carries a positive charge. This unique structure enables it to act as a nucleation site, efficiently binding with negatively charged cfDNA through electrostatic interaction, and actively participating in and mediating the liquid-liquid phase separation process of cfDNA to form stable condensates. The cfDNA captured in the condensates is then efficiently degraded in situ by the loaded DNAse. At the same time, the inherent antioxidant stress resistance of the mesoporous nanoparticle core with nano-enzyme activity and the antibacterial performance brought by the cationic surface synergize with the function of cfDNA clearance, together realizing effective treatment of related diseases.

[0041] Compared with the prior art, the present application has the following advantages.

[0042] 1. Innovative mechanism, high capture efficiency: The present application first proposes a strategy of using nanomaterials to mediate the phase separation of cfDNA. Compared with traditional unstable electrostatic adsorption, the nanotrap of the present application can form more stable condensates with cfDNA, greatly improving the capture and clearance efficiency of cfDNA.

[0043] 2. Functional integration, synergistic treatment: The present application integrates the functions of cfDNA clearance, antioxidant stress resistance and antibacterial properties into one, synergistically intervening in multiple pathological links of sepsis and other complex diseases, and achieving better treatment effect.

[0044] 3. Wide application prospect: The nanotrap constructed by the present application has a stable preparation method and is suitable for large-scale production, and is expected to develop into a new type of nanomedicine for treating sepsis and other inflammatory diseases caused by abnormal elevation of cfDNA. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The Fourier transform infrared spectra of CeO2, CeO2@DNase I and Nano-Traps prepared in Example 1 are shown in the figure, from bottom to top in the figure are CeO2, CeO2@DNase I and Nano-Traps.

[0046] Figure 2 The particle size analysis graphs of CeO2, CeO2@DNase I and Nano-Traps prepared in Example 1 are shown in the figure, from left to right in the figure are CeO2, CeO2@DNase I and Nano-Traps.

[0047] Figure 3Potential analysis chart of CeO2, CeO2@DNase I and Nano-Traps prepared for Example 1, in which CeO2, CeO2@DNase I and Nano-Traps are arranged from left to right.

[0048] Figure 4 Transmission electron micrograph of Nano-Traps prepared for Example 1.

[0049] Figure 5 Quantitative chart of encapsulation effect of Nano-Traps on DNase I detected for Examples 1-3.

[0050] Figure 6 Confocal fluorescence chart of Nano-Traps participating in DNA phase separation detected for Example 4.

[0051] Figure 7 Survival curve of sepsis mice in the sham operation group, PBS, CeO2, CeO2@DNase I and Nano-Traps treatment groups in Application Example 1.

[0052] Figure 8 Weight curve of sepsis mice in the sham operation group, PBS, CeO2, CeO2@DNase I and Nano-Traps treatment groups in Application Example 1.

[0053] Figure 9 cfDNA content in peripheral blood of sepsis mice in the sham operation group, PBS, CeO2, CeO2@DNase I and Nano-Traps treatment groups in Application Example 1 after 5 days of operation.

[0054] Figure 10 Wound healing photo of skin infection mice in the normal saline, CeO2, CeO2@DNase I and Nano-Traps treatment groups in Application Example 1 after modeling.

[0055] Figure 11 HE staining and Masson staining of wound skin tissue sections of skin infection mice in the normal saline, CeO2, CeO2@DNase I and Nano-Traps treatment groups in Application Example 1 after 12 days of modeling.

[0056] Figure 12 cfDNA content in peripheral blood of skin infection mice in the normal saline, CeO2, CeO2@DNase I and Nano-Traps treatment groups in Application Example 1 after 12 days of modeling. DETAILED DESCRIPTION

[0057] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of the present application.

[0058] In some embodiments of the present application, the prepared phase separation mediated nanotrap for removing extracellular free DNA has a structure comprising:

[0059] (a) a hollow mesoporous nanoparticle core;

[0060] (b) a DNA degradation enzyme loaded inside the mesoporous nanoparticle core;

[0061] (c) a surface modification of the mesoporous nanoparticle core to have a cationic layer with a positive zeta potential.

