Polymer nano-carrier as well as preparation method and application thereof

Abstract

The invention relates to a polymer nano-carrier as well as a preparation method and application thereof. The preparation method of the polymer nano-carrier comprises the following step: sequentially reacting polyglutamic acid or a derivative thereof with a compound with a hydrophobic group, a compound with a hydrophilic group and a compound with an oxidative stress group to obtain the polymer nano-carrier. The prepared oxidative stress response polymer nano-carrier can increase the water solubility of a wrapped functional substance and increase the activity of the functional substance. Besides, compared with the traditional complex organic synthesis process, the oxidative stress response polymer nano-carrier prepared by the embodiment of the invention is simple in preparation process and easy to implement.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a polymer nanocarrier and a preparation method and application thereof. Background Art

[0002] Stroke has become a disease that seriously threatens people's life and health due to its high incidence and disability rate. At present, due to the existence of the blood-brain barrier in the brain, it is difficult for drug molecules to enter the lesion site, so there are only a few drugs used to treat stroke, such as t-PA and butylbenzidine.

[0003] Therefore, how to improve the ability of drugs to cross the blood-brain barrier is of great significance for the development of stroke drugs.

[0004] The development of nanomaterials in recent years has made it possible to penetrate the blood-brain barrier. However, due to the oxidative stress response of nanomaterials, there are a large number of reactive oxygen free radicals caused by oxidative stress during the onset of stroke. The presence of these free radicals can lead to the aggravation of stroke symptoms.

[0005] In view of this, finding a nanocarrier based on oxidative stress response for the treatment of stroke has become a hot topic of research. Summary of the invention

[0006] In order to solve at least one of the above technical problems, the present invention provides a polymer nanocarrier and a preparation method and application thereof.

[0007] As one aspect, the present invention provides a method for preparing a polymer nanocarrier, comprising: reacting polyglutamic acid or a derivative thereof with a compound having a hydrophobic group, a compound having a hydrophilic group and a compound having an oxidative stress group in sequence to obtain a polymer nanocarrier.

[0008] In some embodiments of the present invention, the molar ratio of polyglutamic acid or its derivative, the compound with a hydrophilic group, the compound with an oxidative stress group, and the compound with a hydrophobic group is (1:1:1:1) to (1:1:1:5.88), for example, it can be 1:1:1:1.78, 1:1:1:2.69, 1:1:1:3.47, 1:1:1:3.91, 1:1:1:4.58, 1:1:1:4.94, 1:1: 1:5.79, etc.; and / or, the compound with a hydrophobic group includes one or more of amino-modified vitamin E (VE-NH2), palmitic acid, and stearic acid; and / or, the compound with a hydrophilic group includes maleimide or its derivatives, preferably maleimide-polyethylene glycol-hydroxy (MAL-PEG-OH); and / or, the compound with an oxidative stress group includes one or more of 4-(hydroxymethyl)phenylboronic acid pyrrolidone and a compound containing a disulfide bond.

[0009] In some embodiments of the present invention, the amino-modified vitamin E is vitamin E succinate-ethylenediamine (VE-NH2).

[0010] As another aspect, the present invention provides a polymer nanocarrier, which is obtained by the above-mentioned preparation method; wherein the polymer nanocarrier includes a hydrophobic group, a hydrophilic group, and an oxidative stress group at the same time.

[0011] In some embodiments of the present invention, the structural formula of the polymer nanocarrier is as shown in Formula I and Formula II:

[0012]

[0013]

[0014] As another aspect, the present invention provides a polymer nanoformulation, comprising the polymer nanocarrier obtained by the above preparation method, or the above polymer nanocarrier.

[0015] In some embodiments of the present invention, the polymer nanoformulation further comprises tanshinone IIA; preferably, the mass ratio of the polymer nanocarrier to tanshinone IIA is (1:0.001) to (1:0.2). For example, it can be 1:0.023, 1:0.047, 1:0.089, 1:0.109, 1:0.113, 1:0.123, 1:0.147, 1:0.156, 1:0.169, 1:0.178, 1:0.183, 1:0.196, etc.

