Hyperbranched polymer, nano preparation and preparation method and application thereof

Abstract

The invention relates to a hyperbranched polymer, a nano preparation as well as a preparation method and application of the nano preparation, and belongs to the technical field of functional medical materials and nanotechnology. The hyperbranched polymer provided by the invention can break and release dihydrolipoic acid in an ROS environment, exert an anti-oxidation effect, and is reduced into lipoic acid in vivo, thereby exerting an effect of the hyperbranched polymer as a coenzyme, participating in mitochondrial tricarboxylic acid cycle and recovering mitochondrial functions, and further exerting an anti-oxidation effect and an effect of treating cerebral arterial thrombosis. The nano preparation provided by the invention can be quickly distributed to the brain within a short time, BBB cell paracapermeability is temporarily increased by inhibiting expression of multi-drug resistant protein P-gp and ATP-dependent drug efflux protein on BBB, the process of opening BBB by borneol is physiologic and reversible, and the structural integrity of the blood-brain barrier is not damaged.

Description

Technical Field

[0001] The invention belongs to the field of functional medical materials and nanotechnology, and specifically relates to a hyperbranched polymer, a nano preparation, and a preparation method and application thereof. Background Art

[0002] Ischemic stroke (IS) is mainly caused by cerebrovascular obstruction, which leads to a decrease or interruption of blood supply to brain tissue, which in turn leads to a decrease in the delivery of essential nutrients and oxygen, thereby inducing a hypoxic state in brain tissue. Hypoxic conditions promote the recruitment of leukocytes to the lesions, exacerbating oxidative stress and inflammatory damage, further exacerbating brain tissue damage. In addition, recanalization of blocked blood vessels may lead to secondary cerebral ischemia / reperfusion (I / R) injury, thereby expanding the scope of infarction and exacerbating neuronal damage and dysfunction. In addition, damaged neurons and astrocytes produce reactive oxygen species (ROS), which in turn exacerbate damage to neuronal cells and blood vessels. Therefore, long-term oxidative stress and inflammatory responses may eventually lead to the destruction of the blood-brain barrier (BBB), further deteriorating brain tissue, and may eventually lead to brain parenchymal necrosis.

[0003] The blood-brain barrier is a physiological barrier between the blood and brain tissue formed by brain capillary endothelial cells and glial cells, which can effectively prevent toxins and other exogenous harmful substances from entering the brain and damaging nerve cells. It is reported that only small molecules with lipid solubility and molecular weight <400Da can pass through the BBB. About 98% of small molecule drugs and almost 100% of macromolecule drugs cannot enter the brain through peripheral administration. Therefore, the existence of the BBB greatly hinders the development of drugs for central nervous system diseases. At present, there is no good drug that can be widely used to treat stroke. The emergence of thrombolytic agents has epoch-making significance for the research and treatment of stroke. The commonly used drug for the treatment of stroke in clinical practice is tissue plasminogen activator (tPA), which can intravenously dissolve thromboses, but due to the limitations of treatment conditions and drug side effects (increased risk of cerebral hemorrhage), only a few patients can receive effective treatment at present, and most stroke patients do not receive effective treatment. In addition, neuroprotective agents such as glutamate receptor antagonists, antioxidants, apoptosis inhibitors, and cell membrane stabilizers have been found in preclinical trials to be able to antagonize, interfere with, or slow down the main pathophysiological processes of nerve damage, becoming the most promising stroke treatment drugs in the current neuroprotective program. Unfortunately, the vast majority of neuroprotective drugs have been proven to be ineffective in clinical phase II / III trials.

[0004] There are two structures of lipoic acid in the body, one is the oxidized α-lipoic acid (ALA); the other is the reduced dihydrolipoic acid (DHLA). Both have antioxidant capacity in the body, and the two can be converted into each other and work together to achieve antioxidant effects. Lipoic acid is an effective mitochondrial antioxidant. It plays a core role in establishing, maintaining and strengthening the antioxidant defense network in the body by effectively removing ROS and regenerating key antioxidants (such as glutathione, GSH). In addition, lipoic acid is an essential coenzyme for mitochondrial metabolism and is a coenzyme reaction required for the catalysis of the mitochondrial 2-ketoacid dehydrogenase complex, including acting on important enzyme systems such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and branched-chain ketoacid dehydrogenase. The lack of lipoic acid will block the tricarboxylic acid cycle (α-ketoglutarate to succinyl-CoA cycle). When inflammation occurs, lipoic acid synthase (LASY) will be down-regulated and its expression will decrease. The reduction of endogenous lipoic acid caused by the down-regulation of LASY will lead to redox imbalance, inflammation and mitochondrial dysfunction. Compared with traditional nanoformulations, the active ingredients of lipoic acid are easily metabolized rapidly during the first-pass metabolism in the liver after oral administration, and the elimination half-life is within 30 minutes, resulting in insufficient effective concentration in the body and low bioavailability.

[0005] Therefore, it is particularly urgent to develop prodrug nanoformulations based on lipoic acid to improve its bioavailability and enhance its therapeutic effect. Summary of the invention

[0006] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and to provide a hyperbranched polymer, a nanoformulation and a preparation method and application thereof.