[0062] Optionally, the mesoporous nanoparticle core is mesoporous ceria and / or mesoporous manganese dioxide.

[0063] Optionally, the positive zeta potential of the cationic layer is not less than +10 mV, for example, cationic polymers and / or silane coupling agents. Specifically, at least one of polyethyleneimine, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)trimethoxysilane can be selected.

[0064] Optionally, the DNA degradation enzyme can be at least one of type 1 DNA enzyme, type 2 DNA enzyme, and type 3 DNA enzyme.

[0065] In other embodiments of the present application, a preparation method of the above-mentioned phase separation mediated nanotrap for removing extracellular free DNA is provided, comprising the following steps:

[0066] A first dispersion liquid containing the mesoporous nanoparticle core is prepared, a DNA degradation enzyme is added, and a reaction is performed; a precipitate is collected by centrifugation to obtain an enzyme-loaded intermediate;

[0067] A second dispersion liquid containing the enzyme-loaded intermediate is prepared, a modifier is added, and ultrasonic treatment is performed; a precipitate is collected by centrifugation to obtain the nanotrap; the modifier contains a material constituting the cationic layer.

[0068] In the above preparation method, the mass ratio of the mesoporous nanoparticle core to the DNA degradation enzyme can be selected as 1:(1-10); specifically, the mass ratio is 1:10.

[0069] In the above preparation method, the mass ratio of the enzyme-loaded intermediate to the material constituting the cationic layer in the modifier is 1:(1-8); specifically, the mass ratio is 1:8.

[0070] Optionally, the solvent of the first dispersion liquid and the second dispersion liquid can be selected from the commonly used and pharmaceutically acceptable reagents in the art; including but not limited to PBS, physiological saline, deionized water, etc. In the following specific embodiments, commercially available PBS buffer is selected as a representative solvent.

[0071] Optionally, the concentration of the mesoporous nanoparticle core in the first dispersion liquid is 1-10 mg / mL, and specifically 10 mg / mL can be selected.

[0072] Optionally, the DNA degradation enzyme can also be configured into a solution in advance before being added. The solvent can be the same as the solvent of the first dispersion liquid and the second dispersion liquid. Further optionally, the concentration of the DNA degradation enzyme solution is 1-10 mg / mL, and specifically 10 mg / mL can be selected.

[0073] Optionally, the concentration of the enzyme intermediate loaded in the second dispersion liquid is 1-10 mg / mL, and specifically 1 mg / mL can be selected.

[0074] Optionally, the solvent in the modifier also adopts the commonly used and pharmaceutically acceptable reagents in the art; including but not limited to PBS, an ethanol aqueous solution or anhydrous ethanol, physiological saline, deionized water, etc.

[0075] Further optionally, the concentration of the material constituting the cationic layer in the modifier is 1-4 mg / mL, and specifically 4 mg / mL can be selected.

[0076] In the following embodiments of the present application, a sepsis mouse model is selected as the experimental object to verify the treatment effect of the nano-trap.

[0077] Example 1

[0078] The nano-trap provided in this embodiment has the following preparation method.

[0079] 1. Preparation of mesoporous ceria nanoparticles.

[0080] (1) 48 mL of anhydrous ethanol, 8 mL of deionized water and 2 mL of ammonia water were mixed in a round-bottom flask, and 5 mL of tetraethyl orthosilicate (TEOS) was added and stirred at room temperature for 24 h;

[0081] Centrifugation at 8000 rpm for 10 min, washing with deionized water and anhydrous ethanol for 3 times respectively; centrifugal collection of the precipitate, drying in a vacuum drying box at 65℃ for 6 h, to obtain a white powder of silica template.

[0082] (2) 300 mg of the silica template was ultrasonically dispersed in 43 mL of ethylene glycol, 2.25 mL of a 1 M aqueous solution of cerium nitrate hexahydrate (Ce(NO3)3·6H2O) was added, and stirring was performed at room temperature for 30 min;

[0083] The above solution was transferred to a reaction kettle, and reaction was performed at 130°C for 15 h.