[0016] As another aspect, the present invention provides a use of the above-mentioned polymer nanoformulation in the preparation of a targeting compound.

[0017] As another aspect, the present invention provides a method for preparing a targeting compound, comprising: performing a click chemistry reaction on the above-mentioned polymer nanocarrier and a polypeptide to prepare a targeting compound; wherein the molar ratio of the polymer nanocarrier to the polypeptide is (1:0.001) to (1:0.1), for example, it can be 1:0.019, 1:0.037, 1:0.059, 1:0.073, 1:0.089, 1:0.091, 1:0.098, etc.; or, performing a click chemistry reaction on the above-mentioned polymer nanopreparation and a polypeptide to prepare a targeting compound; wherein the molar ratio of the polymer nanopreparation to the polypeptide is (1:0.001) to (1:0.01), for example, it can be 1:0.021, 1:0.043, 1:0.057, 1:0.069, 1:0.079, 1:0.091, 1:0.097, etc.

[0018] As another aspect, the present invention provides a targeting compound, which is obtained by the above-mentioned preparation method.

[0019] As yet another aspect, the present invention provides a use of the above-mentioned polymer nanoformulation or the above-mentioned targeting compound in the preparation of a stroke drug.

[0020] The technical solution of the embodiment of the present invention has the following advantages:

[0021] The preparation method of the polymer nanocarrier of the embodiment of the present invention is to link vitamin E to polyglutamic acid, and then link maleic anhydride and oxidative stress response groups thereto respectively through esterification reaction to obtain oxidative stress response nanocarrier. Specifically, the preparation method of the oxidative stress response nanocarrier of the embodiment of the present invention is: a compound with a hydrophobic group is prepared into an active ester of a compound with a hydrophobic group through an activation reaction, and then an esterification reaction is carried out with polyglutamic acid to obtain a reaction product, and the obtained reaction product is subjected to an esterification reaction with a compound with a hydrophilic group to connect the hydrophilic group to the reaction product; and then an esterification reaction is carried out to obtain a polymer nanocarrier with oxidation response groups and hydrophilic groups linked to the reaction product.

[0022] The oxidative stress responsive polymer nanocarrier obtained by the preparation method of the polymer nanocarrier of the embodiment of the present invention can increase the water solubility of the encapsulated functional substance and increase the activity of the functional substance. In addition, the oxidative stress responsive polymer nanocarrier prepared by the embodiment of the present invention has a simple preparation process and is easy to implement compared with the traditional complex organic synthesis process.

[0023] In addition, the nanocarrier based on oxidative stress response prepared in the embodiment of the present invention encapsulates Tanshinone IIA to prepare a nanoformulation of Tanshinone IIA in response to oxidative stress, and the preparation is used for the treatment of in vitro and in vivo models of stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0026] Figure 1 This is the NMR spectrum of the polymer nanocarrier TPA-PLG-g-PEG-MAL with oxidative stress response characteristics prepared in Example 1 of the present invention;

[0027] Figure 2 The mass spectrum of the polymer nanocarrier TPA-PLG-g-PEG-MAL with oxidative stress response characteristics prepared in Example 1 of the present invention;

[0028] Figure 3 The particle size distribution diagram in the dynamic light scattering diagram of the polymer nanocarrier TPA-PLG-g-PEG-MAL with oxidative stress response characteristics prepared in Example 1 of the present invention;

[0029] Figure 4 A potential diagram in a dynamic light scattering diagram of the polymer nanocarrier TPA-PLG-g-PEG-MAL having oxidative stress response characteristics prepared in Example 1 of the present invention;

[0030] Figure 5 This is a particle size distribution diagram of the dynamic light scattering diagram of TPA-PLG-g-PEG-MAL nanoparticles (TsIIA-NPs) loaded with tanshinone IIA according to Example 2 of the present invention;

[0031] Figure 6 This is a potential diagram in a dynamic light scattering diagram of TPA-PLG-g-PEG-MAL nanoparticles (TsIIA-NPs) loaded with tanshinone IIA according to Example 2 of the present invention;

