Nitric oxide donor type small size nanomicelles, and preparation method and application thereof

Abstract

The application discloses a nitric oxide donor type small-size nanomicelle and a preparation method and application thereof, wherein the small size of the nanomicelle enables the nanomicelle to pass through a blood-brain barrier; the zwitterion enables the nanoparticle to have good anti-protein adsorption and the ability of prolonging blood circulation time; the modification of the nitric oxide enables the nano-drug to relieve oxidative stress, resist brain inflammation and kill brain tumor cells, has good NO delivery stability, and has the ability of anti-protein adsorption, reduction of immunogenicity and prolongation of blood circulation time; and the nanomicelle of the application can pass through the blood-brain barrier, reduce the adhesion of immune cells, effectively treat or relieve brain inflammation, oxidative stress response and brain tumor hypoxia, and has great application prospect in the treatment of brain diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to a small size nanomicelle, in particular to a small size nanomicelle of a nitric oxide donor type, and to a preparation method and application of the nanomicelle. BACKGROUND

[0002] Brain diseases (including intracranial tissue organ inflammation, brain degenerative diseases, brain tumors, etc.) seriously endanger human health, however, the number of clinical drugs for treating brain diseases is relatively small, the main reason is that the blood-brain barrier hinders the drug from reaching the lesion site; at the same time, oxidative stress and inflammatory response are produced after normal brain injury, which will further lead to neural cell apoptosis and brain tissue structure damage.

[0003] Nitric oxide (NO) plays a key role in the balance of the central nervous system. A large amount of evidence shows that supplementing exogenous NO can effectively alleviate cerebral ischemia-reperfusion injury by regulating vascular diastolic tension, inhibiting platelet activation, reducing brain inflammation and reducing adhesion proteins. In addition, NO can activate the immunogenic cell death (ICD) of brain tumors, thereby inhibiting the growth of tumors. Although NO plays a key regulatory role in the treatment of brain diseases, however, NO is easily metabolized during delivery, resulting in poor stability; the efficiency of the carrier for loading NO is poor, and thus the concentration required for treating the brain injury site cannot be reached, and the desired effect cannot be produced; at the same time, the efficiency of NO crossing the blood-brain barrier is low, and it cannot be controlled to be released at the brain lesion site, which makes it impossible to produce a therapeutic effect and has an adverse impact on normal tissues; it is urgent to develop a drug delivery system that can cross the blood-brain barrier and bring more stable therapeutic effect. SUMMARY

[0004] The purpose of the present application is to provide a small size nanomicelle of a nitric oxide donor type which can cross the blood-brain barrier, deliver more stably, has stronger anti-protein adsorption, reduces immunogenicity and prolongs blood circulation time, and to provide a preparation method of the nanomicelle and its application in the synergistic treatment of brain diseases.

[0005] Technical solution: The present application discloses a preparation method of a small size nanomicelle of a nitric oxide donor type, comprising the following steps:

[0006] (1) reacting a cyclic carbonate monomer and a mercapto alcohol compound to obtain a hydroxylated cyclic carbonate, and then ring-opening polymerization to obtain a functional hyperbranched polycarbonate;

[0007] (2) reacting an acrylamide zwitterionic compound and a dimercapto compound to obtain a mercapto zwitterionic compound;

[0008] (3) functional hyperbranched polycarbonate and thiolated zwitterionic compound are subjected to Michael addition reaction to obtain hyperbranched zwitterionic polycarbonate;

[0009] (4) the thiol ethylamine compound is subjected to Michael addition reaction with the hyperbranched zwitterionic polycarbonate, and a small-size nanomicelle with amino functionalization is obtained by using the radical reaction of the double bond;

[0010] (5) finally, the nitroso type nitric oxide donor and the small-size nanomicelle with amino functionalization are subjected to amidation reaction to obtain a nitric oxide donor type hyperbranched zwitterionic polycarbonate micelle, i.e. a nitric oxide donor type small-size nanomicelle;

[0011] The acrylamide type zwitterionic compound has the structure of:

[0012] The dimercapto type compound has the structure of: n = 1-50;

[0013] The thiol ethylamine compound has the structure of: n = 2-20;

[0014] The nitroso type nitric oxide donor has the structure of:

[0015] R3 is selected from H, CH3 or OH.