[0007] The present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a hyperbranched polymer, wherein the hyperbranched polymer is obtained by photoinitiated free radical polymerization of compound A and compound B via thiol-alkyne click chemistry; the structure of compound A is shown in formula I, and the structure of compound B is shown in formula II:

[0009]

[0010] In formula I, R1 and R2 are each independently -OH, -NH2, -O(CH2CH2O) n One of H, where n is 2-44.

[0011] The present invention utilizes the sulfhydryl in compound A and the alkyne of compound B to carry out sulfhydryl-alkynyl click chemistry reaction, and constructs a hyperbranched polymer with thioacetal structure. In ROS environment, the structure can break and release dihydrolipoic acid, play an antioxidant effect, be reduced to lipoic acid in vivo, and then play its role as a coenzyme, participate in the mitochondrial tricarboxylic acid cycle, and restore mitochondrial function. Traditionally, after oral administration of lipoic acid, its active ingredient is easily metabolized rapidly in the first-pass metabolism process of the liver, and the elimination half-life is within 30min, resulting in its insufficient effective concentration in vivo and low bioavailability. And the hyperbranched polymer of the present invention uses lipoic acid as a hyperbranched polymer skeleton after modification and modification, and after arriving at the inflammation site, the hyperbranched polymer first responds to release the lipoic acid drug active ingredient under the stimulation of ROS, which significantly improves the bioavailability of exogenous lipoic acid, plays an antioxidant and plays a role in the treatment of ischemic stroke.

[0012] Preferably, the structure of the hyperbranched polymer of the present invention is as shown in Formula III:

[0013] R is

[0014] The structure shown in formula III is a typical structural unit of the hyperbranched polymer of the present invention.

[0015] In a second aspect, the present invention provides a method for preparing the hyperbranched polymer, comprising the following steps: mixing compound A, compound B and a photoinitiator, and irradiating with ultraviolet light to obtain the hyperbranched polymer.

[0016] Preferably, the photoinitiator includes at least one of benzoin monomethyl ether (DMPA), benzophenone (Benzoin), dibenzophenone (BDME), phenothiazine (Thioxanthone), vinylbenzyl ether (Vinylbenzyl Ether), and cyclohexylaminobenzophenone (Camphorquinone).

[0017] Preferably, the molar ratio of compound A, compound B and photoinitiator is: (4.00-20.00): (1.00-5.00): (0.50-2.50).

[0018] Preferably, the ultraviolet lamp irradiation time is 1h-6h.

[0019] Preferably, the preparation method of the compound A comprises the following steps: subjecting the raw material A to a reduction reaction under the action of a reducing agent to obtain the compound A; the structure of the raw material A is shown in formula A:

[0020] In formula A, R1 is -OH, -NH2, -O(CH2CH2O)n One of H.

[0021] Preferably, the reducing agent includes one of dithiothreitol (DTT), mercaptoethanol (β-ME), thiourea, lithium aluminum hydride (LiAlH4), and sodium borohydride (NaBH4).

[0022] Preferably, the molar ratio of the compound A to the reducing agent is (1-5):(2-11).

[0023] Preferably, when R2 is -OH, the preparation method of compound B comprises the following steps:

[0024] (1) subjecting thioctic acid to an esterification reaction to obtain thioctic acid ester;

[0025] (2) subjecting the lipoic acid ester obtained in step (1) to a reduction reaction under the action of a reducing agent to obtain dihydrolipoic acid ester;

[0026] (3) subjecting the dihydrolipoic acid ester obtained in step (2) to an alkynylation reaction of the thiol group of a compound containing an alkynyl group, followed by hydrolysis to obtain the compound B.

[0027] The preparation method of the hyperbranched polymer of the invention is simple, low in cost and high in reaction efficiency.

[0028] Specifically, in the step (1), there are many ways of esterification, such as dehydration reaction of carboxylic acid and alcohol (methanol or ethanol) under heating conditions and acid catalysis to generate corresponding esters (methyl esters or ethyl esters); under alkaline conditions, carboxylic acid and (Boc)2 anhydride react to generate corresponding tert-butyl esters; under the action of a condensing agent, carboxylic acid and a compound containing a hydroxyl group react to generate corresponding esters; under alkaline conditions, acyl chloride and a compound containing a hydroxyl group react to generate corresponding esters; under alkaline and oxidizing agents, aldehydes and alcohols (such as methanol) react to generate corresponding esters; under alkaline conditions, cyano groups react with alcohols, and then acid hydrolyze to generate corresponding esters. The present invention does not limit the esterification reaction of step (1), as long as lipoic acid can be converted into lipoic acid esters. In a specific embodiment of the present invention, the step (1) is as follows: lipoic acid and anhydrous methanol are mixed under the action of a condensing agent to undergo an esterification reaction to obtain methyl lipoate; further, the molar ratio of lipoic acid to anhydrous methanol is (1.00-5.00): (10-50).

[0029] Preferably, the reducing agent in step (2) can be the reducing agent in the preparation method of compound A, specifically can include one of dithiothreitol (DTT), mercaptoethanol (β-ME), thiourea, lithium aluminum hydride (LAH), sodium borohydride; the molar ratio of lipoic acid ester to reducing agent is (1-5): (2-11).