[0032] Figure 7 This is a particle size distribution diagram in a dynamic light scattering diagram of the targeted compound T-TsIIA-NPs of Example 2 of the present invention;

[0033] Figure 8 This is a potential diagram in a dynamic light scattering diagram of the targeted compound T-TsIIA-NPs of Example 2 of the present invention;

[0034] Fig. 9 The ultraviolet absorption spectrum of tanshinone IIA (TsIIA-NPs) encapsulated in the polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 of the present invention;

[0035] Fig.10 The ultraviolet absorption spectrum of tanshinone IIA (TsIIA-NPs) encapsulated in the polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 of the present invention and the standard curve of tanshinone IIA;

[0036] Fig.11 This is a graph showing the effect of tanshinone IIA (TsIIA-NPs) encapsulated in a polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 of the present invention on cell viability;

[0037] Fig.12 This is a diagram of the administration time of the effect of tanshinone IIA (TsIIA-NPs) encapsulated in the polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 of the present invention on the cell stroke model;

[0038] Fig.13 This is a diagram showing the cell protective effect of tanshinone IIA (TsIIA-NPs) encapsulated in a polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 of the present invention on a cell stroke model. DETAILED DESCRIPTION

[0039] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0040] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0041] Example 1

[0042] Preparation of polymer nanocarrier TPA-PLG-g-PEG-MAL

[0043] 1.1 Preparation of Vitamin E Succinate-Ethylenediamine

[0044] (1) Vitamin E succinate (2.124 g, 4 mmol) was dissolved in 20 mL CH2Cl2, and slowly stirred in an ice-water bath for 20 min. When the temperature dropped to about 0°C, oxalyl chloride (1.02 mL, 12 mmol) was slowly added dropwise, and the mixture was stirred in an ice-water bath overnight. The mixed solution was placed in a rotary evaporator at 40°C and vacuum distilled to dryness to remove dichloromethane and the remaining oxalyl chloride. Dichloromethane was added several times to fully remove the excess oxalyl chloride to obtain the acyl chloride.

[0045] The structural formula of vitamin E succinate is:

[0046]

[0047] The structural formula of the acyl chloride is:

[0048]

[0049] (2) Dissolve N-Boc-ethylenediamine (0.64 mL, 4 mmol) in 15 mL CH2Cl2, and slowly drop NEt3 (1.12 mL, 8 mmol), stir in an ice-water bath for 20 minutes, wait until the temperature drops to about 0°C, then slowly drop the acyl chloride obtained in the previous step (dissolved in 10 mL CH2Cl2), continue to stir in an ice-water bath for 48 hours, and obtain a milky white solution. Wash three times with 1M HCl, saturated NaHCO3 solution and NaCl solution in sequence, and dry over anhydrous MgSO4. Remove the solvent by distillation under reduced pressure to obtain the product vitamin E succinate-ethylenediamine-Boc.

[0050] The structural formula of Vitamin E succinate-ethylenediamine-Boc is:

[0051]

[0052] (3) Removal of Boc protection: dissolve the product obtained in the previous step in 30 mL of ethyl acetate (EtOAc), slowly introduce dry HCl gas into it, and saturate the ethyl acetate (EtOAc) solution; (preparation of dry HCl gas: slowly drip concentrated sulfuric acid into concentrated hydrochloric acid solution, stir to generate HCl gas, and then dry it with CaCl2), stir at room temperature overnight, filter, remove the precipitate, collect the filtrate, rotary evaporate to dryness, remove the solvent, and obtain the product vitamin E succinate-ethylenediamine (VE-NH2).

[0053] The structural formula of vitamin E succinate-ethylenediamine (VE-NH2) is:

[0054]

[0055] 1.2 Preparation of poly-L-glutamate PLG

[0056] PLG was synthesized via the ring-opening polymerization (ROP) of BLG-NCA and subsequent debenzylation reaction using vitamin E succinate-ethylenediamine as an initiator.

