A functional biomimetic nanomaterial and its preparation method and application

Abstract

The present invention discloses a biomimetic nanomaterial, its preparation method, and application. The biomimetic nanomaterial comprises a degradable two-dimensional nanomaterial, a biomimetic biocoating with anti-nonspecific adhesion function bound to the two-dimensional nanomaterial, and a dendritic cationic polymer end-modified with alkyne groups, which is loaded onto the biomimetic biocoating. The biomimetic nanomaterial has the advantages of good tissue compatibility, systemic intravenous administration, high biosafety, simple operation, low cost, minimal toxicity and side effects, and good material stability.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a functionalized biomimetic nanomaterial, its preparation method, and its application. Background Technology

[0002] Cell-free nucleic acids (DNA, mRNA, and miRNA, etc.) in the circulatory system (blood, urine, pleural effusion, and bronchoalveolar lavage fluid) serve as non-invasive markers for the diagnosis and prognostic assessment of various disease states, such as infection, inflammation, malignant tumors, hemodialysis, organ transplantation, and prenatal diagnosis. Increasing evidence suggests that the production of cell-free nucleic acids plays a crucial physiological and pathological role in inflammation-related and autoimmune-related diseases. These include: (1) circulating cell-free nucleic acids as diagnostic markers for tumors; (2) tumor-associated circulating cell-free nucleic acids promoting malignant metastasis of tumor lesions; (3) donor-specific cell-free nucleic acids used to monitor the survival status of recipient grafts; (4) virus- or bacterial-specific cell-free nucleic acids used to monitor infection-related pathology or detect pathogens in sepsis; and (5) fetal-derived cell-free nucleic acids used for prenatal assessment of fetal aneuploidy risk.

[0003] Monitoring circulating cell-free nucleic acids (CFNAs) is an important indicator for disease diagnosis and prognosis. However, CFNAs are also a contributing factor to the development and progression of various diseases, including: (1) Circulating CFNAs can act as an immunostimulant, activating Toll-like receptors (TLRs) and thus the immune system; (2) Circulating CFNAs can promote the growth and metastasis of tumor cells; (3) High levels of CFNAs in the body after trauma exacerbate tissue damage and the release of inflammatory factors; and (4) Circulating CFNAs are also a warning signal for the development of various autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Therefore, neutralizing or clearing circulatory CFNAs can regulate the body's immune response, inhibit the production of inflammatory factors, and mitigate the development and progression of various diseases.

[0004] Currently, methods for neutralizing or clearing free nucleic acids in the circulatory system mainly utilize the electrostatic interaction between negatively charged nucleic acids and positively charged polymers. Since polymers exhibit molecular weight and dose-dependent toxicity, modifying polymers onto the surface of biocompatible nanomaterials using biomimetic chemical methods can construct more effective and safer nanomaterials for neutralizing or clearing free nucleic acids. Summary of the Invention

[0005] The purpose of this invention is to address the problem of molecular weight and dose-dependent toxicity of polymers used in the prior art for neutralizing or clearing free nucleic acids, and to provide a functionalized biomimetic nanomaterial.

[0006] Another object of the present invention is to provide an application of the aforementioned functionalized biomimetic nanomaterial.

[0007] Another objective of this invention is to provide a method for preparing the aforementioned functionalized biomimetic nanomaterials.

[0008] The technical solution adopted to achieve the purpose of this invention is:

[0009] A functionalized biomimetic nanomaterial includes a biodegradable two-dimensional nanomaterial, a biomimetic bio-coating with anti-nonspecific adhesion function bonded to the two-dimensional nanomaterial, and a dendritic cationic polymer with end-modified acetyl groups loaded on the biomimetic bio-coating.

[0010] The biomimetic bio-coating is an antifouling hydrophilic polymer with end-modified amino, phenyl, catechol and azide groups. The amino, phenyl and catechol groups in the biomimetic bio-coating serve as anchoring regions and combine with the two-dimensional nanomaterial. The azide groups in the biomimetic bio-coating react and combine with the alkynyl groups modified at the end of the dendritic cationic polymer.