[0030] Specifically, in step (3), the unsaturated alkynyl group and the thiol group can be subjected to an addition reaction through the thiol alkyne reaction to form a functional group with ROS response. Common alkynylation reactions of thiol (-SH) include: reaction with alkynyl halides, such as reaction of thiol with alkynyl bromide (or alkynyl chloride) to form alkynyl thiol compounds; reaction with alkynyl sulfate, which can also generate alkynyl thiol compounds; reaction with high-valent iodine acetyl reagents to quickly and accurately modify; these reactions are usually carried out under alkaline conditions to ensure that the thiol group effectively participates in the reaction. The present invention does not limit the alkyne reaction of step (3), as long as the thiol group can be converted into an alkynyl group.

[0031] In a specific embodiment of the present invention, the step (3) is as follows: dihydrolipoic acid ester and 1-[(trimethylsilyl)ethynyl]-1,2-benzidoyl-3(1H)-one (TMS-EBX) are subjected to an acetylation reaction in the presence of a catalyst to obtain an intermediate product, trimethylsilyl lipoic acid ester (the molar ratio of the dihydrolipoic acid ester to TMS-EBX is (1-5): (3-15)), which is then hydrolyzed to obtain the compound B.

[0032] Preferably, the hydrolysis can be carried out under alkaline or acidic conditions, such as hydrogen fluoride (HF), ammonium fluoride (NH4F), potassium carbonate, hydrochloric acid (HCl), sulfuric acid (H2SO4); thereby achieving the removal of the trimethylsilyl group to obtain compound B; in a specific embodiment of the present invention, the hydrolysis is: trimethylsilyl lipoic acid ester and potassium carbonate are mixed and hydrolyzed to obtain compound B; the molar ratio of trimethylsilyl lipoic acid ester to potassium carbonate is (1-5): (8-40).

[0033] Preferably, when R2 is -NH2, the preparation method of the compound B comprises the following steps: subjecting the raw material B to an alkynyl-containing compound to an alkynylation reaction of the thiol group to obtain the compound B; the structure of the raw material B is shown in formula B:

[0034]

[0035] When R2 is -NH2, the reaction conditions of the acetylation reaction in the preparation method of compound B are similar to step (3) in the preparation method of compound B when R2 is -OH. There is no limitation on the acetylation reaction as long as the thiol group can be converted into an acetylation group.

[0036] Preferably, when R2 is -O(CH2CH2O) n H, the preparation method of the compound B comprises the following steps: subjecting polyethylene glycol and diacetyl lipoic acid to aldol condensation under the action of a condensing agent to obtain the compound B; the diacetyl lipoic acid has a structural formula C as shown:

[0037]

[0038] Specifically, when R2 is -O(CH2CH2O) n H, in the preparation method of the compound B, what occurs is the aldol condensation between the hydroxyl group in the polyvinyl alcohol and the carboxyl group in the diacetyl lipoic acid, thereby achieving the preparation of the compound B. There is no restriction on the selection of the condensing agent, as long as the above reaction can occur. In a specific embodiment of the present invention, the condensing agent can be 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 4-dimethylaminopyridine (DMAP).

[0039] In a third aspect, the present invention provides a nanoformulation, comprising the hyperbranched polymer and borneol (2-borneol).

[0040] The nano preparation of the present invention includes a hyperbranched polymer and borneol, which is a traditional Chinese medicine for resuscitation, has the effects of awakening the mind and clearing away heat and relieving pain, has anti-inflammatory, antioxidant, anticoagulant neuroprotective effects, can be quickly distributed to the brain in a short time (only 5 minutes), and temporarily increases the paracellular permeability of the BBB by inhibiting the expression of multidrug resistance protein P-gp and ATP-dependent drug efflux protein on the BBB. The process of opening the BBB by borneol is physiological and reversible, and does not destroy the structural integrity of the blood-brain barrier. The nano preparation of the present invention is a condensation-polymerized targeted structure borneol on the surface of a hyperbranched polymer. In an ischemic stroke model, it can achieve rapid and efficient entry into the brain across the BBB to reach the ischemic area, accurately target the lesion site, prolong the circulation time, and improve the bioavailability.

[0041] In a fourth aspect, the present invention provides a method for preparing the nano preparation, comprising the following steps: polycondensing a hyperbranched polymer and borneol under the action of a condensation agent, and then self-assembling to obtain the nano preparation.

[0042] The nano preparation of the present invention is composed of borneol with a "drug-leading upward" targeting structure and a hyperbranched polymer skeleton with ROS response bonds synthesized by click chemistry, and then a nano particle preparation with uniform size is obtained through self-assembly.

[0043] Preferably, the molar ratio of the hyperbranched polymer to borneol is (1.00-5.00):(0.20-1.00).

[0044] Preferably, the condensing agent includes any one of dicyclohexylcarbodiimide (DCC) / N,N-diisopropylethylamine (DIPEA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 4-dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC) / 4-dimethylaminopyridine (DMAP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / triethylamine (TEA).

[0045] In the polycondensation reaction of the present invention, the condensation agent may be any one of the above combinations.

[0046] Preferably, the polycondensation time is 8h-16h.