[0057] (1) Under nitrogen protection, accurately weighed BLG-NCA (6.67 g, 23.35 mmol, 160 equiv) was dissolved in 70 mL of anhydrous DMF, and then vitamin E succinate-ethylenediamine (90.3 mg, 0.158 mmol, 1 equiv) dissolved in 6 mL of anhydrous DMF was added. In order to fully react, stirring was continued at 30°C for three days. After completion, acetic anhydride (150 μL, 1.58 mmol, 10 equiv) and triethylamine (220 μL, 0.158 mmol, 10 equiv) were added, and then the round-bottom flask containing the reaction solution was evacuated for 10 min, and then filled with nitrogen for replacement three times, and the bottle mouth was sealed. The reaction was carried out under nitrogen protection and stirred at 30°C for 24 h. After the reaction solution is naturally cooled to room temperature, it is slowly poured into a large amount of cold ether, and a large amount of precipitation is produced. After high-speed centrifugation, the precipitated white solid is collected by filtration, dialyzed with a large amount of distilled water (MWCO 3500) for 3 days (changing water 5-6 times a day), and vacuum freeze-dried to obtain solid poly (γ-benzyl-L-glutamic acid) (PBLG).

[0058] The structural formula of BLG-NCA is:

[0059]

[0060] The structural formula of poly(γ-benzyl-L-glutamate) (PBLG) is:

[0061]

[0062] (2) Debenzylation of poly(γ-benzyl-L-glutamic acid) (PBLG): Under ventilation conditions, γ-benzyl-L-glutamic acid (PBLG) was dissolved in 60 mL of dichloroacetic acid (DCA), 24 mL of HBr / CH3COOH (33 wt%) was added, and the mixture was stirred at 30°C for two hours. After the reaction solution was cooled to room temperature, it was poured into an excess of cold ether to precipitate the reaction mixture. The precipitated white solid was collected by high-speed centrifugation and filtration, and dialyzed with distilled water (MWCO 3500) for three days (changing water 5-6 times a day), and freeze-dried to obtain white solid polyglutamic acid (PLG). The yield of polyglutamic acid (PLG) was 65%.

[0063] The structural formula of PLG is:

[0064]

[0065] 1.3 Preparation of PLG-g-PEG-MAL

[0066] PLG-g-PEG-MAL nanocarriers were prepared by Steglich esterification reaction using PLG and MAL-PEG3.5k-OH as raw materials, 1,3-diisopropylcarbodiimide (DIC) as condensation agent and 4-dimethylaminopyridine (DMAP) as catalyst.

[0067] (1) PLG (155 mg, 1.2 mmol Glu monomer, 1 equiv) and MAL-PEG3.5k-OH (308 mg, 7.06 mmol EG monomer, 5.88 equiv) were weighed and dissolved in 6 mL of ultra-dry dimethylformamide (DMF) solution, and then DIC (82.1 μL, 0.38 mmol) and 4-dimethylaminopyridine (DMAP) (5.17 mg, 0.038 equiv) were added. The reaction was stirred at room temperature for 3 days, and the solution was precipitated with excess ether and washed twice with ether. Finally, the precipitate was dialyzed in distilled water (MWCO 3500) for three days and freeze-dried to obtain PLG-g-PEG-MAL. The yield of PLG-g-PEG-MAL was 55%.

[0068] The structural formula of PLG-g-PEG-MAL is:

[0069]

[0070] 1.4 Preparation of TPA-PLG-g-PEG-MAL

[0071] TPA-PLG-g-PEG-MAL nanocarriers were prepared by Steglich esterification reaction using PLG-g-PEG-MAL as raw material, 1,3-diisopropylcarbodiimide (DIC) as condensation agent and 4-dimethylaminopyridine (DMAP) as catalyst.