[0016] In step (1), the ring-opening polymerization is to use a hydroxylated cyclic carbonate (molar ratio of 20%-100%) as an initiator for ring-opening polymerization, or to obtain a hydroxylated cyclic carbonate first and then to undergo ring-opening polymerization with a cyclic carbonate monomer.

[0017] In step (1), a polyol molecule can also be added as a co-initiator, and the polyol molecule is selected from trimethylol ethane, glycerol or pentaerythritol.

[0018] The preparation method of the nanomicelle specifically includes the following steps:

[0019] (1) the cyclic carbonate monomer is reacted with a thiol alcohol compound in a certain proportion with chloroform as a solvent to obtain an intermediate product, i.e. a hydroxylated cyclic carbonate, for 4-6 hours, a catalyst (DBU or Sn(Oct)2) is continuously added to the reaction mixture to initiate ring-opening polymerization, and the reaction is transferred to 60℃ for 20-24 hours to obtain a functional hyperbranched polycarbonate;

[0020] (2) the acrylamide type zwitterionic compound is reacted with the dimercapto type compound to obtain a corresponding thiolated zwitterionic TCB;

[0021] (3) The dimethylformamide and methanol of the above hyperbranched polycarbonate are dissolved, the thiolated zwitterion TCB and the catalyst triethylamine are added, and the reaction is carried out at room temperature overnight to obtain a functional hyperbranched zwitterion polymer;

[0022] (4) The dimethylformamide and methanol of the above functional hyperbranched zwitterion polymer are dissolved, the mercaptoethylamine compound and the catalyst triethylamine are added to obtain an amino-functionalized hyperbranched zwitterion polymer;

[0023] (5) The amino-functionalized hyperbranched zwitterion polymer is prepared into a nano-micelle by a solvent exchange method, an ultrasonic water-soluble method or an ultrasonic water-soluble-light crosslinking method; the amino-functionalized nano-micelle is dissolved in water, the nitroso NO donor and the carboxyl activator (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, N-hydroxysuccinimide) are added to obtain a small-size NO nano-micelle.

[0024] The solvent exchange method is that the material is dissolved in N, N-dimethylformamide (DMF), then deionized water is added dropwise to the material under ultrasonic conditions, and finally the DMF is removed by dialysis; the ultrasonic water-soluble method is that deionized water is added to the material under ultrasonic conditions, and the nano-micelle can be obtained after ultrasonic for a certain time; and the ultrasonic water-soluble-light crosslinking method is that deionized water is added to the material under ultrasonic conditions, a photo initiator is added to the solution after ultrasonic for a certain time, the solution is deoxygenated, and then irradiated under a specific ultraviolet wavelength for a period of time to cause crosslinking.

[0025] The application further discloses the small-size zwitterion nano-micelle prepared by the preparation method.

[0026] The mole percentage of the zwitterion unit in the small-size NO nano-micelle is less than or equal to 60%.

[0027] The mole percentage of the mercaptoethylamine unit in the small-size NO nano-micelle is less than or equal to 20%.

[0028] The mole percentage of the nitroso NO donor unit in the small-size NO nano-micelle is less than or equal to 15%.

[0029] The application further discloses application of the small-size zwitterion nano-micelle in preparation of a drug for treating brain diseases.

[0030] The brain diseases are cerebral ischemia-reperfusion injury, brain tumor, Alzheimer's disease or depression.