[0047] Preferably, the self-assembly adopts a fast nanocomplexation (FNC) method; more preferably, in the fast nanocomplexation method, the oil phase solvent includes one of dimethyl sulfoxide (DMSO), acetonitrile, methanol, N,N-dimethylformamide, and tetrahydrofuran; the oil phase and the aqueous phase are nanocomplexed in a ratio of 1:(6-9) (for example, 1:6, 1:7, 1:8, 1:9); the flow rate ratio of oil phase: aqueous phase: aqueous phase is (0.2-5):(0.9-22.5):(0.9-22.5) (0.2:0.9:0.9, 4:1.8:1.8, 1:4.5:4.5, 2:9:9, 4:18:18, 5:22.5:22.5); the polymer dissolved in the oil phase is mixed with the aqueous phase to perform HBP(LA)-Bo nanoprecipitation.

[0048] The present invention adopts a flash nanocomplexation (FNC) platform to regulate the parameters of nanoparticles to obtain nanoparticles with uniform size, good stability under pathological and physiological conditions, and strong pharmacokinetics.

[0049] In a specific embodiment of the present invention, the self-assembly process is as follows: through the FNC nano self-assembly platform, a condensed polymer (20 mg / mL) solution dissolved in the organic phase DMSO is injected into channel 1 of a three-channels confined impinging jet reactor (CIJ), and ultrapure water is injected into the other two channels. Then, a CNC high-pressure injection pump is used to fully mix the solutions in the three channels at a constant preset flow rate to prepare uniform nanoparticles, and then dialyzed in ultrapure water using a dialysis bag (molecular weight cutoff: 14 kDa) to remove the organic phase to obtain the nanoformulation.

[0050] Preferably, the nanoparticles prepared in the present invention are in the shape of spherical particles, the particle size of the nanoparticles is 30nm-100nm, the polydispersity index PDI of the nanoparticles is 0.05-0.35, and the zeta potential of the nanoparticles is -10mV to -40mV.

[0051] In a fifth aspect, the present invention provides use of the hyperbranched polymer and the nanoformulation in preparing a drug for treating ischemic stroke.

[0052] Preferably, the concentration range of the nanoformulation when used for treatment is 10 mg / mL-20 mg / mL.

[0053] The present invention has the following beneficial effects:

[0054] The hyperbranched polymer of the present invention can break and release dihydrolipoic acid in the ROS environment, play an antioxidant role, be reduced to lipoic acid in the body, and then play its role as a coenzyme, participate in the mitochondrial tricarboxylic acid cycle, restore mitochondrial function, thereby playing an antioxidant role and playing a role in treating ischemic stroke. Further, the nano preparation provided by the present invention is composed of a hyperbranched polymer skeleton with a "drug-leading upward" targeting structure and a ROS-responsive key synthesized by click chemistry, which can be quickly distributed to the brain in a short time (only 5 minutes), and temporarily increase the paracellular permeability of the BBB by inhibiting the expression of multidrug resistance protein P-gp and ATP-dependent drug efflux protein on the BBB. The process of opening the BBB by borneol is physiological and reversible, and does not destroy the structural integrity of the blood-brain barrier. In addition, compared with the nanoformulations encapsulating therapeutic agents, the nanoformulations of the present invention have almost pure drug cores, and therefore have higher drug loading and lower toxic side effects. In addition, the nanoformulations not only contain a single drug core, but also have higher drug loading and less side effects. The nanoformulations themselves are drugs, which can effectively solve the problems of early drug leakage and difficult to remove residues after drug release. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The synthetic flow chart of the hyperbranched polymer prepared in Example 1 of the present invention;

[0056] Figure 2 The nuclear magnetic resonance hydrogen spectrum of dihydrolipoic acid prepared in Example 1 of the present invention;

[0057] Figure 3 The nuclear magnetic resonance hydrogen spectrum of methyl lipoate prepared in Example 1 of the present invention;

[0058] Figure 4 The nuclear magnetic resonance hydrogen spectrum of dihydrolipoic acid methyl ester prepared in Example 1 of the present invention;

[0059] Figure 5The hydrogen nuclear magnetic resonance spectrum of methyl trimethylsilyl lipoate prepared in Example 1 of the present invention;

[0060] Figure 6 This is the hydrogen nuclear magnetic resonance spectrum of diacetylene lipoic acid prepared in Example 1 of the present invention;

[0061] Figure 7 HBP prepared in Example 1 of the present invention (LA) NPs and Bo-HBP (LA) Particle size distribution of NPs nanoformulations in aqueous media;

[0062] Figure 8 Bo-HBP prepared in Example 1 of the present invention (LA) The dispersion stability of NPs nanoformulation in two media: water and PBS (10 mM, pH 7.4);

[0063] Fig. 9 HBP prepared in Example 1 of the present invention (LA) NPs and Bo-HBP (LA) Potential diagram of NPs nanoformulations;

[0064] Fig.10 The nuclear magnetic resonance hydrogen spectrum of the hyperbranched polymer prepared in Example 1 of the present invention before and after the addition of hydrogen peroxide;

[0065] Fig.11 The Bo-HBP of Example 1 of the present invention (LA) In vitro ABTS free radical scavenging experimental data of NPs nanoformulations;