[0072] TPA (280 mg, 1.2 mmol Glu monomer, 1 equiv) and PLG-g-PEG-MAL (412 mg, 7.06 mmol EG monomer, 5.88 equiv) were accurately weighed and dissolved in 6 mL of ultra-dry dimethylformamide (DMF) solution, and then DIC (82.1 μL, 0.38 mmol) and 4-dimethylaminopyridine (DMAP) (5.17 mg, 0.038 equiv) were added, and the reaction was stirred at room temperature for 3 days, and the solution was precipitated with excess ether and washed twice with ether. Finally, the precipitate was dialyzed in distilled water (MWCO3500) for three days, and TPA-PLG-g-PEG-MAL was obtained after freeze-drying, wherein the yield of TPA-PLG-g-PEG-MAL was 45%).

[0073] The structural formula of TPA is:

[0074]

[0075] The structural formula of TPA-PLG-g-PEG-MAL is:

[0076]

[0077] Data Analysis:

[0078] Figure 1 This is the nuclear magnetic spectrum of the polymer nanocarrier with oxidative stress response characteristics prepared in Example 1 (ie, polymer nanocarrier TPA-PLG-g-PEG-MAL). Figure 1The top is the H NMR spectrum of TPA-PLG-g-PEG-MAL, the middle is the H NMR spectrum of PLG, and the bottom is the H NMR spectrum of PBLG. All samples were analyzed after treatment with deuterated trifluoroacetic acid, and the solvent peak of deuterated trifluoroacetic acid is represented at δ = 11.67 ppm. First, in the H NMR spectrum of the PBLG molecule, δ=7.52ppm and δ=5.33ppm represent the resonance peaks of the proton H of the phenyl group (C6H5-) and the benzyl group (C6H5CH2-), respectively; the peak of δ=5.33ppm is the resonance peak of the proton H of (-COCH-); the peak of δ=3.58ppm is the resonance peak of the proton H on (-CH2CH2O-); the peak of δ=2.75ppm represents the resonance peak of the proton H of (-CH2CO-); the peaks of δ=2.44ppm and δ=2.23ppm represent the resonance peak of the proton H of (-CH2CH2CH2-). Compared with the 1H NMR spectrum of the PBLG molecule, the 1H NMR signal of the PLG molecule disappears when δ is 5.33ppm (C6H5CH2-) and 7.52ppm (C6H5-), which indicates that the γ-benzyl group has been completely removed and PLG has been successfully prepared. Then, the H NMR spectrum of the PLG-g-PEG-MAL molecule was compared with that of the PLG molecule, and it can be seen that the peaks at δ = 3.93 ppm and δ = 6.93 ppm are assigned to the resonance peaks of the proton H of the methylene (-CH2CH2O-) and the methyne (-CH=CH-CO-) in PEG, respectively.

[0079] Figure 2 The mass spectrum of the polymer nanocarrier with oxidative stress response characteristics (i.e., polymer nanocarrier TPA-PLG-g-PEG-MAL) prepared in Example 1. Specifically, the synthesized TPA-PLG-g-PEG-MAL was characterized by MALDI-TOF-MS mass spectrometry. Figure 2 It can be seen that the molecular weight has reached more than 18000, indicating that the TPA-PLG-g-PEG-MAL polymer with γ-benzyl-L-glutamic acid N-carboxylic anhydride as the monomer has been successfully synthesized. Figure 2 It can be seen that the m / z value decreases regularly, and the decreasing value is 219-220, which is exactly the molecular weight of γ-benzyl-L-glutamic acid monomer.

[0080] Figure 3 The particle size distribution diagram of the polymer nanocarrier with oxidative stress response characteristics prepared in Example 1 (i.e., polymer nanocarrier TPA-PLG-g-PEG-MAL) in the dynamic light scattering diagram; wherein NPs represents the polymer nanocarrier TPA-PLG-g-PEG-MAL. Figure 3From the data analysis in , it can be obtained that the hydrodynamic radius (Rh) of TPA-PLG-g-PEG-MAL in aqueous solution is 270.4±8.0nm.

[0081] Figure 4 The potential diagram in the dynamic light scattering diagram of the polymer nanocarrier with oxidative stress response characteristics prepared in Example 1 (i.e., polymer nanocarrier TPA-PLG-g-PEG-MAL); wherein NPs represents the polymer nanocarrier TPA-PLG-g-PEG-MAL. Figure 4 From the data analysis in , it can be obtained that the Zeta potential of TPA-PLG-g-PEG-MAL in aqueous solution is 76.4±1.069 mV.

