A hyperbranched polymer, nano-preparation and preparation method and application thereof

Abstract

The present application relates to a kind of hyperbranched polymer, nano preparation and its preparation method and application, belong to functional medical material and nanotechnology field.The hyperbranched polymer of the present application can be broken in ROS environment Release dihydrolipoic acid, play the role of antioxidant, be reduced to lipoic acid in vivo, and then play its role as coenzyme, participate in mitochondrial tricarboxylic acid cycle, restore mitochondrial function, thereby play the role of antioxidant and play the role of treating ischemic stroke.The nano preparation provided by the present application can be quickly distributed to brain in a short time, temporarily increase the paracellular permeability of BBB by inhibiting the expression of multi-drug resistance protein P-gp and ATP-dependent drug efflux protein on BBB, and the process of opening BBB by borneol is physiological and reversible, without destroying the structural integrity of blood-brain barrier.

Description

Technical Field

[0001] This invention belongs to the field of functional pharmaceutical materials and nanotechnology, specifically relating to a hyperbranched polymer, nano-formulations, their preparation methods, and applications. Background Technology

[0002] Ischemic stroke (IS) is primarily caused by cerebral vascular occlusion, leading to reduced or interrupted blood supply to the brain tissue. This results in decreased delivery of essential nutrients and oxygen, inducing hypoxia in the brain. Hypoxia promotes leukocyte recruitment to the lesion site, exacerbating oxidative stress and inflammatory damage, further aggravating brain tissue damage. Furthermore, recanalization of the occluded vessel may lead to secondary ischemic / reperfusion (I / R) injury, thereby expanding the infarct area and exacerbating neuronal damage and dysfunction. Damaged neurons and astrocytes also produce reactive oxygen species (ROS), which in turn exacerbate damage to neurons and blood vessels. Therefore, long-term oxidative stress and inflammatory responses may ultimately lead to disruption of the blood-brain barrier (BBB), further deteriorating brain tissue and potentially causing brain parenchymal necrosis.

[0003] The blood-brain barrier (BBB) ​​is a physiological barrier between the blood and brain tissue, formed by cerebral capillary endothelial cells and glial cells. It effectively prevents toxins and other exogenous harmful substances from entering the brain and damaging nerve cells. It has been reported that only lipid-soluble small molecules with a molecular weight <400 Da can cross the BBB. Approximately 98% of small molecule drugs and almost 100% of large molecule drugs cannot reach the brain through peripheral administration. Therefore, the existence of the BBB greatly hinders the development of drugs for central nervous system diseases. Currently, there are no widely applicable drugs for treating stroke. The advent of thrombolytic agents has been epoch-making in the research and treatment of stroke. Clinically, the commonly used drug for stroke treatment is tissue plasminogen activator (tPA), which can dissolve thrombi intravenously. However, due to limitations in treatment conditions and drug side effects (increasing the risk of cerebral hemorrhage), only a small number of patients currently receive effective treatment, and most stroke patients do not receive effective treatment. Furthermore, neuroprotective agents such as glutamate receptor antagonists, antioxidants, apoptosis inhibitors, and cell membrane stabilizers have been found in preclinical trials to antagonize, interfere with, or slow down the main pathophysiological processes of nerve damage, making them the most promising stroke treatments among current neuroprotective strategies. Unfortunately, the vast majority of neuroprotective drugs have proven ineffective in phase II / III clinical trials.

[0004] Two forms of lipoic acid exist in living organisms: oxidized α-lipoic acid (ALA) and reduced dihydrolipoic acid (DHLA). Both possess antioxidant capabilities and can interconvert, working synergistically to achieve antioxidant effects. Lipoic acid is an effective mitochondrial antioxidant, playing a central role in establishing, maintaining, and strengthening the body's antioxidant defense network by effectively scavenging reactive oxygen species (ROS) and regenerating key antioxidants (such as glutathione, GSH). Furthermore, lipoic acid is an essential coenzyme for mitochondrial metabolism, participating in coenzyme reactions essential for the mitochondrial 2-ketoate dehydrogenase complex, including its action on important enzyme systems such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain ketoate dehydrogenase. The absence of lipoic acid blocks the tricarboxylic acid cycle (α-ketoglutarate to succinyl-CoA cycle). When inflammation occurs, the expression of lipoic acid synthase (LASY) is downregulated, leading to a decrease in endogenous lipoic acid. This downregulation of LASY results in a reduction of endogenous lipoic acid, causing redox imbalance, which in turn leads to inflammation and mitochondrial dysfunction. Compared to traditional nanoformulations, lipoic acid undergoes rapid metabolism in the liver during its first pass after oral administration, with an elimination half-life of less than 30 minutes, resulting in insufficient effective concentrations and low bioavailability in the body.