[0011] In the above technical solution, the two-dimensional nanomaterial is a two-dimensional transition metal chalcogenide, graphene and graphene derivatives, black phosphorus, boron nitride, two-dimensional transition metal carbide, two-dimensional transition metal nitride and bimetallic hydroxide, preferably molybdenum disulfide (MoS2), molybdenum diselenide (MoSe2), graphene oxide (GO), hexagonal boron nitride (hBN) and titanium carbide (TiC).

[0012] In the above technical solution, the antifouling hydrophilic polymer is polyglycidyl ether (PG), polyethylene glycol (PEG), poly-2-methacryloyloxyethyl phosphocholine, polybutyl methacrylate, polyhydroxyethyl methacrylate, poly-2-ethylhexyl methacrylate, polynorbornene, or gelatin.

[0013] In the above technical solution, the dendritic cationic polymer is polyethyleneimine (PEI), polyamide-amine (PAMAM), or polylysine (PLL). Preferably, the relative molecular mass of the dendritic cationic polymer is in the range of 200-80000 g / mol.

[0014] In the above technical solution, it is prepared by the following method:

[0015] Step 1: Two-dimensional nanomaterials are prepared from crystalline materials under the protection of a protective gas.

[0016] Step 2: Prepare the antifouling hydrophilic polymer P, modify the end of the antifouling hydrophilic polymer with azide groups to obtain the azide polymer N3-P, and then modify the azide polymer with phenyl, amino and catechol groups to obtain the biomimetic biological coating N3-P-CatPh.

[0017] Step 3: Modify the ends of the dendritic cationic polymer with alkynyl groups to obtain a dendritic cationic polymer with alkynyl groups at the ends.

[0018] Step 4: Mix the biomimetic biological coating and the two-dimensional nanomaterial. The amino, phenyl and catechol groups on the biomimetic biological coating serve as anchoring regions, allowing the biomimetic biological coating to be stably bonded to the surface of the two-dimensional nanomaterial, thus obtaining a coated two-dimensional nanomaterial.

[0019] The coated two-dimensional nanomaterials obtained above are mixed with the dendritic cationic polymer with end-modified alkynyl groups obtained in step 3, and a click chemical reaction is carried out to complete the surface dendritic cationic modification. After purification, the functionalized biomimetic nanomaterials are obtained.

[0020] Alternatively, in step 4, the two-dimensional nanomaterial, the biomimetic coating, and the dendritic cationic polymer are mixed and purified to obtain functionalized biomimetic nanomaterials.

[0021] In the above technical solution, in step 1, the crystal material is prepared into two-dimensional nanomaterials by chemical exfoliation, and the protective gas is argon.

[0022] Preferably, the two-dimensional nanomaterial is prepared by the following method:

[0023] Step S1: Under argon protection, the crystal material and the n-hexane solution of n-butyllithium are mixed and refluxed at 25-80°C with stirring for 0.5-4.5 h.

[0024] After stirring in step S1, add n-hexane to the reaction system in step S2, centrifuge at 500-2500 rpm / min for 5-30 min, and discard the supernatant.

[0025] Step S3: Collect the precipitate from step S2 and disperse and wash it with n-hexane, then centrifuge again. Disperse the precipitate in ultrapure water and centrifuge at 500-2500 rpm / min for 5-30 min.

[0026] Step S4: Add the precipitate from step S3 into a dialysis bag and dialyze for 1-3 days to obtain the two-dimensional nanomaterial.

[0027] In the above technical solution, the biomimetic biological coating is prepared by the following method:

[0028] Step A1: Dissolve the hydrophilic polymer to obtain a hydrophilic polymer solution;

[0029] Step A2: Add sodium azide to the hydrophilic polymer solution obtained in step A1, heat to 25-80℃ and react for 0.5-4.5 hours to modify the azide group by substitution reaction to obtain a solution of azide polymer;

[0030] Step A3: Add benzoin dimethyl ether, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3,4-dihydroxyphenylpropionic acid and phenylpropionic acid to the solution of the azide polymer obtained in step A2, and stir at 25-80°C for 0.5-4.5 hours to further modify the phenyl, amino and catechol groups to obtain the biomimetic biological coating.

[0031] In the above technical solution, the terminally modified alkynyl dendritic cationic polymer is prepared by the following method:

[0032] Step B1: Dissolve the dendritic cationic polymer in a solvent to obtain a dendritic cationic polymer solution;

[0033] Step B2: Add DIPEA to the dendritic cationic polymer solution from Step B1, heat, and then add dibenzocyclooctyn-C6-active ester.