[0084] After the reaction was completed, the residual solution was centrifuged at 12500 rpm for 10 min, and washed with anhydrous ethanol for 3 times; the precipitate was collected by centrifugation, and freeze-dried for 12 h to obtain a mesoporous cerium oxide powder with a silica template.

[0085] (3) 70 mg of the mesoporous cerium oxide powder with a silica template was ultrasonically dispersed in 30 mL of deionized water, 1 mL of a 2.5 M aqueous solution of sodium hydroxide was added, and stirring was performed at 50°C for 2 h to remove the silica template;

[0086] Centrifugation was performed at 3200 rpm for 10 min, and washing was performed with deionized water and anhydrous ethanol for 3 times, respectively; the precipitate was collected and freeze-dried for 12 h to obtain mesoporous cerium oxide (CeO2) nanoparticles with an average particle size of 180-200 nm.

[0087] 2. Preparation of Nano-Traps.

[0088] (1) 10 mg of the mesoporous cerium oxide nanoparticles were dispersed in 10 mL of PBS to obtain a first dispersion liquid;

[0089] 100 mg of DNAse I of type 1 was dispersed in 10 mL of the first dispersion liquid, so that the mass ratio of the mesoporous cerium oxide nanoparticles and the DNAse I of type 1 was 1:10; and overnight at 4°C.

[0090] (2) Centrifugation was performed at 12000 rpm for 15 min, and the precipitate was collected as mesoporous cerium oxide nanoparticles loaded with DNAse (CeO2@DNase I, enzyme loading intermediate).

[0091] (3) 1 mg of the mesoporous cerium oxide nanoparticles loaded with DNAse was dispersed in 1 mL of PBS to obtain a second dispersion liquid;

[0092] 4 mg of polyethyleneimine (PEI) was dispersed in 1 mL of PBS, and the two were mixed in a volume ratio of 1:2 after ultrasonic treatment for 10 min; and overnight at 4°C, centrifugation was performed at 12000 rpm for 15 min, and the precipitate was collected as Nano-Traps, that is, the final nano-trap was obtained.

[0093] Example 2

[0094] The preparation method of the nanotrap of this example is basically the same as that of Example 1, except that when loading the DNA enzyme, 10 mg / mL of type 1 DNA enzyme solution is mixed with 1 mg / mL of mesoporous cerium dioxide solution at a volume ratio of 1:5, i.e. the mass ratio of mesoporous cerium dioxide nanoparticles to type 1 DNA enzyme is 1:2; 4°C overnight.

[0095] Example 3

[0096] The preparation method of the nanotrap of this example is basically the same as that of Example 1, except that when loading the DNA enzyme, 10 mg / mL of type 1 DNA enzyme solution is mixed with 1 mg / mL of mesoporous cerium dioxide solution at a volume ratio of 1:10, i.e. the mass ratio of mesoporous cerium dioxide nanoparticles to type 1 DNA enzyme is 1:1; 4°C overnight.

[0097] Example 4

[0098] The preparation method of the nanotrap of this example is basically the same as that of Example 1, except that the hollow mesoporous cerium dioxide nanoparticles are replaced by hollow mesoporous manganese dioxide nanoparticles.

[0099] The preparation method of the mesoporous manganese dioxide nanoparticles is as follows.

[0100] (1) The silica template in the form of white powder was prepared by the same method as step (1) of Example 1.

[0101] (2) 50 mg of the silica template was stirred with 500 mg of polyvinylpyrrolidone (PVP) at room temperature overnight, centrifuged at 8000 rpm for 10 min, and washed with deionized water for 3 times to obtain SiO2@PVP particles.

[0102] (3) 25.5 mL of deionized water, 20 mg of methyldopa and 28 μL of formaldehyde were added to the SiO2@PVP particles, mixed thoroughly, and 100 μL of ammonia water (concentration 2.8%) was added;

[0103] After warming to 60°C, stirring for 2 h, and then warming to 100°C, stirring for 2 h;

[0104] After cooling, centrifugation at 8000 rpm for 10 min, and washing with deionized water for 3 times, the intermediate product was obtained.

