Nano material for inhibiting aseptic inflammation as well as preparation method and application of nano material

Abstract

The invention discloses a nano material for inhibiting aseptic inflammation as well as a preparation method and application of the nano material. The nano material is hybrid liposome nano vesicles modified by a platelet membrane, and the mass ratio of protein to lipid is 1: 2. The nano vesicles are loaded with sylvite compounds, can target in-vivo tissue injury parts, release potassium ions under the action of ultrasound, realize intracellular pH regulation and control through hydrogen-potassium exchange, and inhibit aseptic inflammation. The hybrid liposome is formed by mixing phospholipid and cholesterol, and the potassium salt compound is potassium carbonate or potassium bicarbonate. The preparation method comprises the following steps: preparing a hybrid liposome membrane and a platelet membrane, respectively adding a sylvite compound solution to carry out ultrasonic hydration and resuspension washing, preparing hybrid liposome vesicles and platelet membrane vesicles entrapped with sylvite compounds, and carrying out ultrasonic treatment, size stabilization and dialysis to obtain the platelet membrane modified hybrid liposome nano-vesicles. Inflammation can be inhibited in the early stage of injury, and side effects of traditional drugs are avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical configuration bodies, and relates to materials for treating inflammation after tissue damage, and specifically to a nanomaterial for inhibiting aseptic inflammation, and a preparation method and application thereof. Background Art

[0002] In severely damaged tissues, the chronic accumulation of inflammatory macrophages can significantly delay tissue repair. Therefore, regulating the activity of inflammatory macrophages and terminating the proinflammatory response at critical moments is crucial for promoting effective repair of severe tissue damage.

[0003] Potassium ions play a central role in maintaining biological activities, regulating cell membrane potential and maintaining the acid-base balance of organisms. Most metabolic processes of organisms are directly or indirectly affected by potassium ions. + -K + -ATPase; NKA) and sodium hydrogen exchanger (NHE) mediated potassium ion (K + ) and hydrogen ions (H + ) are constantly exchanged between the intracellular and extracellular spaces, maintaining cellular metabolism and biological function. Potassium ions surrounding immune cells can regulate the pH within these cells and activate the AMPK / Nrf2 signaling pathway, thereby suppressing inflammation. Therefore, potassium ion concentration may influence the transformation and polarization of immune cells. During tissue injury, potassium ion homeostasis is easily disrupted, causing immune dysregulation, leading to localized excessive inflammation, and hindering tissue repair. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention proposes a nanomaterial for inhibiting aseptic inflammation and its preparation method and application, based on the K + The trend characteristics of the concentrations suggest a K + -H + Coupling strategy to modulate K through nanomaterials to inhibit sterile inflammation + concentration, while suppressing early excessive inflammation and promoting repair, it avoids drug damage to other systems in the body.

[0005] A nanomaterial for inhibiting aseptic inflammation is a nanovesicle formed by encapsulating a potassium salt compound in a hybrid liposome membrane modified with a platelet membrane. The nanovesicle inhibits aseptic inflammation by targeting tissue damage sites in the body.

[0006] Preferably, the nanovesicles include nanoplatelet membranes and nanoliposomes.

[0007] Preferably, the average particle size of the nanomaterial is 50 to 200 nanometers.

[0008] Preferably, the potassium salt compound is potassium carbonate or potassium bicarbonate.

[0009] Preferably, the hybrid liposome membrane is a liposome membrane hybridized with phospholipids and cholesterol.

[0010] Preferably, the phospholipid is a mixture of one or more of natural phospholipids, semi-synthetic phospholipids and fully synthetic phospholipids.

[0011] Preferably, the phospholipid is a mixture of one or more of lecithin, hydrogenated lecithin and cephalin.

[0012] A method for preparing a nanomaterial for inhibiting aseptic inflammation comprises the following steps:

[0013] Step 1: Dissolve phospholipids and cholesterol in an organic solvent to prepare a hybrid liposome membrane.

[0014] Step 2: adding the hybrid liposome membrane to the potassium salt compound solution, and obtaining an emulsion after ultrasonic hydration, which is the hybrid liposome vesicle encapsulating the potassium salt compound.

[0015] Step 3: prepare platelet membranes, resuspend and wash them with a potassium salt compound solution to obtain platelet membrane vesicles encapsulating potassium salt compounds.

[0016] Step 4: The hybrid liposome vesicles are mixed with platelet membrane vesicles, and after ultrasonic disruption, the granules are shaped and dialyzed to obtain platelet membrane-modified hybrid liposome nanovesicles.