[0066] Fig.12 The Bo-HBP of Example 1 of the present invention (LA) In vitro hydroxyl radical scavenging experimental data of NPs nanoformulations;

[0067] Fig.13 The Bo-HBP of Example 1 of the present invention (LA) Diagram of the in vivo therapeutic effect of NPs nanoformulations. DETAILED DESCRIPTION

[0068] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0069] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0070] Example 1

[0071] The schematic flow diagram of the method for preparing the hyperbranched polymer of this embodiment is as follows Figure 1 As shown, the specific steps include:

[0072] (1) Thioctic acid (5.10 g, 24.72 mmol, 1.00 eq) was suspended in 125 mL of 0.2 M NaHCO3 aqueous solution and stirred to obtain a light yellow solution. A small amount of sodium borohydride (1.90 g, 50.22 mmol, 2.03 eq) was added under ice bath conditions and stirred for 30 min. The mixture was then stirred at room temperature for another 30 min. The mixture was ice bathed again and the pH was adjusted to 1 with 2 M HCl. The mixture was extracted three times with chloroform (50 mL). The organic layer was retained and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography to obtain dihydrolipoic acid as a light yellow oily liquid. The NMR spectrum is shown in FIG. Figure 2 ; The synthesis path is as follows:

[0073]

[0074] (2) Thioctic acid (5.00 g, 24.20 mmol, 1.00 eq) and anhydrous methanol 10 mL (7.75 g, 242.50 mmol, 10.00 eq) were dissolved in 75 mL of dichloromethane solution, and a mixed solution of 4-dimethylaminopyridine (1.48 g, 12.10 mmol, 0.50 eq) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (5.57 g, 29.00 mmol, 1.20 eq) dissolved in dichloromethane was slowly added dropwise. After the reaction was completed, the solvent and unreacted methanol were removed by evaporation under reduced pressure, and the mixture was redispersed in ethyl acetate. The organic phase of the reaction solution was washed with 1 M hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride respectively. The organic phase was separated and dried with anhydrous sodium sulfate. After drying, the solid was removed by filtration and purified by column chromatography to obtain the product methyl thioate. The NMR spectrum is shown in FIG. Figure 3 ; The synthesis path is as follows:

[0075]

[0076] (3) Methyl lipoate (5.00 g, 22.69 mmol, 1.00 eq) was dissolved in methanol. A small amount of sodium borohydride (1.72 g, 45.38 mmol, 2.00 eq) was added under ice bath conditions. The mixture was stirred for 30 min and then stirred for another 30 min at room temperature. The mixture was ice bathed again and the pH was adjusted to 1 with 2M HCl. The mixture was extracted three times with chloroform (50 mL), dried over anhydrous sodium sulfate overnight, and purified by column chromatography to obtain methyl dihydrolipoate as a light yellow oily liquid. The NMR spectrum is shown in FIG. Figure 4 ; The synthesis path is as follows:

[0077]

[0078] (4) Methyl dihydrolipoate (2.00 g, 8.99 mmol, 1.00 eq) was added to 145 mL of tetrahydrofuran, 1,1,3,3-tetramethylguanidine (2.07 g, 17.99 mmol, 2.00 eq) was added and the reaction was continued for 5 min, TMS-EBX (9.34 g, 26.98 mmol, 3.00 eq) was added and the reaction was continued for 5 min, tetrahydrofuran was removed by rotary evaporation under reduced pressure, the mixture was dissolved in ethyl acetate, washed three times with an appropriate amount of saturated NaCl solution, the organic layer was collected, and purified by TLC column chromatography to obtain methyl trimethylsilyl lipoate (TMS-alk-DHLA), the NMR spectrum of which is shown in FIG. Figure 5 ; The synthesis path is as follows:

[0079]

[0080] (5) Methyl trimethylsilyl lipoate (1.00 g, 9.64 mmol, 1.00 eq) was dissolved in 10 mL of methanol. Potassium carbonate (2.67 g, 19.29 mmol, 8.00 eq) was added under ice bath conditions and reacted for 30 min. The ice bath was removed and the reaction was continued at room temperature for 2 h. The reaction mixture was quenched with H2O. The methanol was rotary evaporated and extracted with ethyl acetate. The mixture was washed with saturated NaCl three times and dried over anhydrous sodium sulfate. The mixed organic layer was concentrated in vacuo and purified by TLC column chromatography to obtain diacetylene lipoic acid (Alk-DHLA). The NMR spectrum is shown in FIG. Figure 6 ; The synthesis path is as follows:

[0081]

[0082] (6) Add dihydrolipoic acid (420.00 mg, 2.02 mmol, 4.00 eq) obtained in step (1), diacetylene lipoic acid (129.21 mg, 504.00 μmol, 1.00 eq) obtained in step (5) and photoinitiator DMPA (64.59 mg, 252.00 μmol, 0.50 eq) into a Shrek bottle, remove oxygen three times by double removal, place under a 365 nm ultraviolet lamp, illuminate for 3 h, and collect by precipitation with ice ether to obtain a solid hyperbranched polymer HBP. (LA) ; The synthesis path is as follows:

[0083]

[0084] The obtained hyperbranched polymer is prepared into a nano preparation, and the preparation method of the nano preparation comprises the following steps:

[0085] S1. HBP (LA)The polymer (160 mg, 0.71 mmol, 1.00 eq), borneol (22.01 mg, 0.142 mmol, 0.20 eq), and DMAP (8.61 mg, 0.071 mmol, 0.10 eq) were dissolved in 1.5 mL DMSO, and EDCI·HCl (40.83 mg, 0.213 mmol, 0.30 eq) was added dropwise under ice bath conditions. After the reaction was carried out for 12 h, the product was dialyzed with DMSO for 4 h (molecular weight cutoff: 14 kDa), and then dialyzed with water for 12 h (molecular weight cutoff: 14 kDa), and then freeze-dried to obtain solid Bo-HBP. (LA) ; The synthesis path is as follows:

[0086]

[0087] S2. Bo-HBP dissolved in organic phase DMSO was injected into channel 1 of a three-channel confined impinging jet reactor through the FNC nano-self-assembly platform. (LA) (20 mg / mL) solution was prepared, and ultrapure water was injected into the other two channels. The solutions in the three channels were then fully mixed at a constant preset flow rate by a digitally controlled high-pressure injection pump to prepare uniform nanoparticles. Then, the organic solvent DMSO was removed by dialyzing in ultrapure water using a dialysis bag (molecular weight cutoff: 14 kDa) to obtain the nanoparticle preparation (Bo-HBP (LA) NPs); similarly, the hyperbranched polymer HBP prepared in step (6) (LA) Nanoformulation HBP prepared by FNC nano self-assembly platform (LA) NPs.

[0088] Example 2

[0089] The preparation method of the hyperbranched polymer of the present embodiment comprises the following steps:

[0090] (1) Thioctic acid (4.80 g, 23.00 mmol, 1.00 eq) was dissolved in 200 mL CH3CN, di-(N, N'-succinimidyl) carbonate (7.20 g, 28.00 mmol, 1.20 eq) and triethylamine (3.00 eq, 10.0 mL) were added to the above solution, stirred at room temperature for 2 h, the mixture was concentrated under reduced pressure at 35°C, and then a 5% NaHCO3 aqueous solution was added to produce a yellow precipitate, the precipitate was filtered, washed with water, and then dissolved in 200 mL CH3CN;

[0091] 15 mL of 25% ammonia solution (14.00 g, 0.20 mol, 9.00 eq) was added dropwise to the above solution, and the mixture was stirred at room temperature for 5 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure, and then crystallized twice from CH3CN at -25°C to obtain yellow solid monomer lipoic acid amide; the synthesis route is as follows:

[0092]

[0093] (2) Disperse lipoamide (1.04 g, 5.00 mmol, 1.00 eq) in 50 mL MeOH, add NaBH4 (0.38 g, 10.00 mmol, 2.00 eq), stir at room temperature for 1 h, and then rotary evaporate the reaction mixture to obtain transparent solid dihydrolipoamide; the synthesis route is as follows:

[0094]

[0095] (3) Dihydrolipoamide (1.87 g, 9.04 mmol, 1.00 eq) was added to 145 mL of tetrahydrofuran, 1,1,3,3-tetramethylguanidine (2.07 g, 17.99 mmol, 2.00 eq) was added and reacted for 5 min, TMS-EBX (9.34 g, 26.98 mmol, 3.00 eq) was added and continued to react for 5 min, tetrahydrofuran was removed by rotary evaporation under reduced pressure, dissolved in ethyl acetate, washed three times with an appropriate amount of saturated NaCl solution, the organic layer was collected, and purified by TLC column chromatography to obtain trimethylsilyl lipoamide; the synthesis route is as follows:

[0096]

[0097] (4) Dissolve trimethylsilyl lipoic acid amide (1.00 g, 2.50 mmol, 1.00 eq) in 10 mL of methanol, add potassium carbonate (2.67 g, 20.01 mmol, 8.00 eq) under ice bath conditions, react for 30 min, remove the ice bath and react for 2 h at room temperature, quench the reaction mixture with H2O, rotary evaporate the methanol, extract with ethyl acetate, wash with saturated NaCl three times, dry with anhydrous sodium sulfate, concentrate the mixed organic layer in vacuo, and purify by TLC column chromatography to obtain diacetyl lipoic acid amide; the synthetic route is as follows:

[0098]

[0099] The dihydrolipoamide (100.00 mg, 0.48 mmol, 4.00 eq) obtained in step (2), the diacetylene lipoamide (30.79 mg, 0.12 mmol, 1.00 eq) obtained in step (4) and the photoinitiator DMPA (15.38 mg, 0.06 mmol, 0.50 eq) were added into a Shrek bottle, and the mixture was placed under a 365 nm ultraviolet lamp for 3 h after double oxygen removal. The solid hyperbranched polymer HBP was obtained by precipitation with ether. (LA-NH2) ; The synthesis path is as follows:

[0100]

[0101] Example 3

[0102] The preparation method of the hyperbranched polymer of the present embodiment comprises the following steps:

[0103] (1) Suspend lipoic acid (5.10 g, 24.72 mmol, 1.00 eq) in 125 mL of 0.2 M NaHCO3 aqueous solution and stir to obtain a light yellow solution. Add a small amount of sodium borohydride (1.90 g, 50.22 mmol, 2.03 eq) under ice bath conditions and stir for 30 min. Then, stir at room temperature for another 30 min. Ice bath again, adjust pH to 1 with 2 M HCl, extract the mixture three times with chloroform (50 mL), retain the organic layer and dry it with anhydrous sodium sulfate, purify it by column chromatography, and obtain dihydrolipoic acid as a light yellow oily liquid. The synthesis route is as follows:

[0104]

[0105] (2) Diacetyl lipoic acid (5.00 g, 24.20 mmol, 1.00 eq) and PEG 1k -OH (24.96g, 24.20mmol, 1.0eq) was dissolved in 75mL dichloromethane solution, and a mixed solution of 4-dimethylaminopyridine (1.48g, 12.10mmol, 0.50eq) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (5.57g, 29.00mmol, 1.20eq) dissolved in dichloromethane was slowly dripped into it. After the reaction was completed, the solvent dichloromethane was evaporated under reduced pressure to remove it, and the mixture was redispersed in ethyl acetate. The reaction solution was washed with 1M hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride respectively. The organic phase was separated and dried with anhydrous sodium sulfate. After drying, the solid was filtered out and purified by column chromatography to obtain the product diacetylene lipoic acid polyethylene glycol ester; the synthesis route is as follows:

[0106]

[0107] (3) Add dihydrolipoic acid (100.00 mg, 0.48 mmol, 4.00 eq) obtained in step (1), diacetylene lipoic acid polyethylene glycol ester (152.35 mg, 0.12 mmol, 1.00 eq) obtained in step (2) and photoinitiator DMPA (15.38 mg, 0.06 mmol, 0.50 eq) into a Shrek bottle, remove oxygen three times by double removal, place under a 365 nm ultraviolet lamp, illuminate for 3 h, and collect by precipitation with ice ether to obtain a solid hyperbranched polymer HBP (LA-PEG) ; The synthesis path is as follows:

[0108]

[0109] Nano-preparation HBP prepared in Example 1 (LA) NPs and Bo-HBP (LA) The particle size distribution of NPs nanoformulations in water is shown in the figure below: Figure 7 As shown, from Figure 7 It can be seen that in water environment, nano-preparation HBP (LA) The particle size of NPs is about 29.7nm, and the PDI is about 0.153; the nanoformulation Bo-HBP (LA) The particle size of NPs is approximately 35.88 nm, and the PDI is approximately 0.157.

[0110] 2 mg / mL of Bo-HBP prepared in Example 1 (LA) The dispersion stability of NPs nanoformulations in water and PBS (10 mM, pH 7.4) was Figure 8 As shown, from Figure 8 As can be seen, 2 mg / mL of Bo-HBP (LA) During the 7 days of monitoring, the particle size and PDI of NPs did not change much in either PBS (10 mM, pH 7.4) or water, indicating that Bo-HBP (LA) NPs can be stably present in PBS (10mM, pH 7.4) and water.

[0111] Fig. 9 HBP prepared in Example 1 (LA) NPs and Bo-HBP (LA) Potential diagram of NPs nanoformulations, from Fig. 9 It can be seen that HBP (LA) The potential value of NPs is -33.56, and that of Bo-HBP (LA)The potential of NPs is -27.50, which indicates that the attached borneol structure shields some of the negative charges on the periphery of the nanoparticles, resulting in a slight increase in the potential. The electronegativity makes the surface of the nanoparticles negatively charged, which helps to increase the electrostatic repulsion between the particles and prevent the particles from aggregating in the solution, thereby improving their dispersibility and stability.

[0112] The nuclear magnetic resonance proton spectra of the hyperbranched polymer prepared in Example 1 before and after the addition of hydrogen peroxide are as follows: Fig.10 As shown, from Fig.10 It can be seen that the bond of the thioacetal disappears, indicating that the hyperbranched polymer prepared in Example 1 of the present invention can be effective under the H2O2 condition.

[0113] Performance Testing:

[0114] Fig.11 The Bo-HBP of Example 1 of the present invention (LA) In vitro ABTS free radical scavenging experimental data of NPs nanoformulations; ABTS is oxidized to green ABTS under the action of oxidants. + , has characteristic absorption peaks at 734nm or 405nm. When antioxidants are present, ABTS· + The generation of ABTS is inhibited, causing the reaction system to fade and the absorbance at 405nm to decrease. Within a certain range, the change in absorbance is proportional to the degree to which free radicals are scavenged. The degree of absorbance decrease can reflect the ability of the sample to scavenge ABTS free radicals. Fig.11 It can be seen from the test results that with the increase of the concentration of the nanoformulation, the ability to scavenge ABTS free radicals increases. When the polymer concentration reaches 2.0 mg / mL, the ability to scavenge ABTS free radicals is equivalent to 0.58 mM Trolox standard solution.