[0082] Example 2

[0083] Preparation of polymer nanoformulation TSIIA-NPs

[0084] 10 mg of the TPA-PLG-g-PEG-MAL prepared in Example 1 after freeze drying was added to 5 mL of ultrapure water, stirred at 1000 rpm to completely dissolve it, and 2 mg of tanshinone IIA (dissolved in 1 mL of ethanol) was added, and stirred overnight under ventilation conditions to allow the ethanol solution to evaporate completely, thereby obtaining TPA-PLG-g-PEG-MAL nanoparticles (TsIIA-NPs) loaded with tanshinone IIA. The obtained product TsIIA-NPs nanoparticles were stored in a 4°C refrigerator for use.

[0085] Preparation of targeted compound T-TsIIA-NPs

[0086] Accurately weigh 0.1 g of TPA-PLG-g-PEG-MAL nanoparticles loaded with tanshinone IIA prepared in the step of "Preparation of polymer nanoformulation TSIIA-NPs" in Example 2, dissolve it in 10 mL PBS (pH = 7), stir at 1000 rpm to mix it evenly, then add 1 mg of blood-brain barrier targeting peptide RVG 29 (dissolved in 2 mL PBS, pH = 7), stir overnight at room temperature, freeze-dry to obtain T-TsIIA-NPs, and store in a 4°C refrigerator for use.

[0087] Data Analysis:

[0088] Figure 5 This is the particle size distribution diagram of the dynamic light scattering diagram of TPA-PLG-g-PEG-MAL nanoparticles (TsIIA-NPs) loaded with tanshinone IIA in Example 2.

[0089] Figure 6This is a potential diagram in the dynamic light scattering diagram of TPA-PLG-g-PEG-MAL nanoparticles (TsIIA-NPs) loaded with tanshinone IIA in Example 2.

[0090] Figure 7 This is the particle size distribution diagram in the dynamic light scattering diagram of the targeted compound T-TsIIA-NPs in Example 2.

[0091] Figure 8 This is a potential diagram in the dynamic light scattering diagram of the targeted compound T-TsIIA-NPs of Example 2.

[0092] according to Figures 5 to 8 From the data analysis, we can draw the following conclusions: the hydrated particle sizes of NPs, TsIIA-NPs and T-TsIIA-NPs nanoparticles are 270.4±8.0nm, 325.2±19.2nm and 349.3±17.2nm, and the Zeta potentials are 76.4±1.069mV, 17.3±0.472mV and 28.2±0.253mV, respectively. The increase in the particle size of the nanoparticles indicates that the PLG-g-PEG-MAL nanocarriers are successfully encapsulated with tanshinone IIA through hydrophilic and hydrophobic interactions, and the blood-brain barrier targeting peptide RVG 29 is linked to the surface of the nanocarriers.

[0093] Fig. 9 The ultraviolet absorption spectrum of the polymer nanocarrier with oxidative stress response characteristics encapsulating Tanshinone IIA (TsIIA-NPs) prepared in Example 2. Fig. 9 The three broken lines in the figure correspond to tanshinone-NPs, tanshinone, and NPs respectively from top to bottom.

[0094] Fig.10 The ultraviolet absorption spectrum of tanshinone IIA (TsIIA-NPs) encapsulated in the polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 and the standard curve of tanshinone IIA.

[0095] Example 3

[0096] 3.1 PC12 cell culture

[0097] Immediately place the previously frozen PC12 cells in a 37°C constant temperature water bath, then centrifuge, remove the supernatant, add complete culture medium, and mix well. Then transfer to a sterile culture dish and place in an incubator at 37°C and 5% (v / v) CO2. Continue to observe and replace the culture medium every 24 hours. After 24 hours, spray the ultraviolet disinfected clean bench with 75% alcohol, take out the culture dish, carefully remove the slightly yellow culture medium, wash three times with PBS, add trypsin, digest for about 35 to 55 seconds, stop digestion, transfer to a centrifuge tube, centrifuge, remove the supernatant, add fresh complete culture medium, and blow evenly. Move to the culture dish and shake the culture dish 20 times back and forth and left and right to evenly disperse the cells. Take 4μL of cell suspension and 16μL PBS (diluted 5 times), mix well, move to a cell counting plate, and count the cells under a biological microscope. According to 8×10 per well 3 The medium required for each cell count.