[0034] Step B3: After the reaction is complete, the product is purified and dried to obtain the dendritic cationic polymer with terminal alkynyl groups.

[0035] Another aspect of the present invention is the application of the functionalized biomimetic nanomaterials in the preparation of drugs containing specific neutralizing and circulating free nucleic acids.

[0036] Another aspect of the present invention provides a method for preparing functionalized biomimetic nanomaterials, comprising the following steps:

[0037] Step C1: Two-dimensional nanomaterials are prepared from crystalline materials under the protection of a protective gas.

[0038] Step C2: Prepare a hydrophilic polymer, modify the end of the hydrophilic polymer with an azide group to obtain an azide polymer, and then modify the azide polymer with amino, phenyl and catechol groups to obtain a biomimetic biological coating.

[0039] Step C3: Modify the end of the dendritic cationic polymer with alkynyl groups to obtain a dendritic cationic polymer with alkynyl groups at the end.

[0040] Step C4: Mix the biomimetic biological coating and the two-dimensional nanomaterial. The amino, phenyl, and catechol groups on the biomimetic biological coating serve as anchoring regions, allowing the biomimetic biological coating to be stably bonded to the surface of the two-dimensional nanomaterial, thus obtaining a coated two-dimensional nanomaterial.

[0041] The coated two-dimensional nanomaterials obtained above are mixed with the dendritic cationic polymer with end-modified alkynyl groups obtained in step 4, and a click chemical reaction is carried out to complete the surface cationization modification. After purification, the functionalized biomimetic nanomaterials are obtained.

[0042] Alternatively, the two-dimensional nanomaterial, the biomimetic coating, and the dendritic cationic polymer can be mixed and purified to obtain functionalized biomimetic nanomaterials.

[0043] Compared with the prior art, the beneficial effects of the present invention are:

[0044] 1. In this invention, biodegradable two-dimensional nanomaterials are used as carriers, and antifouling polymers and cationic dendritic polymers are modified on the surface of the nanomaterials in a "layer-by-layer" manner. The biomimetic biological coating can avoid non-specific adsorption of nanomaterials to blood cells, proteins or other biomolecules in the blood. The biotechnology of modifying nanomaterials by coating method utilizes hydrophilic polymers to modify the surface of nanomaterials, so that the nanomaterials not only have excellent biocompatibility, but also reduce non-specific adsorption of substances other than the target substances in the blood, thereby enabling clinical intravenous drug delivery therapy.

[0045] 2. Dendritic cationic polymers are used for efficient neutralization of circulating free nucleic acids. The dendritic cationic polymers utilize the strategy of adsorbing circulating free nucleic acids by combining negatively charged nucleic acids with positively charged polymer materials. Different molecular weight dendritic small molecule cationic polymers can achieve different binding efficiencies, thereby screening out the dendritic small molecule cationic polymers with the best binding efficiency and the least toxic side effects.

[0046] 3. The biomimetic nanomaterials of the present invention have the advantages of good tissue compatibility, systemic intravenous administration, high biosafety, simple operation, low cost, low toxicity and side effects, and good material stability.

[0047] 4. The biomimetic nanomaterials of the present invention can be used to neutralize or clear circulating free nucleic acids in various trauma patients, circulating free nucleic acids in sepsis patients, circulating tumor free nucleic acids in cancer patients, donor-derived free nucleic acids in organ transplant recipients, or circulating free nucleic acids in patients with autoimmune diseases, and have broad clinical application prospects.

[0048] 5. The biomimetic nanomaterials of this invention can greatly improve the specific binding ability of nanomaterials to free nucleic acids, while reducing the adhesion ability of non-specific substances in the circulatory system. This material is highly efficient and sensitive, and has broad clinical application prospects. Attached Figure Description

[0049] Figure 1 This is a flowchart illustrating the preparation process of the biomimetic nanomaterials that specifically neutralize circulating free nucleic acids in this invention.

[0050] Figure 2 Morphological characterization of the binding of MoS2, MoS2-PG-PEI, and MoS2-PG-PEI nanomaterials with free DNA.

[0051] Figure 3 This is a particle size distribution diagram of MoS2-PG-PEI nanomaterials.