[0105] (4) 10 mg of the intermediate product and 5 mL of deionized water were mixed thoroughly, 3 mL of KMnO4 solution (concentration 0.2M) was added, stirring at room temperature for 4 h, centrifugation at 8000 rpm for 10 min, and washing with deionized water for 3 times to obtain mesoporous manganese dioxide powder with silica template.

[0106] (5) 20 mL of NaOH aqueous solution (1 M) and 50 mg of mesoporous manganese dioxide powder with silica template were stirred at room temperature overnight, centrifuged at 8000 rpm for 10 min, washed with deionized water for 3 times, to obtain hollow mesoporous manganese dioxide (MnO2) nanoparticles.

[0107] Test Example 1: Physical and chemical index detection of intermediate products (CeO2, CeO2@DNase I) and product (Nano-Traps) prepared in Example 1

[0108] The Fourier transform infrared spectrograms were used to characterize the intermediate products (CeO2, CeO2@DNase I) and product (Nano-Traps) prepared in Example 1, and the results are shown in Figure 1 It can be seen that the change of the peak at 900-1000 cm -1 indicates that DNase I is successfully loaded inside the cerium dioxide through the coordination bond (such as Ce-O-C, Ce-N) between the amino group (-NH2) and carboxyl group (-COOH) of DNase I and the Ce 3+ / Ce 4+ on the surface of cerium dioxide; the change of the peak at 2800-3000 cm -1 indicates the successful modification of PEI on the surface of cerium dioxide.

[0109] The particle size analysis results of the intermediate products (CeO2, CeO2@DNase I) and product (Nano-Traps) prepared in Example 1 are shown in Figure 2 It can be seen that the average particle size of the initially prepared CeO2 is about 180-200 nm, the particle size increases to 240 nm after loading DNase I, and the particle size increases to 280 nm after PEI modification.

[0110] The Zata potential results of the intermediate products (CeO2, CeO2@DNase I) and product (Nano-Traps) prepared in Example 1 are shown in Figure 3 It can be seen that the initially prepared CeO2 is negatively charged, the negative charge decreases after loading DNase I, and the PEI modification changes to positive charge.

[0111] The transmission electron micrograph of the product (Nano-Traps) prepared in Example 1 is shown in Figure 4 It can be seen that the mesoporous nanoparticle core has a significant hollow structure, which provides sufficient space for the internal loading of DNA degrading enzyme, combined with the high drug encapsulation rate of 78% measured in the test example, further confirming that the DNA degrading enzyme is effectively encapsulated inside the nano-trap, rather than simply adsorbed on the surface, which is conducive to protecting the biological activity of the enzyme and realizing the controllable degradation function.

[0112] The DNAse encapsulation effect of the Nano-Traps prepared in Examples 1-3 was determined by BCA protein assay, and the results are shown in Figure 5 The data in the figure is the DNAse concentration loaded on the mesoporous ceria nanoparticles. It can be seen that, compared with the mass ratio of mesoporous ceria nanoparticles and type 1 DNAse, a lower mass ratio of Example 2 (mass ratio 1:2) and Example 3 (mass ratio 1:1), a higher ratio of Example 1 has a better encapsulation effect.

[0113] Test Example 2: Test of the process of cfDNA phase separation involving Nano-Traps

[0114] This test example is used to detect the process of cfDNA phase separation involving Nano-Traps, and the specific steps are as follows.

[0115] (1) Disperse 1 mg of mesoporous ceria nanoparticles in 1 mL of PBS, disperse 2.5 mg of polyethyleneimine (PEI) in 5 mL of anhydrous ethanol, and ultrasonic for 10 min. Mix the two according to the volume ratio of 1:2, and incubate at 4°C overnight.

[0116] After centrifugation at 12000 rpm for 15 minutes, collect the precipitate as CeO2@PEI.

[0117] (2) Take 1 mg / mL Rhodamine B (Rhodamine B) solution, and mix with 1 mg / mL mesoporous ceria nanoparticles (CeO2) and 1 mg / mL CeO2@PEI, respectively, with a mixing volume ratio of 1:1000.