[0098] 3.2 Building the OGDR model

[0099] The oxygen glucose deprivation reperfusion (OGDR) model simulates cerebral ischemia. In the incubator, N2 is introduced to adjust the CO2 and O2 content, and the sugar in the cells is deprived, resulting in cellular hypoxia and glucose deficiency.

[0100] OGDR cell experiment: After the cell culture medium was replaced with DMEM culture medium, the incubator culture conditions were changed to: 37°C, 95% N2+5% CO2 for 4 hours. After the end of OGD, the cell culture medium was changed to complete culture medium (DMEM containing serum) and cultured again at 37°C, 5% CO2 to construct the OGDR model.

[0101] 3.3 Safety evaluation of nanomaterials

[0102] PC12 cells were used as an experimental cell model by MTT method to detect the biological safety of the nanomaterial PLG-g-PEG-MAL. The control group was the experimental cell model PC12, and DMEM complete culture medium was used as a blank control group.

[0103] (1) PC12 cells were cultured at a density of 8×10 3 Each well was inoculated into a 96-well plate and incubated at 37°C and 5% CO2 for 12-24 h to allow the cells to fully adhere to the plate.

[0104] (2) Discard the original culture medium and add 100 μL of basic culture medium DMEM containing different concentrations of PLG-g-PEG-MAL (0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 800 μg / mL) and culture for 24 h.

[0105] (3) The culture medium containing PLG-g-PEG-MAL was removed, 100 μL MTT (1 mg / mL) was added, and the culture was continued for 4 h.

[0106] (4) Discard the MTT reagent and add 150 μL DMSO to each well. Then use an ELISA reader to measure the absorbance at 490 nm and calculate the cell survival rate based on the absorbance of formazan in each well.

[0107] (5) Calculation formula:

[0108] Cell viability = (OD value of control group - OD value of experimental group) / OD value of control group × 100%.

[0109] 3.4 Drug concentration screening

[0110] PC12 cells were treated with oxygen glucose deprivation and reperfusion (OGDR) to simulate cerebral ischemia in vitro. Different concentrations of tanshinone IIA (0 ng / mL, 0.00128 ng / mL, 0.0064 ng / mL, 0.032 ng / mL, 0.16 ng / mL, 0.8 ng / mL, 4 ng / mL, 20 ng / mL, 100 ng / mL) were added to the cell model. The activity of cells in different groups was detected by MTT method, and the drug concentration with low cytotoxicity and high biosafety was screened out.

[0111] Data Analysis:

[0112] Fig.11 This is a graph showing the effect of tanshinone IIA (ie, polymer nanoformulation TSIIA-NPs) encapsulated in the polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 on cell viability.

[0113] Fig.11 The data in the study have the following characteristics: as the concentration of Tanshinone IIA increases, its cytotoxicity is greater than 80%. Fig.11 The data analysis in can show that when the concentration of Tanshinone IIA varies in the range of 0 to 100 ng / mL, the polymer nanocarrier encapsulated with Tanshinone IIA (i.e., polymer nanoformulation TSIIA-NPs) with oxidative stress response characteristics prepared in Example 2 of the present invention does not show toxicity.

[0114] 3.5 Damage protection assessment

[0115] PC12 cells injured by OGDR were used as an in vitro stroke model. The experiment was divided into five groups according to Control, OGDR, NPs, Ts IIA and Ts IIA-NPs. Among them, PC12 cells in OGDR, NPs, Ts IIA and TsIIA-NPs groups needed to be treated with OGDR. After PC12 cells were treated with OGD for 4 hours, they were cultured under normal culture conditions for 24 hours. Then, NPs, Ts IIA or Ts IIA-NPs nanomaterials were added to the culture medium in turn according to the grouping. After 24 hours, the MTT method was used to detect the recovery of the viability of PC12 cells by the prepared different nanocarriers, so as to evaluate the protective effect of Ts IIA-NPs on OGDR injury.