[0017] Preferably, the organic solvent is one or more of an alcohol solvent, an ester solvent, a halogenated hydrocarbon, a nitrile solvent, and an ether solvent.

[0018] Preferably, the organic solvent is one or more of methanol, ethanol, methyl acetate, ethyl acetate, dichloromethane, chloroform, acetonitrile, and diethyl ether.

[0019] Preferably, the mass ratio of the platelet membrane, phospholipid and cholesterol is (80-120):100:(10-25).

[0020] Preferably, the mass ratio of protein to lipid in the mixed hybrid liposome vesicles and platelet membrane vesicles is 1:2.

[0021] A method for applying a nanomaterial for inhibiting aseptic inflammation comprises administering the nanomaterial as an intravenous drug, targeting a tissue injury site, and releasing potassium ions under the action of ultrasound to inhibit aseptic inflammation.

[0022] The present invention has the following beneficial effects:

[0023] 1. Hybrid liposome vesicles are modified with platelet membranes, enabling the nanomaterials to precisely target sites of tissue damage.

[0024] 2. The nanomaterial regulates the potassium ion concentration in the microenvironment by releasing potassium ions in a targeted manner to the tissue damage area. + -H + Coupled immunomodulatory therapy regulates the intracellular pH through the physiological process of "potassium hydrogen exchange", inhibits inflammation in the early stages of injury, avoids the side effects of traditional drugs, and provides more ideas for the development of tissue damage repair drugs, which has broad significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a confocal fluorescence image of KHCO3@PLV prepared in Example 1.

[0026] Figure 2 These are cryo-electron microscopy images of the three nanovesicle materials in Test Example 1.

[0027] Figure 3 This is the electrophoresis and Coomassie Brilliant Blue staining image in Test Example 1.

[0028] Figure 4 This is the Fourier transform infrared spectrum of KHCO3@PLV in test example 1.

[0029] Figure 5 is the FRET fluorescence intensity of the three nanovesicle materials in Test Example 1.

[0030] Figure 6 The particle size changes of KHCO3@PLV before and after ultrasound in Test Example 1.

[0031] Figure 7 This is the in vivo imaging and statistical results in Test Example 2.

[0032] Figure 8 This is the change in local potassium ion concentration after ultrasonic release of KHCO3@PLV in test example 2.

[0033] Figure 9 This is the flow cytometry result in Example 2.

[0034] Figure 10 Micro-CT bone morphology statistics of different nanovesicle materials used to treat critical fracture healing in Test Example 2. DETAILED DESCRIPTION

[0035] The present invention will be further explained below with reference to the accompanying drawings. It should be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make appropriate selections based on the description herein, and are not necessarily limited to the specific values ​​​​in the following examples.

[0036] Example 1

[0037] This embodiment provides a method for preparing a nanomaterial for inhibiting aseptic inflammation. The method uses a liposome membrane hybridized with platelet membrane-modified lecithin and cholesterol, and then encapsulates potassium bicarbonate to prepare nanovesicles. The specific steps are as follows:

[0038] Step 1: Weigh 100 mg of lecithin and 16 mg of cholesterol and dissolve them in 10 mL of chloroform. Place the solution in a round-bottom flask and dry it in a rotary evaporator at 37° C. to prepare a hybrid liposome membrane.

[0039] Step 2: Add 10 mL of 150 mM potassium bicarbonate solution into the round-bottom flask and ultrasonically hydrate to obtain an emulsion, which is the hybrid liposome vesicles encapsulating potassium bicarbonate.

[0040] Step 3, with 100G, 5min, DEC5 deceleration centrifugation of platelet-rich plasma, after taking the supernatant, and then with 800G, 20min, DEC5 deceleration centrifugation, take the precipitate, and obtain platelets. The platelet suspension was frozen at -80 ° C, thawed at room temperature, and then centrifuged at 4000g for 3 minutes to obtain a precipitate. 10mg / ml of pH-responsive fluorescent agent fluorescein isothiocyanate (FITC) was added to a 150mM potassium bicarbonate solution. The precipitate was resuspended and washed with 10mL of potassium bicarbonate. After repeated three times, platelet membrane nanovesicles (KHCO3@PMV) loaded with potassium bicarbonate were obtained and stained with lipid affinity dye DiD.