[0031] Invention principle: the nitric oxide donor type small size zwitterionic nanomicelles of the application have the size advantage that the nanomicelles can pass through the blood-brain barrier; the zwitterion as a hydrophilic unit module endows the nanocarrier with a more compact hydration layer through electrostatic interaction with water molecules, has excellent anti-protein adsorption performance, reduces the clearance of the reticuloendothelial system (RES) in the blood circulation; at the same time, the zwitterionic nanocarrier can be mediated by the high expression of choline transporters or betaine / gamma-aminobutyric acid transporter-1 (BGT-1) on the blood-brain barrier, without destroying tight junctions, to promote the efficient crossing of drugs through the blood-brain barrier. The modification of nitric oxide enables the nanodrug to relieve oxidative stress, resist brain inflammation, kill brain tumor cells and improve drug sensitivity by improving brain tumor hypoxia;

[0032] Specifically, the zwitterionic surface prolongs the half-life of the nanocarrier in the blood, and cooperates with the size advantage to enhance the ability of the nanocarrier to cross the blood-brain barrier and enhance the enrichment in the brain; solve the problem of short drug half-life, difficult to cross the blood-brain barrier, and unable to enrich in the brain; at the same time, the addition of nitric oxide can relieve oxidative stress, reduce brain inflammation, resist tumor growth and improve hypoxia to overcome multidrug resistance, integrally and synergistically improve the efficacy of the treatment of brain diseases. On the basis of promoting the crossing of drugs through the blood-brain barrier and enhancing the enrichment in the brain, the application maintains the healthy state of the normal brain in multiple dimensions, resists the invasion of brain tumors and reverses hypoxia to improve the brain drug resistance microenvironment, thereby achieving efficient treatment of brain diseases.

[0033] Advantages: compared with the prior art, the application has the following obvious advantages: (1) the nitric oxide donor type small size zwitterionic nanomicelles of the application have the size advantage that the nanomicelles can pass through the blood-brain barrier; the zwitterion as a hydrophilic unit module endows the nanocarrier with a more compact hydration layer through electrostatic interaction with water molecules, has excellent anti-protein adsorption performance, reduces the clearance of the reticuloendothelial system (RES) in the blood circulation; at the same time, the zwitterionic nanocarrier can be mediated by the high expression of choline transporters or betaine / gamma-aminobutyric acid transporter-1 (BGT-1) on the blood-brain barrier, without destroying tight junctions, to promote the efficient crossing of drugs through the blood-brain barrier. The modification of nitric oxide enables the nanodrug to relieve oxidative stress, resist brain inflammation, kill brain tumor cells and improve drug sensitivity by improving brain tumor hypoxia; BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the nitric oxide type polyester (HP-HAC 10％ -CB 60％ -SNO) in Example 2;

[0035] Figure 2 Particle size chart of small size nanomicelles in Example 2;

[0036] Figure 3 Stability of small size nanomicelles in Example 3;

[0037] Figure 4 Toxicity of small size nanomicelles in Example 4 to PC12 cells;

[0038] Figure 5 Toxicity of small size nanomicelles in Example 5 to U251 cells;

[0039] Figure 6 Penetration ratio of small size nanomicelles in Example 6 through in vitro BBB model;

[0040] Figure 7 Inflammatory alleviating ability of small size nanomicelles in Example 7;

[0041] Figure 8 Oxidative stress alleviating ability of small size nanomicelles in Example 8;

[0042] Figure 9 Brain tumor hypoxia alleviating ability of small size nanomicelles in Example 9. DETAILED DESCRIPTION

[0043] The technical solutions of the present application are further described below in combination with examples, and the test materials used in the examples can be purchased through conventional channels.

[0044] Example 1

[0045] Synthesis of HP-HAC 10％ CB 60％ AC 30％

[0046] (1) Synthesis of hydroxylated carbonate HAC:

[0047]

[0048] Acrylate carbonate AC (2 g, 10 mmol) was dissolved in 5 ml of dichloromethane, and mercaptoethanol (0.82 g, 10.50 mmol) and a catalytic amount of triethylamine were added under nitrogen protection. The reaction was carried out at room temperature for 4 h. After the reaction was completed, the reaction solution was added dropwise into a sufficient amount of ice ethyl ether to precipitate and separate out, and the precipitate was dried under reduced pressure to obtain a colorless oily compound HAC;

[0049] (2) Synthesis of hyperbranched polycarbonate HP-HAC:

[0050] ​In the glove box, 400 mg HAC monomer was dissolved in 2 ml chloroform, added to the sealed reactor, then added catalyst 1, 8-diazabicycloundec-7-ene (DBU), then the reactor was sealed, taken out of the glove box, and placed in a 60°C oil bath for 20 h. After the reaction was completed, 2 drops of glacial acetic acid were added to terminate the reaction, and the reaction was precipitated in ice ethyl ether. The supernatant was discarded, and the bottom oily viscous liquid was collected and vacuum dried to obtain the product.