[0052] Figure 4 The NMR spectrum of the N3-PG-CatPh polymer bio-coating with anti-nonspecific adhesion function.

[0053] Figure 5 To verify the biosafety of MoS2-PG-PEI nanomaterials.

[0054] Figure 6 Verification of the binding ability of MoS2 sheet nanomaterials modified with dendritic cationic polymers (PEI) of different molecular weights to free DNA.

[0055] Figure 7 To investigate the inhibitory effect of MoS2-PG-PEI nanomaterials on TLR3 receptor activation.

[0056] Figure 8 To investigate the inhibitory effect of MoS2-PG-PEI nanomaterials on TLR9 receptor activation.

[0057] Figure 9 To reduce the level of free DNA in peripheral blood of mice after skin transplantation using MoS2-PG-PEI nanomaterials.

[0058] Figure 10 To alleviate acute rejection and prolong graft survival time using MoS2-PG-PEI nanomaterials.

[0059] Figure 11 The MoS2-PG-PEI nanomaterials reduce the number of innate immune cells in an inflammatory state by clearing circulating free DNA.

[0060] Figure 12 The flowchart shows the preparation process of N3-PG-CatPh. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0062] Example 1

[0063] A functionalized biomimetic nanomaterial that specifically neutralizes circulating free nucleic acids, such as Figure 1 As shown, it is prepared by the following method:

[0064] Step 1: Under argon protection, molybdenum disulfide (MoS2) lamellar molybdenum disulfide crystals are prepared by chemical exfoliation. The lamellar molybdenum disulfide is a two-dimensional nanomaterial with a large specific surface area.

[0065] Step 2: Polyethoxyethyl glycidyl ether-b-polyallyl glycidyl ether (PEEGE-b-PAGE) was prepared by anionic polymerization. Then, the PEEGE-b-PAGE was modified with azide groups through deacetal protection and substitution reactions to obtain a brown paste polymer (N3-PG-b-PAGE).

[0066] The N3-PG-b-PAGE was then modified with phenyl, amino, and catechol groups to obtain a biomimetic biological coating, N3-PG-CatPh. The N3-PG-CatPh coating was then analyzed using proton nuclear magnetic resonance spectroscopy. Figure 3 As shown.

[0067] Step 3: Modify the terminal alkynyl groups of PEI with different molecular weights to prepare PEI-DBCO.

[0068] Step 4: N3-PG-CatPh and MoS2 nanoparticles were mixed and stirred overnight at room temperature. PEI-DBCO was then added, and the mixture was stirred at room temperature for 1-12 hours. The mixture was washed again to obtain MoS2-PG-PEI nanomaterials modified with different amounts of PEI. The morphology of the MoS2-PG-PEI nanomaterials was characterized using transmission electron microscopy (TEM). Figure 2 As shown in Figure B, the diameter of the MoS2-PG-PEI nanomaterial was characterized using a dynamic light scattering (DLS) system. Figure 4 As shown. In this invention, transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the morphology and particle size of the nanomaterials. The results showed that the MoS2 nanosheets had a large surface area, uniform particle size, and good dispersion.

[0069] MoS2 is prepared by the following method:

[0070] Step S1: Under argon protection, a solution of n-butyllithium in n-hexane is added to a Schlenk flask containing MoS2 crystals, and the mixture is refluxed at 60°C with stirring for 48 hours.

[0071] After stirring in step S1, add n-hexane to the reaction system, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant.

[0072] Step S3: Collect the precipitate from step S2 and disperse and wash it with n-hexane, then centrifuge again. Disperse the precipitate in ultrapure water and centrifuge at 10000 rpm / min for 10 min.

[0073] Step S4: The precipitate from step S3 is added to a dialysis bag (molecular weight cutoff of 50 kDa) and dialyzed for 3 days to obtain the sheet-like MoS2, which is then set aside. The morphology of the MoS2 nanoparticles is characterized using transmission electron microscopy (TEM) as follows: Figure 2 As shown in Figure A.

[0074] like Figure 12 As shown, the N3-PG-CatPh is prepared by the following method:

[0075] Step A1: Polymer PEEGE-b-PAGE is prepared from ethoxyethyl glycidyl ether and allyl glycidyl ether by anionic ring-opening polymerization.

