Nitric oxide donor type small-size nano-micelle as well as preparation method and application thereof

Abstract

The invention discloses a nitric oxide donor type small-size nano-micelle as well as a preparation method and application thereof, the nano-micelle has the advantage of small size, so that the nano-micelle can pass through a blood brain barrier, and zwitter-ions enable the nano-micelle to have good capabilities of resisting protein adsorption and prolonging blood circulation time; through modification of nitric oxide, the nano-drug can relieve oxidative stress, resist brain inflammation and kill brain tumor cells, has good NO delivery stability, and has the capabilities of resisting protein adsorption, reducing immunogenicity and prolonging blood circulation time; as a drug carrier, the nano-micelle disclosed by the invention can penetrate through a blood brain barrier, reduce the adhesion of immune cells and effectively treat or relieve brain inflammation, oxidative stress response and hypoxia of brain tumors, and has a great application prospect in treatment of brain diseases.

Description

Technical Field

[0001] The invention relates to a small-sized nano micelle, in particular to a nitric oxide donor type small-sized nano micelle, and also to a preparation method and application of the nano micelle. Background Art

[0002] Brain diseases (including inflammation of intracranial tissues and organs, 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 prevents drugs from reaching the lesion site. At the same time, normal brain damage produces oxidative stress and inflammatory response, which will further lead to neuronal 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 vasodilation tension, inhibiting platelet activation, reducing brain inflammation and reducing adhesion proteins. In addition, NO can activate immunogenic cell death (ICD) of brain tumors, thereby inhibiting tumor growth. Although NO plays a key regulatory role in the treatment of brain diseases, NO is easily metabolized during the delivery process, resulting in poor stability; the efficiency of NO encapsulation in the carrier is poor, and thus the concentration required for treating brain injury sites cannot be reached, and the ideal effect cannot be produced; at the same time, NO has low efficiency in crossing the blood-brain barrier and cannot be controlled to release at the brain lesion site, resulting in no therapeutic effect and adverse effects on normal tissues; it is urgent to develop a drug delivery system that crosses the blood-brain barrier and brings more stable therapeutic effects. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a nitric oxide donor type small-sized nanomicelle that can cross the blood-brain barrier, deliver more stably, have stronger resistance to protein adsorption, reduce immunogenicity and prolong blood circulation time, and also provide a method for preparing the above-mentioned nanomicelle and its application in the synergistic treatment of brain diseases.

[0005] Technical solution: The present invention discloses a method for preparing a nitric oxide donor type small-sized nano-micelle, comprising the following steps:

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

[0007] (2) reacting an acrylamide zwitterionic compound with a dithiol compound to obtain a thiol zwitterionic compound;

[0008] (3) subjecting the functional hyperbranched polycarbonate and the thiol zwitterionic compound to a Michael addition reaction to obtain a hyperbranched zwitterionic polycarbonate;

[0009] (4) subjecting mercaptoethylamine compounds to Michael addition reaction with hyperbranched zwitterionic polycarbonate to obtain amino-functionalized small-sized nanomicelles by utilizing the free radical reaction of double bonds;

[0010] (5) finally, the nitroso-type nitric oxide donor and the amino-functionalized small-sized nano-micelles undergo an amidation reaction to obtain nitric oxide donor-type hyperbranched zwitterionic polycarbonate micelles, i.e., nitric oxide donor-type small-sized nano-micelles;

[0011] Wherein, the structure of the acrylamide zwitterionic compound is:

[0012] The dithiol compound structure is: n = 1-50;

[0013] The structure of the mercaptoethylamine compound is: n = 2-20;

[0014] The structure of the nitroso-type nitric oxide donor is: R3 is selected from H, CH3 or OH.

[0015] Wherein, in step (1), the ring-opening polymerization is carried out by using hydroxylated cyclic carbonate (molar ratio is 20%-100%) as the initiator of the ring-opening polymerization, or the hydroxylated cyclic carbonate is first obtained and then ring-opening polymerization is carried out with the cyclic carbonate monomer.

[0016] In step (1), a polyol molecule may be added as a co-initiator, wherein the polyol molecule is selected from trimethylolethane, glycerol or pentaerythritol.