[0051] (3) Synthesis of zwitterionic hyperbranched polycarbonate HP-HAC10%-CB60%-AC30%:

[0052] The hyperbranched polycarbonate HP-HAC with acrylate reacted with mercapto carboxy betaine to produce multifunctional biodegradable hyperbranched polycarbonate zwitterionic polymer through Michael addition reaction under the catalysis of triethylamine.

[0053]

[0054] Example 2

[0055] HP-HAC 10％ -CB 60％ Synthesis of HP-HAC-SNO:

[0056] (1) Synthesis of amino-functionalized micelles:

[0057] The zwitterionic hyperbranched polycarbonate HP-HAC10%-CB60%-AC30% reacted with cysteamine through Michael addition reaction under the catalysis of triethylamine. Under ultrasonic conditions, 2 ml of purified water was added to the polymer, and ultrasonic was performed for half an hour. Then a catalytic amount of photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl benzophenone (I2959) was added to the micelle solution, and nitrogen was blown into the solution to exclude air. It was placed under 580 nm ultraviolet light for 20 min to obtain amino-functionalized small-sized nanomicelles;

[0058] (2) Synthesis of NO donor type small-sized nanomicelles:

[0059] The NO donor (nitroso-N-acetyl penicillamine) was dissolved in methanol, and the carboxyl group was activated by adding 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide. The amino-functionalized small-sized nanomicelles were reacted to obtain HP-HAC 10％ -CB 60％ -SNO micelles. The nuclear magnetic resonance chart is shown in Figure 1 ; and the micelle particle size was measured by dynamic light scattering instrument, and the particle size chart is shown in Figure 2 .

[0060] Example 3

[0061] Verification of the stability of small size NO nanomicelles

[0062] The stability of the micelles was evaluated by turbidity method, observing the increase of absorbance at 500 nm (indicative of aggregation). The micelles (1.0 wt% concentration) were dispersed in 10% fetal bovine serum buffer at 37°C and the absorbance was monitored with a UV-Vis spectrophotometer.

[0063] The results are shown in Table 1 Figure 3 The change in turbidity of the micelles within 24 h was negligible, proving that the small size NO nanomicelles are effectively resistant to protein adsorption and have excellent colloidal stability.

[0064] Example 4

[0065] Cell toxicity test of small size NO nanomicelles (MTT)

[0066] The cell toxicity test of small size NO nanomicelles was performed using the MTT method. Rat pheochromocytoma cells (PC12) were used in the cell toxicity test. The rat pheochromocytoma cells (PC12) were cultured in DMEM medium containing 10% serum at 37°C in a 5% carbon dioxide atmosphere, at a cell density of 3500 cells / well. After 12 hours, 10 μL of PBS and different concentrations of small size nanomicelles of the type of nitric oxide (50, 100, 200, 400 and 800 μg / mL) were added, and incubated for 48 h, followed by the addition of 10 μL of MTT (5 mg / mL). The incubation was continued for 4 h, the medium was removed and 100 μL of DMSO was added to each well. After the violet crystals had completely dissolved, the absorbance was measured at 490 nm using an enzyme marker.

[0067] The results are shown in Table 2 Figure 4 At the maximum micelle concentration, the survival rate of the PC12 cells was still above 80%, proving that the micelles have essentially no cytotoxicity.