[0076] Step A2: Remove the acetal protecting group from the side chain of the polymer PEEGE-b-PAGE obtained in step A1 to generate hydroxyl groups, thus obtaining the polymer PG-b-PAGE.

[0077] Step A3: Add sodium azide to the polymer solution obtained in step A2, heat to 40°C and react for 72 hours to modify the azide group by substitution reaction to obtain polymer N3-PG-b-PAGE;

[0078] Step A4: Add benzoin dimethyl ether as a catalyst to the polymer N3-PG-b-PAGE solution obtained in step A3, and modify the amino group using the "thiol-ene" reaction to obtain polymer N3-PG-A;

[0079] Step A5: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (activator), 3,4-dihydroxyphenylpropionic acid and phenylpropionic acid to the polymer solution obtained in step A4, stir overnight at room temperature, and further modify the phenyl and catechol groups to obtain polymer N3-PG-CatPh.

[0080] The PEI-DBCO is prepared by the following method:

[0081] Step B1: Add DMF to a round-bottom flask containing PEI with different molecular weights (Mw = 0.8k, 2k, 5k, 15k and 25k) and stir until the polymer is dissolved.

[0082] Step B2: Add DIPEA to the mixture from step B1, heat to 37°C, and then add dibenzocyclooctyn-C6-active ester (DBCO-C6-NHS).

[0083] Step B3: After the reaction in step B2 is completed, the PEI-DBCO polymer is obtained, purified by dialyzing in methanol, and dried under high vacuum for later use.

[0084] The structural formula of PEI-DBCO is as follows:

[0085]

[0086] Example 2

[0087] Characterization and biological properties of the MoS2-PG-PEI nanomaterials obtained in Example 1

[0088] The specific implementation method for evaluating the biocompatibility of the nanomaterial MoS2-PG-PEI is as follows:

[0089] Different doses (5, 15, 30, 60, 120, 240, and 480 μg / mL) of PEI with different molecular weights (Mw = 0.8k, 2k, 5k, 15k, and 25k) functionalized nanomaterials MoS2-PG-PEI and cationic polymers were co-incubated with 4T1 mouse breast cancer cells for 24 h. Cell viability was detected by CCK-8 assay, and the IC50 value of cell growth inhibition and its effect on apoptosis rate were calculated to investigate the cytotoxicity of MoS2 sheet nanomaterials modified with PEI of different molecular weights.

[0090] like Figure 5 As shown, the biosafety assessment results indicate that low molecular weight PEI polymers exhibit lower cytotoxicity; MoS2 nanomaterials have low toxicity and can serve as a carrier for PEI polymers to improve biosafety; the results demonstrate that MoS2-PG-PEI... 800 PEI has relatively low cytotoxicity and high biosafety. 800 It can be used for subsequent research.

[0091] The specific implementation method for evaluating the ability of MoS2-PG-PEI nanomaterials to scavenge free DNA is as follows:

[0092] In 96-well plates, MoS2 nanomaterials modified with different proportions and molecular weights of PEI (Mw = 0.8k, 2k, 5k, 15k, and 25k) were mixed with denatured salmon sperm DNA solutions (1:10, 1:5, 1:1, 2:1, 5:1, and 10:1). After incubation for 30 min, the mixture was centrifuged and treated with Quant-iT PicoGreen. TM A dsDNA kit was used to detect the content of free DNA in the supernatant. The binding affinity of PEI-modified MoS2 nanomaterials of different molecular weights to free DNA was also determined.

[0093] like Figure 6 As shown, the evaluation results of the binding ability of PEI-modified MoS2 nanomaterials to free DNA indicate that the higher the molecular weight of PEI, the higher the binding rate of free DNA. PEI-modified MoS2 nanomaterials have similar free DNA binding rates, suggesting that this polymer-modified nanomaterial can be used for the development of novel free DNA scavengers.

[0094] The morphology of MoS2-PG-PEI nanomaterials bound to free DNA was characterized using transmission electron microscopy (TEM). Figure 2 As shown in C.