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

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

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a hyperbranched polymer, which is obtained by free radical polymerization of compound A and compound B via a mercapto-alkynyl click chemiluminescence; 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 independently -OH, -NH2, and -O(CH2CH2O), respectively. n One of H, where n is 2-44.

[0011] This invention utilizes a thiol-alkynyl click chemistry reaction between the thiol group in compound A and the alkyne in compound B to construct a hyperbranched polymer with a thioacetal structure. In a ROS environment, this structure can cleave to release dihydrolipoic acid, exerting an antioxidant effect. In vivo, it is reduced to lipoic acid, which then acts as a coenzyme, participating in the mitochondrial tricarboxylic acid cycle and restoring mitochondrial function. Traditionally, after oral administration, lipoic acid undergoes rapid metabolism in the liver during first-pass metabolism, with an elimination half-life within 30 minutes, resulting in insufficient effective concentrations and low bioavailability. In contrast, the hyperbranched polymer of this invention uses modified lipoic acid as its backbone. Upon reaching the site of inflammation, under ROS stimulation, the hyperbranched polymer first responds by releasing the active pharmaceutical ingredient of lipoic acid. This significantly improves the bioavailability of exogenous lipoic acid, exerting antioxidant effects and contributing to the treatment of ischemic stroke.

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

[0013] R is

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

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

[0016] Preferably, the photoinitiator includes at least one of benzoin monomethyl ether (DMPA), benzoin, benzophenone (BDME), thioxanthone, vinylbenzyl ether, and 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 irradiation time of the ultraviolet lamp is 1-6 hours.

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

[0020] In formula A, R1 is -OH, -NH2, or -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 compound A to reducing agent is (1-5):(2-11).

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

[0024] (1) The esterification reaction of lipoic acid yields lipoic acid ester;

[0025] (2) The thioctic acid ester obtained in step (1) is reduced under the action of a reducing agent to obtain dihydrothioctic acid ester;

[0026] (3) The dihydrothioctic acid ester obtained in step (2) is reacted with an alkynyl group-containing compound to undergo a thiol alkynylation reaction, followed by hydrolysis to obtain the compound B.

[0027] The preparation method of the hyperbranched polymer of this invention is simple, low-cost, and highly efficient.

[0028] Specifically, in step (1), there are many ways to perform esterification. For example, under heating conditions and acid catalysis, carboxylic acid and alcohol (methanol or ethanol) undergo a dehydration reaction to generate the corresponding ester (methyl ester or ethyl ester); under alkaline conditions, carboxylic acid and (Boc)2 anhydride react to generate the corresponding tert-butyl ester; under the action of a condensing agent, carboxylic acid and a compound containing a hydroxyl group react to generate the corresponding ester; under alkaline conditions, acyl chloride and a compound containing a hydroxyl group react to generate the corresponding ester; under alkaline conditions and an oxidizing agent, aldehyde and alcohol (such as methanol) react to generate the corresponding ester; under alkaline conditions, cyano reacts with an alcohol and then undergoes acid hydrolysis to generate the corresponding ester. This invention does not limit the esterification reaction in step (1), as long as it can convert lipoic acid into lipoic acid ester. In one specific embodiment of the present invention, step (1) is as follows: under the action of a condensing agent, thioctic acid and anhydrous methanol are mixed to undergo an esterification reaction to obtain methyl thioctic acid; further, the molar ratio of thioctic acid and anhydrous methanol is (1.00-5.00):(10-50).

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

[0030] Specifically, in step (3), the unsaturated alkynyl group and the thiol group can undergo an addition reaction through the alkynylation reaction of the thiol group to jointly form a functional group with ROS response. Common alkynylation reactions of the thiol group (-SH) include: reactions with alkynyl halides, such as the reaction of the thiol group with alkynyl bromine (or alkynyl chloride) to form alkynyl thiol compounds; reactions with alkynyl sulfates, which can also generate alkynyl thiol compounds; and reactions with high-valent iodoalkynyl reagents for rapid and precise modification. These reactions are usually carried out under alkaline conditions to ensure that the thiol group effectively participates in the reaction. This invention does not limit the alkynylation reaction in step (3), as long as the thiol group can be converted into an alkynyl group.

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

[0032] Preferably, hydrolysis can be carried out under alkaline or acidic conditions, such as hydrogen fluoride (HF), ammonium fluoride (NH4F), potassium carbonate, hydrochloric acid (HCl), or 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: mixing trimethylsilyl thiocate with potassium carbonate for hydrolysis to obtain compound B; the molar ratio of trimethylsilyl thiocate to potassium carbonate is (1-5):(8-40).