[0068] Example 5

[0069] Cell toxicity test of small size NO nanomicelles (MTT)

[0070] Cell toxicity experiment of small size NO nanomicelles was performed by MTT method. Human glioma cells (U251) were used in the cell toxicity experiment. Human glioma cells (U251) were cultured in DMEM medium containing 10% serum at 37°C in 5% CO2, and the cell density was 3500 cells per well. After 12 hours, 10 μL PBS and different concentrations of small size nanomicelles of NO (concentrations were 3, 5, 7, 10 and 15 mg / mL, respectively) were added, and incubated for 48 h, followed by adding 10 μL MTT (5 mg / mL). After 4 h of incubation, the medium was removed, 100 μL DMSO was added to each well, and the absorbance was measured at 490 nm after the purple crystals were completely dissolved.

[0071] The results are shown in Figure 5 At the maximum micelle concentration, the survival rate of U251 cells was more than 35%, which proved that the micelles could efficiently kill brain tumor cells.

[0072] Example 6

[0073] Materials Small size NO nanomicelles for in vitro BBB penetration experiment

[0074] Human brain microvascular endothelial cells (bEnd.3) were cultured in DMEM medium containing 10% serum at 37°C in 5% CO2. To establish a BBB model in vitro, bEnd.3 cells (5×10 4 cells per well) were seeded in the upper chamber of a transwell plate, and 800 μL of DMEM medium containing 10% serum was added to the lower chamber. The medium was replaced every two days. A transendothelial electrical resistance (TEER) instrument was used to monitor the tightness of the cell monolayer. When the TEER value of the bEnd.3 cell monolayer was greater than 200 Ω·cm 2 Small size nanomicelles of NO were linked to Cy5-NH2 by amide reaction, and the proportion of fluorescence passing through was measured at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h to determine the penetration rate of the material.

[0075] The results are shown in Figure 6 Small size NO nanomicelles can efficiently cross the in vitro blood-brain barrier, and 35.0% of the nanomicelles can penetrate the in vitro blood-brain barrier model after 24 h.

[0076] Example 7

[0077] Materials Small size NO nanomicelles for reducing inflammation

[0078] Mouse microglial cells (BV2) were cultured in DMEM medium containing 10% serum at 37°C in 5% CO2. The cell density was 4×10 5Cells were seeded at a density of 100 cells / mL in a 24-well plate and incubated for 12 hours. Small-sized NO nano-micelles were added to the wells for 6 hours. Then, after 3 hours of hypoxia (OGD), reoxygenation was performed for 24 hours, i.e., a cellular ischemia-reperfusion model (OGD / R). The cells were fixed and blocked with 5% bovine serum albumin (BSA). The cells were then incubated overnight at 4°C with CD16 / 32 antibodies (marking pro-inflammatory) and CD206 antibodies (marking anti-inflammatory). The next day, the cells were incubated with goat anti-mouse immunoglobulin G (IgG) Alexa Fluor 594 for 1 hour. Images were taken using a fluorescence microscope, and the fluorescence intensity of the images was quantified using Image J software.

[0079] The results are as follows Figure 7 As shown, small-sized NO nanomicelles can alleviate inflammatory responses.

[0080] Example 8

[0081] Small-sized NO nanomicelles can alleviate oxidative stress

[0082] PC12 cells were grown at 4 × 10 4 Cells were seeded at a density of 100 cells / mL in a 24-well plate and incubated for 12 hours. Small-sized NO nanomicelles were added to the wells for 6 hours. After 3 hours of oxygen deprivation (OGD), reoxygenation was performed for 24 hours, creating a cellular ischemia-reperfusion model (OGD / R). The cells were washed and incubated with a reactive oxygen species (ROS) dye for 20 minutes in a 37°C cell culture incubator. Images were taken using a fluorescence microscope, and fluorescence intensity was quantified using Image J software.

[0083] The results are as follows Figure 8 As shown in the results, small-sized NO nanomicelles can effectively scavenge reactive oxygen species, thereby alleviating oxidative stress.