[0095] Example 3

[0096] The study investigated the impact of the MoS2-PG-PEI nanomaterials obtained in Example 1 on the immune microenvironment. The specific implementation method for the evaluation is as follows:

[0097] Evaluation of the ability of MoS2-PG-PEI nanomaterials to inhibit the activation of TLR3 receptor by free nucleic acids:

[0098] In a 96-well plate, Poly(I:C) was added to HEK-Blue. TM TLR3 reporter cells were then incubated with MoS2-PG-PEI nanomaterials for 24 hours, and the supernatant was collected and mixed with QUANTI-Blue. TM The reagents were incubated, and the absorbance at 620 nm was detected using an ELISA reader. The alkaline phosphatase activity in the supernatant was measured to evaluate whether the TLR3 receptor was activated.

[0099] like Figure 7 As shown, the activation results of the TLR3 receptor indicate that Poly(I:C) activates HEK-Blue. TM TLR3 reports TLR3 in cells, while MoS2-PG-PEI nanomaterials inhibit this activation process.

[0100] Evaluation of the ability of MoS2-PG-PEI nanomaterials to inhibit the activation of TLR9 receptor by free nucleic acids:

[0101] In a 96-well plate, CpG 1826 was added to HEK-Blue. TM TLR9 reporter cells were then incubated with PEI-modified MoS2 nanomaterials for 24 hours, and the supernatant was collected along with QUANTI-Blue. TM The reagents were incubated, and the absorbance at 620 nm was detected using an ELISA reader. The alkaline phosphatase activity in the supernatant was measured to evaluate whether the TLR9 receptor was activated.

[0102] like Figure 8 As shown, the activation results of the TLR9 receptor indicate that CpG 1826 activates HEK-Blue. TM TLR9 reports the TLR9 receptor in cells, while MoS2-PG-PEI nanomaterials inhibit this activation process.

[0103] Example 4

[0104] The therapeutic effect of the MoS2-PG-PEI nanomaterial obtained in Example 1 on allogeneic mouse skin transplant rejection.

[0105] This embodiment provides an experiment demonstrating how the nanomaterial alleviates rejection by neutralizing free nucleic acids in an allogeneic mouse skin transplant rejection model:

[0106] The specific neutralizing effect of MoS2-PG-PEI nanomaterials on free nucleic acids was evaluated using a tail-back mouse skin transplant rejection model. Tail skin from BALA / c mice was transplanted to the back of C57BL / 6 mice, with the day of transplantation designated as day 0. MoS2-PG-PEI was injected once daily via tail vein into the treatment group from day 0 to day 7 at a dose of 5 mg / kg. The control group received the same dose of PBS buffer using the same method.

[0107] Mouse skin grafts were observed for two weeks post-surgery, with the following indicators: ① Blood supply: Red and soft transplanted skin indicates good subcutaneous blood circulation and graft survival; blackening and hardening of the graft indicates rejection and graft death. ② Hair growth: New, bright-colored hair growth indicates graft survival; lack of new hair growth or loss of luster indicates rejection and graft death. The endpoint of graft rejection was determined when over 90% of the grafts died and detached; observation was then discontinued.

[0108] Four weeks after skin transplantation, mice were euthanized by cervical dislocation. Peripheral blood was collected using the orbital blood sampling method. The blood was centrifuged at 3000 rpm / min for 15 min at 4°C, and the supernatant serum was collected.

[0109] Cell-free DNA was extracted from mouse peripheral serum using the QIAamp DNA Blood Mini Kit.

[0110] Utilizing Quant-iT PicoGreen TM The dsDNA kit was used to detect the level of cell-free DNA in the peripheral serum of mice in the Control and Treatment groups.

[0111] like Figure 9 As shown, the detection results indicate that, compared with the Control group, the MoS2-PG-PEI nanomaterial treatment group can significantly reduce the level of free DNA in peripheral blood of mice after skin transplantation.

[0112] like Figure 10 As shown in the photographic record of skin grafts, the circulating free DNA adsorbent material MoS2-PG-PEI can alleviate acute rejection and prolong graft survival time compared to the control group.

[0113] Four weeks after skin transplantation, mice were euthanized by cervical dislocation, and the spleen, an immune organ, was dissected. Flow cytometry was used to detect changes in immune cell subsets in the mouse spleen.

[0114] Changes in macrophages (F4 / 80+CD11c+) and dendritic cells (CD11b+CD11c+) in mouse spleen were detected using flow cytometry.