[0118] 4°C rotary incubation overnight, and perform fluorescence labeling.

[0119] (3) The phase separation experiment was carried out in 25 mM Tris-HCl buffer (pH=7.4), and the final concentrations of the components were as follows: 20 μM 6-carboxyfluorescein (6-FAM) labeled DNA (sequence (TTAGGG)4, synthesized by Shengong Bioengineering Co., Ltd.), 10 μM FUS protein (purchased from MCE Company, item number: P702630), 50 μM poly-L-lysine (purchased from Shengong Bioengineering Co., Ltd., item number: A600751).

[0120] Add Rhodamine B-labeled CeO2 and CeO2@PEI to the above Tris-HCl buffer system at a final concentration of 10 μM, prepare in a 96-well confocal culture plate, and use a confocal microscope to image.

[0121] The droplet formation and the localization distribution of the material were observed by monitoring the 6-FAM fluorescence signal of DNA and the Rhodamine B fluorescence signal of the material. The results are shown in Figure 6 It can be seen that the CeO2@PEI and DNA phase separation aggregate has the co-localization characteristics, while CeO2 does not have this characteristic, which indicates that cfDNA is wrapped in the aggregate and has a stable binding state.

[0122] Test Example 3: Verification of the therapeutic effect of nano-traps on sepsis

[0123] In this test example, the nano-traps prepared in Example 1 were used to verify their effect on treating sepsis. The specific treatment process is as follows.

[0124] 1. Constructing a sepsis mouse model

[0125] The experimental animals were 6-8 week old male C57BL / 6 mice. Isoflurane inhalation anesthesia was used, and then the mouse abdomen was shaved and disinfected; then a 1 cm long incision was made on the midline of the abdomen, the cecum was carefully pulled out of the body, and the cecum was ligated at a specific position with a 4-0 silk thread; then a 21-gauge needle was used to puncture the cecum, and the cecal contents were squeezed out from the puncture; finally, the cecum was returned to the abdominal cavity, and the abdominal incision was sutured.

[0126] The sham group mice only received abdominal open surgery and did not undergo subsequent cecal ligation and puncture operation.

[0127] The CLP group was injected with PBS buffer for control at a dose of 10 mg / kg through the tail vein at 12 h and 24 h after the operation.

[0128] The nano-material treatment group was injected with CeO2, CeO2@DNase I and Nano-Traps prepared in Example 1 at a dose of 10 mg / kg through the tail vein at 12 h and 24 h after the operation.

[0129] The survival rate and body weight of the mice were monitored continuously for 7 days during the experiment, and blood was collected through the retro-orbital venous plexus on the 5th day after the operation for detection of peripheral blood cfDNA content.

[0130] The experimental results are shown in Figures 7-9 It can be seen that: compared with the CLP group, the CeO2 group and the CeO2@DNase I group, the Nano-Traps group can significantly improve the survival rate of mice ( Figure 7 ) and restore body weight ( Figure 8 ); the cfDNA content in the peripheral blood of the Nano-Traps group mice is significantly lower than that of the PBS group ( Figure 9 ).

[0131] 2. Construction of a mouse model of skin infection

[0132] The experimental animals were 6-8 week old female C57BL / 6 mice. The method of anesthesia was isoflurane inhalation anesthesia. The hair on the back of the mouse was first removed with a hair clipper, and then further removed with depilatory cream. 5 x 10 6 The staphylococcus aureus solution of 5 x 105CFU was diluted to 100 μL with normal saline (CTL group) or 1 mg / mL CeO2, CeO2@DNase I or Nano-Traps solution prepared in Example 1, and subcutaneously injected into the back of the mouse for 3 consecutive days.

[0133] On the 3rd, 4th, 6th, 9th and 12th day of modeling, the infection site of the mouse was photographed. On the 12th day of modeling, blood was collected from the eye orbit for cfDNA content analysis; then the mouse was euthanized by carbon dioxide inhalation, and the infected tissue was cut off for subsequent staining experiments.