[0017] The method for preparing the nano micelles specifically comprises the following steps:

[0018] (1) Cyclic carbonate monomers are reacted with mercapto alcohol compounds in a certain ratio using chloroform as solvent for 4-6 hours to generate an intermediate product, hydroxylated cyclic carbonate, and a catalyst (DBU or Sn(Oct)2) is added to the reaction mixture to initiate ring-opening polymerization, and the reaction is transferred to 60° C. for 20-24 hours to obtain a functional hyperbranched polycarbonate;

[0019] (2) acrylamide zwitterionic compounds react with dithiol compounds to obtain the corresponding thiolated zwitterionic TCB;

[0020] (3) the hyperbranched polycarbonate is dissolved in dimethylformamide and methanol, and the thiolated zwitterionic TCB and catalyst triethylamine are added, and the reaction is carried out overnight at room temperature to obtain a functional hyperbranched zwitterionic polymer;

[0021] (4) dissolving the functional hyperbranched zwitterionic polymer in dimethylformamide and methanol, adding mercaptoethylamine compounds and catalyst triethylamine to obtain amino-functionalized hyperbranched zwitterionic polymer;

[0022] (5) The amino-functionalized hyperbranched zwitterionic polymer is prepared into nano-micelles by solvent exchange method, ultrasonic water dissolution method or ultrasonic water dissolution-photocrosslinking method; the amino-functionalized nano-micelles are dissolved in water, and a nitroso NO donor and a carboxyl activator (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide) are added to obtain small-sized NO nano-micelles.

[0023] Among them, the solvent exchange method is: first dissolve the material in N,N-dimethylformamide (DMF), then add deionized water dropwise under ultrasonic conditions, and finally dialyze to remove DMF; the ultrasonic water dissolution method is: under ultrasonic conditions, add deionized water to the material, and ultrasonicate for a certain time to obtain nano micelles; the ultrasonic water dissolution-photocrosslinking method is: under ultrasonic conditions, add deionized water to the material, and after ultrasonicating for a certain time, add a photoinitiator to the solution, deoxygenate the solution, and then irradiate it at a specific ultraviolet wavelength for a period of time to cause crosslinking.

[0024] The invention also discloses nitric oxide donor type small-size zwitterionic nano micelles prepared by the preparation method, the size of which is 1-50nm.

[0025] The molar percentage of the zwitterionic unit on the small-sized NO nano-micelles is less than or equal to 60%.

[0026] The molar percentage of mercaptoethylamine units in the small-sized NO nano-micelles is less than or equal to 20%.

[0027] The molar percentage of the nitroso NO donor unit on the small-sized NO nano-micelles is less than or equal to 15%.

[0028] The invention also discloses the application of the nitric oxide donor type small-size zwitterionic nano micelles in preparing medicines for treating brain diseases.

[0029] Wherein, the brain disease is cerebral ischemia-reperfusion injury, brain tumor, Alzheimer's disease or depression.

[0030] Principle of the invention: The nitric oxide donor type small-sized zwitterionic nanomicelles of the present invention have the advantage of small size, which enables the nanomicelles to pass through the blood-brain barrier; zwitterions, as hydrophilic unit modules, give the nanocarriers a denser hydration layer through electrostatic interaction with water molecules, have excellent anti-protein adsorption performance, and reduce the clearance of the reticuloendothelial system (RES) in the blood circulation; at the same time, zwitterionic nanocarriers can be mediated by choline transporters or betaine / γ-aminobutyric acid transporter-1 (BGT-1) highly expressed on the blood-brain barrier, without destroying tight junctions, to enable drugs to efficiently cross the blood-brain barrier. The modification of nitric oxide enables nanomedicines to relieve oxidative stress, resist brain inflammation, kill brain tumor cells, and improve drug sensitivity by improving brain tumor hypoxia;