[0084] Example 9

[0085] Small-sized NO nanomicelles alleviate hypoxia in brain tumors

[0086] U251 cells (3×10 5 Cells (cells / well) were seeded overnight in 6-well culture plates. After 3 hours of hypoxia, they were treated with various concentrations of small-sized NO nanomicelles for 12 hours. The cells were then lysed, and the resulting protein was collected by centrifugation. Its concentration was determined using a protein assay kit. Western blotting was performed according to standard methods to monitor changes in the concentration of the hypoxia factor (HIF-1α).

[0087] The results are as follows Figure 9 As shown, small-sized NO nanomicelles can significantly reduce the expression of hypoxia factors and effectively alleviate hypoxia.

[0088] Therefore, the nitric oxide donor type small size nanomicelles have good NO delivery stability, and the abilities of resisting protein adsorption, reducing immunogenicity and prolonging blood circulation time; as a drug carrier, the nanomicelles can pass through the blood-brain barrier, reduce the adhesion of immune cells, alleviate brain inflammation, relieve brain oxidative stress, relieve brain tumor hypoxia and activate the immunogenic cell death of brain tumor, efficiently kill brain tumor cells, and have great application prospects in the treatment of brain diseases.

Claims

1. A method for preparing nitric oxide donor type small-sized nanomicelles, characterized in that: The preparation method comprises the following steps: (1) Cyclic carbonate monomers and mercapto alcohol compounds are reacted to generate hydroxylated cyclic carbonates, which are then subjected to ring-opening polymerization to obtain functional hyperbranched polycarbonates; (2) Acrylamide zwitterionic compounds react with dithiol compounds to obtain thiol zwitterionic compounds; (3) A functionalized hyperbranched polycarbonate and a thiol-modified zwitterionic compound are subjected to a Michael addition reaction to obtain a hyperbranched zwitterionic polycarbonate; (4) The mercaptoethylamine compounds were subjected to Michael addition reaction with hyperbranched zwitterionic polycarbonate to obtain amino-functionalized small-sized nanomicelles by utilizing the free radical reaction of the double bond; (5) A nitroso-type nitric oxide donor and an amino-functionalized small-sized nanomicelle are subjected to an amidation reaction to obtain nitric oxide donor-type hyperbranched zwitterionic polycarbonate micelles, i.e., nitric oxide donor-type small-sized nanomicelles; Wherein, the structure of the acrylamide zwitterionic compound is: 、 or ; The dithiol compound structure is: 、 or , n=1-50; The structure of the mercaptoethylamine compound is: , n=2-20; The structure of the nitroso nitric oxide donor is: 、 or ; R is selected from H, CH3 or OH; The cyclic carbonate monomer is , the mercapto alcohol compound is .

2. The preparation method according to claim 1, characterized in that In step (1), the ring-opening polymerization is carried out by using a hydroxylated cyclic carbonate as an initiator, or by first obtaining a hydroxylated cyclic carbonate and then performing ring-opening polymerization with a cyclic carbonate monomer.

3. The preparation method according to claim 1, characterized in that In step (1), a polyol molecule is added as a co-initiator, wherein the polyol molecule is selected from trimethylolethane, glycerol or pentaerythritol.

4. A nitric oxide donor type small-sized nanomicelle prepared by the preparation method according to any one of claims 1 to 3, characterized in that: The size of the nanomicelles is 1-50 nm.

5. The nitric oxide donor type small-sized nanomicelle according to claim 4, characterized in that: The molar percentage of the zwitterionic unit in the nitric oxide donor type small-sized nanomicelles is ≤60%.

6. The nitric oxide donor type small-sized nanomicelle according to claim 4, characterized in that: The molar percentage of mercaptoethylamine units in the nitric oxide donor type small-sized nanomicelles is ≤20%.

7. The nitric oxide donor type small-sized nanomicelle according to claim 4, characterized in that: The molar percentage of the nitroso NO donor unit in the nitric oxide donor small-sized nanomicelles is ≤15%.

8. Use of the nitric oxide donor small-sized nanomicelles according to any one of claims 4 to 7 in the preparation of a drug for treating cerebral ischemia-reperfusion injury or brain tumor.

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