[0041] Step 4: Hybrid liposome vesicles were mixed with platelet membrane nanovesicles at a lipid-to-protein ratio of 2:1. The vesicles were then ultrasonicated for 10–20 minutes to disrupt and fuse the vesicles. The vesicles were then repeatedly extruded using a 100 nm extruder. Finally, the mixture was dialyzed against 0.9% saline at 4°C in the dark for 24–48 hours to obtain platelet membrane-modified, potassium bicarbonate-loaded hybrid liposome nanovesicles (KHCO3@PLV).

[0042] The obtained nanovesicles were observed under a confocal fluorescence microscope. Figure 1 As shown, the scale bar is 1 μm, and it can be seen that the platelet membrane-modified hybrid liposome nanovesicles encapsulate the alkaline potassium bicarbonate solution.

[0043] Comparative Example 1

[0044] This comparative example provides a method for preparing potassium bicarbonate-loaded lecithin and cholesterol hybrid liposome nanovesicles, the specific steps of which are as follows:

[0045] Step 1: Weigh 100 mg of lecithin and 16 mg of cholesterol and dissolve them in 10 mL of chloroform. Place the solution in a round-bottom flask and dry it in a rotary evaporator at 37° C. to prepare a hybrid liposome membrane.

[0046] Step 2: Add 10 mL of 150 mM potassium bicarbonate solution into the round-bottom flask and ultrasonically hydrate to obtain an emulsion, which is the hybrid liposome vesicles encapsulating potassium bicarbonate.

[0047] Step 3: Repeated extrusion was performed using a 100 nm extruder, and finally dialyzed with 0.9% saline in the dark at 4° C. for 24 to 48 h to obtain hybrid liposome nanovesicles (KHCO 3 @LNP) loaded with potassium bicarbonate.

[0048] Test Example 1

[0049] This test example tests the physicochemical properties of platelet membrane nanovesicles (KHCO3@PMV), platelet membrane modified hybrid liposome nanovesicles (KHCO3@PLV), and hybrid liposome nanovesicles (KHCO3@LNP) prepared in Example 1 and Comparative Example 1:

[0050] Step 1: After diluting the three nanovesicle samples to appropriate concentrations, the morphology of the nanovesicles was observed using a 200 kV cryo-transmission electron microscope. Figure 2 As shown, the scale bar is 100 nm.

[0051] Step 2: SDS-PAGE electrophoresis was performed on the platelet and three nanovesicle samples, and Coomassis Brilliant Blue staining was performed respectively. The results are as follows: Figure 3 As shown, platelet membrane proteins can be observed in the platelet membrane modified hybrid liposome nanovesicles (KHCO3@PLV), confirming that the platelet membrane has successfully modified the hybrid liposomes.

[0052] Step 3: Detect the three nanovesicle samples by Fourier transform infrared spectroscopy. Figure 4 As shown, the platelet membrane modified hybrid liposome nanovesicles (KHCO3@PLV) have characteristic peaks such as C=O and protein-lipid.

[0053] Step 4: The hybrid liposome nanovesicles (KHCO3@LNP) were stained with the lipid affinity dye DiI, and the platelet membrane nanovesicles (KHCO3@PMV) stained with DiD were crushed and mixed. After extrusion, the fluorescence spectra of the three nanovesicles were detected under a fluorescence spectrometer, as shown in FIG. Figure 5 As shown, the platelet-modified hybrid nanoliposome vesicles (KHCO3@PLV) exhibited an obvious fluorescence resonance shift, proving that the platelet membrane was successfully modified on the liposomes.

[0054] Step 5: Use nanoparticle size potential analyzer to measure the particle size of platelet membrane modified hybrid liposome nanovesicles (KHCO3@PLV), recorded as Control, after the sound intensity is 2.5W / cm 2 After 5 minutes of ultrasonic treatment, the vesicle size was measured again and recorded as Ultrasound. Figure 6 As shown, it can be seen that the vesicles are broken under the action of ultrasound.

[0055] Test Example 2

[0056] In this test example, a femoral bone defect model was established in 10-week-old C57BL / 6 mice to test the targeting and release properties of the platelet membrane-modified hybrid liposome nanovesicles (KHCO3@PLV) and hybrid liposome nanovesicles (KHCO3@LNP) prepared in Example 1 and Comparative Example 1 at the tissue defect site:

[0057] Step 1: In vivo imaging and tracing

[0058] During the ultrasonic hydration process of the hybrid liposome membranes of Example 1 and Comparative Example 1, 0.5 mg / mL indocyanine green (ICG) was added for in vivo tracing, and the obtained platelet membrane-modified hybrid liposome nanovesicles (KHCO3@PLV) and hybrid liposome nanovesicles (KHCO3@LNP) were respectively recorded as ICG@PLV and ICG@LNP. ICG@PLV and ICG@LNP were respectively injected into the bone defect model of C57BL / 6 mice via the tail vein at a dose of 10 mL / kg. The distribution of ICG@PLV and ICG@LNP in the mice was observed by IVIS in vivo imaging at 30 minutes, 3 hours, and 6 hours, as shown in FIG. Figure 7 As shown in the figure, it can be seen that platelet membrane-modified hybrid liposome nanovesicles (KHCO3@PLV) can be effectively enriched in the bone defect site.