[0031] Specifically, zwitterion surface treatment prolongs the half-life of nanocarriers in the blood, and synergizes with the advantage of small size to enhance the ability of nanocarriers to cross the blood-brain barrier and enhance their accumulation in the brain; it solves the problem of short drug half-life, difficulty in crossing the blood-brain barrier, and inability to accumulate in the brain; at the same time, the addition of nitric oxide can alleviate oxidative stress, reduce brain inflammation, resist tumor growth, and improve hypoxia to overcome multidrug resistance, and treat brain diseases in an integrated and multi-effect manner, synergistically bringing about an improvement in efficacy. On the basis of promoting drugs to cross the blood-brain barrier and enhance brain accumulation, this application maintains the healthy state of the normal brain in multiple dimensions, resists the invasion of brain tumors, and reverses hypoxia, thereby improving the brain's drug-resistant microenvironment and achieving efficient treatment of brain diseases.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The nitric oxide donor type small-sized zwitterionic nanomicelles of the present invention have size advantages, and the cross-linked nanoparticles improve the stability of the NO delivery process, and have good anti-protein adsorption, immunogenicity reduction and blood circulation time prolonging capabilities; the existing nanocarriers are mostly enriched in the brain at less than 3% ID / g, while the nanomicelles of the present invention are enriched in the brain at up to 6.4 ID / g, and the enrichment is significantly improved; (2) The preparation method of the nanomicelles of the present invention is based on a one-pot method and Michael addition reaction, which is easy to operate, has high reaction efficiency and is suitable for large-scale production; (3) The nanomicelles of the present invention are used as drug carriers, especially drug carriers for the treatment of brain diseases, which can cross the blood-brain barrier, reduce the adhesion of immune cells, reduce brain inflammation, relieve brain oxidative stress response, relieve brain tumor hypoxia and activate immunogenic cell death of brain tumors, and have great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The nitric oxide polyester (HP-HAC 10％ -CB 60％ -SNO) H NMR spectrum;

[0034] Figure 2 is a particle size diagram of small-sized nanomicelles in Example 2;

[0035] Figure 3 The stability of small-sized nanomicelles in Example 3;

[0036] Figure 4 The cytotoxicity of small-sized nanomicelles to PC12 cells in Example 4;

[0037] Figure 5 The cytotoxicity of small-sized nanomicelles to U251 cells in Example 5;

[0038] Figure 6 is the proportion of small-sized nanomicelles penetrating the in vitro BBB model in Example 6;

[0039] Figure 7 The ability of small-sized nanomicelles in Example 7 to reduce inflammation;

[0040] Figure 8 The ability of small-sized nanomicelles in Example 8 to alleviate oxidative stress;

[0041] Fig. 9 The small-sized nanomicelles in Example 9 can alleviate brain tumor hypoxia. DETAILED DESCRIPTION

[0042] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0043] Example 1

[0044] HP-HAC 10％ -CB 60％ -AC 30％ Synthesis of:

[0045] (1) Synthesis of hydroxylated carbonate HAC:

[0046]

[0047] Acrylic carbonate AC (2g, 10mmol) was dissolved in 5ml of dichloromethane, and mercaptoethanol (0.82g, 10.50mmol) and a catalytic amount of triethylamine were added under nitrogen protection, and the reaction was carried out at room temperature for 4h. After the reaction was completed, the reaction solution was added dropwise to a sufficient amount of icy ether to precipitate, and the precipitate was dried under reduced pressure to obtain a colorless oily compound HAC;

[0048] (2) Synthesis of hyperbranched polycarbonate HP-HAC:

[0049] In a glove box, 400 mg of HAC monomer was dissolved in 2 ml of chloroform and added to a sealed reactor, and then the catalyst 1,8-diazabicycloundec-7-ene (DBU) was added. Then the reactor was sealed, transferred out of the glove box, and placed in a 60°C oil bath for reaction for 20 hours. After the reaction was completed, 2 drops of glacial acetic acid were used to terminate the reaction, and precipitation was carried out in glacial ether. The supernatant was discarded, the oily viscous liquid at the bottom was collected, and the product was obtained by vacuum drying;

[0050] (3) Synthesis of zwitterionic hyperbranched polycarbonate HP-HAC10%-CB60%-AC30%

[0051] Hyperbranched polycarbonate HP-HAC with acrylate undergoes Michael addition reaction with mercapto-carboxylic acid betaine under the catalysis of triethylamine to obtain multifunctional biodegradable hyperbranched polycarbonate zwitterionic polymer.

[0052]

[0053] Example 2

[0054] HP-HAC 10％ -CB 60％ Synthesis of -SNO:

[0055] (1) Synthesis of amino-functionalized micelles:

[0056] The zwitterionic hyperbranched polycarbonate HP-HAC10%-CB60%-AC30% reacts with cysteamine under the catalysis of triethylamine to undergo Michael addition reaction; under ultrasonic conditions, 2 ml of purified water is added to the polymer and ultrasonicated for half an hour. Then a catalytic amount of photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959) is added to the micelle solution, and nitrogen is blown into the solution to exclude air. After being placed under 580 nm ultraviolet light for 20 minutes, amino-functionalized small-sized nanomicelles are obtained;

[0057] (2) Synthesis of NO donor small-sized nanomicelles:

[0058] NO donor (nitroso-N-acetylpenicillamine) was dissolved in methanol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to activate the carboxyl group; small-sized nano-micelles functionalized with amino groups were added to react to obtain HP-HAC. 10％ -CB 60％ -SNO micelles. NMR images are shown in Figure 1 ; The micelle size was measured by dynamic light scattering instrument, and the particle size diagram is shown in Figure 2 .