[0134] The experimental results are shown in Figures 10-12 It can be seen that the Nano-Traps group can significantly reduce the infection area and accelerate the healing of the infection wound compared with the CTL group, the CeO2 group and the CeO2@DNase I group Figure 10 ); the epithelial structure of the Nano-Traps group is relatively complete, the amount of inflammatory cell infiltration is less, and the skin repair effect is best Figure 11 ); the cfDNA content of the peripheral blood of the mouse in the Nano-Traps group is significantly lower than that in the CTL group Figure 12 ).

[0135] These results show that the present application successfully constructs a phase separation mediated nanotrap for removing extracellular free DNA, which has good DNA adsorption and degradation effect, and has good therapeutic effect on sepsis; at the same time, the nanomaterial is durable and suitable for large-scale production, and is expected to develop into a new biomaterial for more effective treatment of bacterial infectious diseases.

[0136] The above specific embodiments describe the implementation of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the present application, the technical solutions of the present application can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A phase separation-mediated nanotraps for clearance of extracellular free DNA, characterized in that, The structure of the nanotrap comprises: (a) a hollow mesoporous nanoparticle core; (b) a DNA degradation enzyme loaded inside the mesoporous nanoparticle core; (c) a surface of the mesoporous nanoparticle core modified to have a cationic layer with a positive zeta potential.

2. The phase separation-mediated nanotraps for clearance of extracellular free DNA according to claim 1, wherein, The mesoporous nanoparticle core is mesoporous ceria and / or mesoporous manganese dioxide.

3. The phase separation-mediated nanotraps for clearance of extracellular free DNA according to claim 1, wherein, The positive zeta potential of the cationic layer is not less than +10 mV.

4. The phase separation-mediated nanotraps for clearance of extracellular free DNA according to claim 3, wherein, The material constituting the cationic layer is a cationic polymer and / or a silane coupling agent; Further, the material constituting the cationic layer is at least one of polyethyleneimine, (3-aminopropyl)triethoxysilane, and (3-aminopropyl)trimethoxysilane.

5. The phase separation-mediated nanotraps for clearance of extracellular free DNA according to claim 1, wherein, The average particle size of the mesoporous nanoparticle core is 180-200 nm, and the average particle size of the nanotrap is 240-280 nm.

6. A method for preparing the phase separation-mediated nanotraps for clearing extracellular free DNA according to any one of claims 1-5, characterized in that, The method comprises the following steps: A first dispersion liquid containing the mesoporous nanoparticle core is prepared, a DNA degradation enzyme is added, and a reaction is performed; a precipitate is collected by centrifugation to obtain an enzyme-loaded intermediate; A second dispersion liquid containing the enzyme-loaded intermediate is prepared, a modifier is added, and ultrasonic treatment is performed to react; a precipitate is collected by centrifugation to obtain the nanotrap; and the modifier contains the material constituting the cationic layer.

7. The method of claim 6, wherein the phase separation-mediated clearance of extracellular free DNA nanotraps is prepared by, The mass ratio of the mesoporous nanoparticle core to the DNA degradation enzyme is 1:(1-10); Further, the mass ratio of the mesoporous nanoparticle core to the DNA degradation enzyme is 1:

10.

8. The method of claim 6, wherein the phase separation-mediated clearance of extracellular free DNA nanotraps is prepared by, The mass ratio of the enzyme-loaded intermediate to the material constituting the cationic layer in the modifier is 1:(1-8); Further, the mass ratio of the enzyme-loaded intermediate to the material constituting the cationic layer in the modifier is 1:

8.

9. Use of the phase separation mediated nanotraps for the removal of extracellular free DNA according to any one of claims 1 to 5, characterized in that, A medicament for preventing or treating systemic inflammatory response syndrome mediated by abnormally elevated cfDNA levels.

10. A composition characterized in that, The composition comprises the phase separation-mediated nanotrap for removing extracellular free DNA according to any one of claims 1-5, and the composition is used for preventing or treating systemic inflammatory response syndrome mediated by abnormally elevated cfDNA levels. Further, the composition further comprises a pharmaceutically acceptable carrier or excipient.

Images