[0059] Step 2: In vivo release experiment

[0060] Eight hours after surgery, platelet membrane-modified hybrid liposome nanovesicles (KHCO3@PLV) were injected into the tail vein of C57BL / 6 mice with a dose of 10 mL / kg. Five minutes after the injection, an ultrasonic coupling agent was applied to the defect site and an ultrasonic therapeutic device was used at 2.5 W / cm 2 Ultrasound was performed for 5 minutes, and then at 2.5 W / cm 2 Ultrasound was performed for 5 minutes at a high intensity, which was recorded as the KHCO3@PLV+Ultrasound group. The control group was injected with platelet membrane-modified hybrid liposome nanovesicles (KHCO3@PLV) at a dose of 10 mL / kg without ultrasonic treatment, which was recorded as KHCO3@PLV. The hind limbs on the modeling side were removed every 1 hour to obtain interstitial fluid and measure the potassium ion concentration of the interstitial fluid. Figure 8 As shown, it can be seen that the potassium ion concentration in the KHCO3@PLV+Ultrasound group increased significantly after ultrasound.

[0061] Comparative Example 2

[0062] In this comparative example, based on Example 1, a platelet membrane-modified hybrid liposome nanovesicle loaded with sodium bicarbonate was prepared. The specific steps are as follows:

[0063] Step 1: Weigh 100 mg of lecithin and 16 mg of cholesterol and dissolve them in 10 mL of chloroform. Place the solution in a round-bottom flask and dry it in a rotary evaporator at 37° C. to prepare a hybrid liposome membrane.

[0064] Step 2: Add 10 mL of 150 mM sodium bicarbonate solution into the round-bottom flask and ultrasonically hydrate to obtain an emulsion, which is the hybrid liposome vesicles encapsulating potassium bicarbonate.

[0065] Step 3: Centrifuge the platelet-rich plasma at 100g for 5 minutes at a deceleration rate of 5°C. The supernatant is then centrifuged again at 800g for 20 minutes at a deceleration rate of 5°C to obtain the precipitate, thereby obtaining platelets. The platelet suspension is frozen at -80°C, thawed at room temperature, and then centrifuged at 4000g for 3 minutes. The precipitate is then resuspended and washed with 10 mL of 150 mM sodium bicarbonate. This is repeated three times to obtain potassium bicarbonate-loaded platelet membrane vesicles.

[0066] Step 4: Hybrid liposome vesicles were mixed with platelet membrane vesicles at a lipid-to-protein ratio of 2:1. The vesicles were then sonicated for 10–20 minutes to disrupt and fuse the vesicles. The vesicles were then repeatedly extruded using a 100 nm extruder. Finally, the vesicles were dialyzed against 0.9% saline at 4°C in the dark for 24–48 hours to obtain platelet membrane-modified, sodium bicarbonate-loaded hybrid liposome nanovesicles (NaHCO3@PLV).

[0067] Comparative Example 3

[0068] In this comparative example, based on Example 1, a hybrid liposome nanovesicle (NaCl3@PLV) modified with platelet membrane and loaded with sodium chloride was prepared.

[0069] Example 2

[0070] This embodiment provides an application method of a nanomaterial for inhibiting aseptic inflammation. The nanomaterial prepared in Example 1 was injected into mice with femoral bone defects via the tail vein to inhibit inflammation and promote repair at the injury site. The nanomaterial was compared with NaHCO3@PLV and NaCl3@PLV prepared in Comparative Examples 2 and 3 to verify the inhibitory effect of platelet membrane-modified hybrid liposome nanovesicles (KHCO3@PLV) on aseptic inflammation at the injury site and the promotion of injury repair.