[0059] Example 3

[0060] Verification of the stability of small-sized NO nanomicelles

[0061] The stability of micelles was evaluated by turbidimetry by observing the increase in absorbance at 500 nm (indicating aggregation). Micelles (concentration 1.0 wt%) were dispersed in 10% fetal bovine serum buffer at 37°C and the absorbance was monitored by UV-Vis spectrophotometer.

[0062] The results are as follows Figure 3 , the change of turbidity of micelles within 24 h was negligible, which proved that small-sized NO nanomicelles could effectively resist protein adsorption and had excellent colloidal stability.

[0063] Example 4

[0064] Cytotoxicity test of small-sized NO nanomicelles (MTT)

[0065] The cytotoxicity experiment of small-sized NO nano-micelles was carried out using the MTT method. Rat adrenal medullary tumor cells (PC12) were used in the cytotoxicity test. Rat adrenal medullary tumor cells (PC12) were cultured in DMEM medium containing 10% serum at 37°C and 5% carbon dioxide, with a cell density of 3500 cells / well. After 12 hours, 10 μL PBS and different concentrations of nitric oxide-type small-sized nano-micelles (concentrations of 50, 100, 200, 400 and 800 μg / mL) were added, incubated for 48 hours, and then 10 μL MTT (5 mg / mL) was added. After continuing to culture for 4 hours, the culture medium was removed, 100 μL DMSO was added to each well, and the absorbance was measured at 490 nm using an enzyme reader after the purple crystals were completely dissolved.

[0066] The results are as follows Figure 4 As shown, at the maximum micelle concentration, the survival rate of PC12 cells is still above 80%, proving that the micelles have basically no cytotoxicity.

[0067] Example 5

[0068] Cytotoxicity test of small-sized NO nanomicelles (MTT)

[0069] The cytotoxicity experiment of small-sized NO nano-micelles was carried out using the MTT method. Human glioma cells (U251) were used in the cytotoxicity test. Human glioma cells (U251) were cultured in DMEM medium containing 10% serum at 37°C and 5% carbon dioxide, with a cell density of 3500 cells / well. After 12 hours, 10μL PBS and different concentrations of nitric oxide-type small-sized nano-micelles (concentrations were 3, 5, 7, 10 and 15 mg / mL, respectively) were added, incubated for 48 hours, and then 10μL MTT (5 mg / mL) was added. After continuing to culture for 4 hours, the culture medium was removed, 100μL DMSO was added to each well, and the absorbance was measured at 490nm using an enzyme reader after the purple crystals were completely dissolved.

[0070] The results are as follows Figure 5 As shown, at the maximum micelle concentration, the survival rate of U251 cells was above 35%, proving that the micelles can effectively kill brain tumor cells.

[0071] Example 6

[0072] In vitro BBB penetration experiment of small-sized NO nanomicelles

[0073] Human brain microvascular endothelial cells (bEnd.3) were cultured in DMEM medium containing 10% serum at 37°C and 5% carbon dioxide. In order to establish the BBB model in vitro, bEnd.3 cells (5×10 4 / well) were inoculated in the transwell plate chamber, and 800μL of DMEM medium containing 10% serum was added to the lower chamber. The medium was renewed every two days. The 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 200Ω·cm 2 In vitro BBB penetration experiments were performed. Nitric oxide-type small-sized nanomicelles were bonded to Cy5-NH2 through an amidation reaction, and the ratio of fluorescence passing through was measured at 1h, 2h, 4h, 6h, 8h, 12h, and 24h to determine the transmittance of the material.

[0074] The results are as follows Figure 6 As shown, small-sized NO nanomicelles can efficiently cross the in vitro blood-brain barrier, and 35.0% of the nanomicelles permeate the in vitro blood-brain barrier model after 24 hours.