[0115] Fig.12 The Bo-HBP of Example 1 of the present invention (LA) In vitro hydroxyl radical scavenging test data of NPs nanoformulations; Hydroxyl radical scavenging test is a commonly used experimental method to determine the scavenging ability of substances to hydroxyl radicals. The experimental principle is to use the Fenton reaction to generate hydroxyl radicals, and then add the test substance with hydroxyl radical scavenging function to reduce the generated hydroxyl radicals, thereby reducing the amount of colored compounds generated accordingly. Fig.12 The test results show that when the concentration of the nanoformulation reaches 1.5 mg / mL, the hydroxyl radical scavenging rate is close to 100%.

[0116] Fig.13 The Bo-HBP of Example 1 of the present invention (LA)In vivo therapeutic effect diagram of NPs nanoformulation; TTC (Tunica Tecta Catalase) staining for stroke is a method used to identify stroke. It uses TTC (triphenyltetrazolium chloride), a redox indicator, to detect the active enzyme in the tissue - catalase (Catalase). Under normal circumstances, catalase exists in brain tissue, which can reduce the dye TTC, making the tissue appear red or orange. When a stroke occurs, the cells in the damaged brain tissue will lose their activity, and catalase will decrease, so TTC cannot be reduced, resulting in the damaged tissue being stained white or grayish white. The experimental method is: after 1 hour of ischemia, reperfusion is immediately administered, and α-lipoic acid (LA) and edaravone (Eda) are used as controls; when the injection dose of the nanoformulation reaches 20mg / kg, it is killed at 24h, and the brain is collected and placed in a special slice mold. It is cut into 2mm thick coronal slices, immediately stained with 1% TTC for 15min at 37°C, and then fixed with 4% paraformaldehyde. Non-ischemic areas are shown in red, and infarcted areas are shown in white. Fig.13 It can be seen from the test results that there is almost no infarction area in TTC after being treated with the nano-preparation of the present invention, indicating that the mice have been effectively treated.

[0117] The hyperbranched polymers of Examples 2 and 3 have similar skeleton structures and response groups to those of Example 1. It can be expected that if they are made into nanoformulations, the nanoformulations can release the active pharmaceutical ingredient of lipoic acid through response under the stimulation of ROS, similar to the nanoformulation of Example 1, to play an antioxidant role and a role in treating ischemic stroke.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A hyperbranched polymer, characterized in that The hyperbranched polymer is obtained by photoinitiated free radical polymerization of compound A and compound B through thiol-alkyne click chemistry; the structure of compound A is shown in formula I, and the structure of compound B is shown in formula II: Wherein, R1 and R2 are each independently -OH, -NH2, -O(CH2CH2O) n One of H, where n is 2-44.

2. A method for preparing a hyperbranched polymer according to claim 1, characterized in that: The following steps are involved: Compound A, compound B and a photoinitiator are mixed and irradiated with an ultraviolet lamp to obtain the hyperbranched polymer.

3. The method for preparing a hyperbranched polymer according to claim 2, characterized in that The photoinitiator includes at least one of benzoin monomethyl ether, benzophenone, benzophenone, phenothiazine, vinyl styrene, and cyclohexylaminobenzophenone.

4. The method for preparing a hyperbranched polymer according to claim 2, wherein The molar ratio of the compound A, the compound B and the photoinitiator is: (4.00-20.00): (1.00-5.00): (0.50-2.50).

5. The method for preparing a hyperbranched polymer according to claim 2, characterized in that: The preparation method of the compound A comprises the following steps: subjecting the raw material A to a reduction reaction under the action of a reducing agent to obtain the compound A; the structure of the raw material A is shown in formula A: In formula A, R1 is -OH, -NH2, -O(CH2CH2O) n One of H.

6. The method for preparing a hyperbranched polymer according to claim 2, characterized in that: When R2 is -OH, the preparation method of compound B comprises the following steps: (1) subjecting thioctic acid to an esterification reaction to obtain thioctic acid ester; (2) subjecting the lipoic acid ester obtained in step (1) to a reduction reaction under the action of a reducing agent to obtain dihydrolipoic acid ester; (3) subjecting the dihydrolipoic acid ester obtained in step (2) to an alkynylation reaction of the thiol group of a compound containing an alkynyl group, followed by hydrolysis to obtain the compound B; When R2 is -NH2, the preparation method of the compound B comprises the following steps: subjecting the raw material B to an alkynyl-containing compound to an alkynylation reaction of the thiol group, followed by hydrolysis to obtain the compound B; the structure of the raw material B is shown in formula B: When R2 is -O(CH2CH2O) n H, the preparation method of the compound B comprises the following steps: subjecting polyethylene glycol and diacetyl lipoic acid to aldol condensation under the action of a condensing agent to obtain the compound B; the diacetyl lipoic acid has a structural formula C as shown:

7. A nano preparation, characterized in that: The nanoformulation comprises the hyperbranched polymer according to claim 1 and borneol.

8. A method for preparing the nanoformulation according to claim 7, characterized in that: The method comprises the following steps: polycondensing the hyperbranched polymer and borneol under the action of a condensation agent, and then self-assembling to obtain the nano preparation.

9. The method for preparing the nanoformulation according to claim 8, characterized in that: The molar ratio of the hyperbranched polymer to borneol is (1.00-5.00):(0.20-1.00).

10. Use of the hyperbranched polymer according to claim 1 and the nanoformulation according to claim 7 in the preparation of a drug for treating ischemic stroke.

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