[0071] Step 1: Verification of the inhibitory effect on critical bone defect inflammation

[0072] Femoral bone defect models were established in 10-week-old C57BL / 6 mice. On the third day after surgery, a blank control group was set up, denoted as Control. A treatment group was set up, and KHCO3@PLV, NaHCO3@PLV, and NaCl3@PLV prepared in Example 1 and Comparative Examples 2 and 3 were injected through the tail vein at a dose of 10 mL / kg, and 5 minutes after injection, the KHCO3@PLV, NaHCO3@PLV, and NaCl3@PLV were injected through the tail vein at a dose of 2.5 W / cm 2 Ultrasonic intensity was 5 minutes, and after 3 hours, it was 2.5W / cm 2 The mice were sacrificed 6 hours after injection, and tissue samples were collected and stained with CD11b, Ly6C, and Ly6G antibodies for flow cytometry. Figure 9 As shown in the figure, it can be seen that KHCO3@PLV has a significant inhibitory effect on local CD11b, Ly6G-positive and Ly6C-highly expressed monocytes in the tissue.

[0073] Step 2: Verification of critical bone defect healing effect

[0074] Femoral bone defect models were established in 10-week-old C57BL / 6 mice. On the third day after surgery, a blank control group was set up, denoted as Control. A treatment group was set up, and KHCO3@PLV, NaHCO3@PLV, and NaCl3@PLV prepared in Example 1 and Comparative Examples 2 and 3 were injected through the tail vein at a dose of 10 mL / kg, and 5 minutes after injection, the KHCO3@PLV, NaHCO3@PLV, and NaCl3@PLV were injected through the tail vein at a dose of 2.5 W / cm 2 Ultrasonic intensity was 5 minutes, and after 3 hours, it was 2.5W / cm 2 Ultrasonication for 5 minutes.

[0075] Mice were sacrificed 14 days after injection, tissue samples were collected, and femurs were scanned by Micro-CT and decalcified for immunohistochemistry. Figure 10 As shown, it can be seen that KHCO3@PLV has a significant promoting effect on the repair of critical bone defects.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention and do not deviate from the scope defined by the claims of the present invention.

Claims

1. A nanomaterial for inhibiting aseptic inflammation, characterized in that: The nano material is a hybrid liposome nanovesicle modified with a platelet membrane and loaded with potassium salt compounds.

2. The nanomaterial for inhibiting aseptic inflammation according to claim 1, characterized in that: The nanovesicles are mixed platelet membranes and hybrid liposome membranes, with a mass ratio of protein to lipid of 1:

2.

3. The nanomaterial for inhibiting aseptic inflammation according to claim 2, characterized in that: The hybrid liposome membrane is a liposome membrane hybridized with phospholipid and cholesterol.

4. The method for preparing a nanomaterial for inhibiting aseptic inflammation according to claim 3, characterized in that: The mass ratio of the platelet membrane, phospholipid and cholesterol is (80-120):100:(10-25).

5. The nanomaterial for inhibiting aseptic inflammation according to claim 3, characterized in that: The phospholipid is a mixture of one or more of natural phospholipids, semi-synthetic phospholipids and fully synthetic phospholipids.

6. The nanomaterial for inhibiting aseptic inflammation according to claim 1, characterized in that: The potassium salt compound is potassium carbonate or potassium bicarbonate.

7. A method for preparing a nanomaterial for inhibiting aseptic inflammation according to claims 1 to 6, characterized in that: The specific steps include: Step 1: dissolving phospholipids and cholesterol in an organic solvent to prepare a hybrid liposome membrane; Step 2: adding the hybrid liposome membrane to the potassium salt compound solution, and ultrasonically hydrating to obtain an emulsion, which is the hybrid liposome vesicles encapsulating the potassium salt compound; Step 3: preparing platelet membranes, resuspending and washing them with a potassium salt compound solution to obtain platelet membrane vesicles encapsulating the potassium salt compound; Step 4: The hybrid liposome vesicles are mixed with platelet membrane vesicles, and after ultrasonic disruption, the granules are shaped and dialyzed to obtain platelet membrane-modified hybrid liposome nanovesicles.

8. The method for preparing a nanomaterial for inhibiting aseptic inflammation according to claim 7, characterized in that: The organic solvent is one or more of an alcohol solvent, an ester solvent, a halogenated hydrocarbon, a nitrile solvent, and an ether solvent.

9. The method for preparing a nanomaterial for inhibiting aseptic inflammation according to claim 7, characterized in that: Preferably, the organic solvent is one or more of methanol, ethanol, methyl acetate, ethyl acetate, dichloromethane, chloroform, acetonitrile, and diethyl ether.

10. The method for using a nanomaterial for inhibiting aseptic inflammation according to claims 1 to 6, characterized in that: The nanomaterial is used as an intravenous drug to target the tissue damage site, and then releases potassium ions under the action of ultrasound to inhibit aseptic inflammation.

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