[0115] like Figure 11 As shown, flow cytometry results indicate that, compared with the Control group, the MoS2-PG-PEI nanomaterial treatment group significantly reduced macrophages and dendritic cells in the spleen during skin transplant rejection in mice, suggesting that the clearance of circulating free DNA by MoS2-PG-PEI nanomaterials can reduce innate immune cells in an inflammatory state.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made 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. A functionalized biomimetic nanomaterial, characterized in that, The invention includes biodegradable two-dimensional nanomaterials, a biomimetic bio-coating with anti-nonspecific adhesion function bonded to the two-dimensional nanomaterials, and a dendritic cationic polymer with end-modified acetyl groups loaded on the biomimetic bio-coating. The biomimetic bio-coating is an antifouling hydrophilic polymer with phenyl, amino, catechol and azide groups modified at the ends. The phenyl, amino and catechol groups in the biomimetic bio-coating serve as anchoring regions and combine with the two-dimensional nanomaterial. The azide groups in the biomimetic bio-coating react and combine with the alkyne groups modified at the ends of the dendritic cationic polymer. The functionalized biomimetic nanomaterials are prepared by the following method: Step 1: Under argon protection, lamellar molybdenum disulfide (MoS2) is prepared from molybdenum disulfide crystals by chemical exfoliation. The specific steps are as follows: Step S1: Under argon protection, add a hexane solution of n-butyllithium to a Schlenk flask containing MoS2 crystals, and reflux and stir the mixture at 60°C for 48 h. After stirring in step S1, add n-hexane to the reaction system, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant. Step S3: Collect the precipitate from step S2 and disperse and wash it with n-hexane, then centrifuge again. Disperse the precipitate in ultrapure water and centrifuge at 10,000 rpm for 10 min. Step S4: Add the precipitate from step S3 into a dialysis bag and dialyze for 3 days. The molecular weight cutoff of the dialysis bag is 50 kDa, and the sheet MoS2 is obtained for later use. Step 2: Polyethoxyethyl glycidyl ether-b-polyallyl glycidyl ether (PEEGE-b-PAGE) was prepared by anionic polymerization. Then, the PEEGE-b-PAGE was deacetalized and modified with azide groups via substitution to obtain a brown paste polymer, N3-PG-b-PAGE. The specific steps are as follows: Step A1: Polymer PEEGE-b-PAGE is prepared from ethoxyethyl glycidyl ether and allyl glycidyl ether by anionic ring-opening polymerization. Step A2: Remove the acetal protecting group from the side chain of the polymer PEEGE-b-PAGE obtained in step A1 to generate hydroxyl groups, thus obtaining the polymer PG-b-PAGE. Step A3: Add sodium azide to the polymer solution obtained in step A2, heat to 40°C and react for 72 hours to modify the azide groups by substitution reaction to obtain polymer N3-PG-b-PAGE; Step A4: Add benzoin dimethyl ether as a catalyst to the polymer N3-PG-b-PAGE solution obtained in step A3, and modify the amino group using the "thiol-ene" reaction to obtain polymer N3-PG-A; Step A5: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3,4-dihydroxyphenylpropionic acid and phenylpropionic acid to the polymer solution obtained in step A4, stir overnight at room temperature, and further modify the phenyl and catechol groups to obtain polymer N3-PG-CatPh. Step 3: Modify the ends of PEI molecules with different molecular weights to obtain PEI-DBCO. The specific steps are as follows: Step B1: Add DMF to a round-bottom flask containing PEI with different molecular weights and stir until the polymer dissolves. The molecular weight of the PEI is 0.8k, 2k, 5k, 15k or 25k. Step B2: Add DIPEA to Step B1, heat to 37°C, and then add dibenzocyclooctyn-C6-active ester DBCO-C6-NHS; Step B3: After the reaction in step B2 is completed, the PEI-DBCO polymer is obtained, purified by dialysis in methanol, and dried under high vacuum for later use. Step 4: Mix N3-PG-CatPh and MoS2 nanoparticles, stir overnight at room temperature, add PEI-DBCO, stir at room temperature for 1-12 hours, and wash again to obtain MoS2-PG-PEI nanomaterials modified with different amounts of PEI.

2. The use of the functionalized biomimetic nanomaterial as described in claim 1 in the preparation of a drug for inhibiting skin graft rejection.

Images