[0116] Fig.12 This is a diagram showing the administration time of the effect of tanshinone IIA (ie, Ts IIA-NPs) encapsulated in the polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 on the cell stroke model.

[0117] Fig.13 This is a diagram showing the cell protective effect of tanshinone IIA (ie, TsIIA-NPs) encapsulated in the polymer nanocarrier with oxidative stress response characteristics prepared in Example 2 on a cell stroke model.

[0118] In summary, the oxidative stress responsive polymer nanocarrier obtained by the preparation method of the polymer nanocarrier of the embodiment of the present invention can increase the water solubility of the encapsulated functional substance and increase the activity of its functional substance. In addition, the oxidative stress responsive polymer nanocarrier prepared by the embodiment of the present invention has a simple preparation process and is easy to implement compared with the traditional complex organic synthesis process. In addition, the nanocarrier based on oxidative stress response prepared by the embodiment of the present invention, by encapsulating Tanshinone IIA, prepared a nanoformulation of Tanshinone IIA with oxidative stress response, and the preparation was used for the treatment of in vitro and in vivo models of stroke.

[0119] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0120] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a polymer nanocarrier, characterized in that: include: The polymer nanocarrier is obtained by reacting polyglutamic acid or its derivatives with a compound having a hydrophobic group, a compound having a hydrophilic group and a compound having an oxidative stress group in sequence.

2. The preparation method according to claim 1, characterized in that: The molar ratio of the polyglutamic acid or its derivative, the compound with a hydrophilic group, the compound with an oxidative stress group, and the compound with a hydrophobic group is (1:1:1:1) to (1:1:1:5.88); And / or, the compound with a hydrophobic group includes one or more of amino-modified vitamin E, palmitic acid, and stearic acid; And / or, the compound with a hydrophilic group comprises maleimide or its derivatives, preferably maleimide-polyethylene glycol-hydroxyl; And / or, the compound with oxidative stress group includes one or more of 4-(hydroxymethyl)phenylboronic acid pyrophenol ester and a compound containing a disulfide bond.

3. A polymer nanocarrier, characterized in that: The polymer nanocarrier is obtained by the preparation method according to claim 1 or 2; Wherein, the polymer nanocarrier includes hydrophobic groups, hydrophilic groups and oxidative stress groups at the same time.

4. The polymer nanocarrier according to claim 3, characterized in that The structural formula of the polymer nanocarrier is shown in Formula I and Formula II:

5. A polymer nanoparticle preparation, characterized in that: The invention comprises a polymer nanocarrier obtained by the preparation method according to claim 1 or 2, or a polymer nanocarrier according to claim 3 or 4.

6. The polymer nanoformulation according to claim 5, characterized in that The polymer nanoformulation also includes Tanshinone IIA; Preferably, the mass ratio of the polymer nanocarrier to tanshinone IIA is (1:0.001) to (1:0.2).

7. Use of the polymer nanoformulation according to claim 5 or 6 in the preparation of a targeting compound.

8. A method for preparing a targeting compound, characterized in that: include: Conducting a click chemistry reaction between the polymer nanocarrier according to claim 3 or 4 and the polypeptide to prepare a targeting compound; Wherein, the molar ratio of the polymer nanocarrier to the polypeptide is (1:0.001) to (1:0.1); Or, the polymer nanoformulation according to claim 5 or 6 is subjected to a click chemistry reaction with a polypeptide to prepare a targeting compound; Wherein, the molar ratio of the polymer nano preparation to the polypeptide is (1:0.001) to (1:0.01).

9. A targeting compound, characterized in that The targeting compound is obtained by the preparation method described in claim 8.

10. Use of the polymer nanoformulation according to claim 5 or 6 or the targeted compound according to claim 8 or 9 in the preparation of a stroke drug.

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