[0075] Example 7

[0076] Small-sized NO nanomicelles reduce inflammation

[0077] Mouse microglial cells (BV2) were plated at 4×10 per well. 5The cells were seeded at a density of 100 cells / mL in a 24-well plate and incubated for 12 h. Small-sized NO nano-micelles were added to the wells for 6 h. Then, after 3 h of hypoxia (OGD), reoxygenation was performed for 24 h, i.e., the cellular ischemia-reperfusion model (OGD / R). The cells were fixed and blocked with 5% bovine serum albumin (BSA). Then, the cells were incubated with CD16 / 32 antibodies (marking pro-inflammatory) and CD206 antibodies (marking anti-inflammatory) at 4 °C overnight. The next day, the cells were incubated with goat anti-mouse immunoglobulin G (IgG) Alexa Fluor 594 for 1 h. The images were taken using a fluorescence microscope, and the fluorescence intensity of the images was quantified using Image J software.

[0078] The results are as follows Figure 7 As shown, small-sized NO nanomicelles can alleviate inflammatory responses.

[0079] Example 8

[0080] Small-sized NO nanomicelles can alleviate oxidative stress

[0081] PC12 cells were grown at 4 × 10 4 The cells 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., the cellular ischemia-reperfusion model (OGD / R). The cells were washed and incubated with reactive oxygen species (ROS) dye for 20 minutes in a cell culture incubator at 37°C. The images were taken using a fluorescence microscope, and the fluorescence intensity of the images was quantified using Image J software.

[0082] The results are as follows Figure 8 As shown, small-sized NO nanomicelles can effectively scavenge reactive oxygen species, thereby alleviating oxidative stress.

[0083] Example 9

[0084] Small-sized NO nanomicelles can alleviate hypoxia in brain tumors

[0085] U251 cells (3×10 5 Cells / well) were seeded on 6-well culture plates overnight, and after 3 hours of hypoxia, they were treated with small-sized NO nano-micelles of different concentrations for 12 hours. Then, the cells were lysed, the final protein was collected by centrifugation, and its concentration was detected by a protein assay kit. Western blotting was performed according to standard methods to monitor the changes in the concentration of hypoxia factor (HIF-1α).

[0086] The results are as follows Fig. 9 As shown, small-sized NO nanomicelles can significantly reduce the expression of hypoxia factors and effectively alleviate hypoxia.

[0087] Therefore, the nitric oxide donor type small-sized nano-micelles of the present invention have good NO delivery stability, and the ability to resist protein adsorption, reduce immunogenicity and prolong blood circulation time; and can be used as a drug carrier.

Claims

1. A method for preparing nitric oxide donor type small-sized nanomicelles, characterized in that: The preparation method comprises the following steps: (1) reacting a cyclic carbonate monomer with a mercapto alcohol compound to generate a hydroxylated cyclic carbonate, and then performing ring-opening polymerization to obtain a functional hyperbranched polycarbonate; (2) reacting an acrylamide zwitterionic compound with a dithiol compound to obtain a thiol zwitterionic compound; (3) subjecting the functional hyperbranched polycarbonate and the thiol zwitterionic compound to a Michael addition reaction to obtain a hyperbranched zwitterionic polycarbonate; (4) subjecting mercaptoethylamine compounds to Michael addition reaction with hyperbranched zwitterionic polycarbonate to obtain amino-functionalized small-sized nanomicelles by utilizing the free radical reaction of double bonds; (5) subjecting the nitroso-type nitric oxide donor and the amino-functionalized small-sized nanomicelles 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: The dithiol compound structure is: The structure of the mercaptoethylamine compound is: The structure of the nitroso-type nitric oxide donor is: R3 is selected from H, CH3 or OH.

2. The preparation method according to claim 1, characterized in that: In step (1), the ring-opening polymerization is carried out by using hydroxylated cyclic carbonate as an initiator for the ring-opening polymerization, or by first obtaining hydroxylated cyclic carbonate and then carrying out ring-opening polymerization with 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 any one of the preparation methods of claims 1 to 3, characterized in that: The size of the nano micelles is 1-50 nm.

5. The nitric oxide donor type small-sized nanomicelle according to claim 4, characterized in that: The molar percentage of zwitterionic units in the small-sized NO nanomicelles is less than or equal to 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 small-sized NO nanomicelles is less than or equal to 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 on the small-sized NO nanomicelles is ≤15%.

8. Use of the nitric oxide donor type small-sized nanomicelles according to any one of claims 4 to 7 in the preparation of drugs for treating brain diseases.

9. The use according to claim 8, characterized in that: The brain disease is cerebral ischemia-reperfusion injury, brain tumor, Alzheimer's disease or depression.

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