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

[0034]

[0035] When R2 is -NH2, the reaction conditions for the alkynylation 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 are no restrictions on the alkynylation reaction, as long as the thiol group can be converted into an alkynyl group.

[0036] Preferably, when R2 is -O(CH2CH2O) n At time H, the preparation method of compound B includes the following steps: polyethylene glycol and diacetylacetic acid undergo aldol condensation under the action of a condensing agent to obtain compound B; the structural formula of diacetylacetic acid is shown in C:

[0037]

[0038] Specifically, R2 is -O(CH2CH2O). n In step H, the preparation method of compound B involves an aldol condensation between the hydroxyl groups in polyvinyl alcohol and the carboxyl groups in diacetylacetic acid, thereby achieving the preparation of compound B. The choice of condensing agent is not limited, as long as the above reaction can occur. In one specific embodiment of the invention, the condensing agent may be 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 4-dimethylaminopyridine (DMAP).

[0039] Thirdly, the present invention provides a nano-formulation comprising the hyperbranched polymer and borneol (2-carboxyol).

[0040] This invention's nano-formulation comprises a hyperbranched polymer and borneol. Borneol is a traditional Chinese medicine used for resuscitation, possessing the functions of opening the orifices, reviving the mind, clearing heat, and relieving pain. It also exhibits anti-inflammatory, antioxidant, anticoagulant, and neuroprotective effects, and can rapidly distribute to the brain within a short time (only 5 minutes). By inhibiting the expression of multidrug resistance protein P-gp and ATP-dependent drug efflux proteins on the blood-brain barrier (BBB), it temporarily increases the BBB cell permeability. The process of borneol opening the BBB is physiological and reversible, without disrupting the integrity of the blood-brain barrier structure. This invention's nano-formulation condenses and targets borneol on the surface of the hyperbranched polymer. In an ischemic stroke model, it can achieve rapid and efficient trans-BBB entry into the brain to reach the ischemic area, precisely targeting the lesion site, prolonging circulation time, and improving bioavailability.

[0041] Fourthly, the present invention provides a method for preparing the nano-formulation, comprising the following steps: polycondensing hyperbranched polymer and borneol under the action of a condensing agent, and then performing self-assembly to obtain the nano-formulation.

[0042] The nano-formulation of the present invention consists of borneol with a "drug-guided upward" targeting structure and a hyperbranched polymer backbone with ROS-responsive bonds synthesized by click chemistry, and then obtains a uniformly sized nanoparticle formulation 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 comprises any one of dicyclohexylcarbodiimide (DCC) / N,N-diisopropylethylamine (DIPEA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / 4-dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC) / 4-dimethylaminopyridine (DMAP), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) / triethylamine (TEA).

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

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

[0047] Preferably, the self-assembly is performed using the rapid nanocomplexing (FNC) method; more preferably, in the rapid nanocomplexing 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) (e.g., 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] This invention utilizes a Flash Nanocomplexation (FNC) platform to control the parameters of nanoparticles, resulting in nanoparticles with uniform size, good stability under pathophysiological conditions, and strong pharmacokinetic properties.

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

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

[0051] Fifthly, the present invention provides the application of the hyperbranched polymer and the nano-formulation in the preparation of drugs for treating ischemic stroke.

[0052] Preferably, the concentration range of the nano-formulation for treatment is 10 mg / mL to 20 mg / mL.

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

[0054] The hyperbranched polymer of this invention can cleave to release dihydrolipoic acid in a ROS environment, exerting an antioxidant effect. In vivo, it is reduced to lipoic acid, which then acts as a coenzyme, participating in the mitochondrial tricarboxylic acid cycle and restoring mitochondrial function, thereby playing an antioxidant role and treating ischemic stroke. Furthermore, the nano-formulation provided by this invention consists of borneol with a "drug-guided upward" targeting structure and a hyperbranched polymer backbone with ROS-responsive bonds synthesized using click chemistry. It can rapidly distribute to the brain in a short time (only 5 minutes). By inhibiting the expression of multidrug resistance protein P-gp and ATP-dependent drug efflux proteins on the blood-brain barrier (BBB), it temporarily increases the permeability of BBB cells. The process of borneol opening the BBB is physiological and reversible, without disrupting the structural integrity of the blood-brain barrier. Furthermore, compared to nano-formulations that encapsulate therapeutic agents, the nano-formulations of the present invention have an almost pure drug core, thus exhibiting higher drug loading capacity and lower toxicity. In addition, the nano-formulations not only contain a single drug core but also have higher drug loading capacity and fewer side effects. The nano-formulations themselves are drugs, effectively solving the problems of early drug leakage and difficulty in removing residues after drug release. Attached Figure Description

[0055] Figure 1 This is a flowchart illustrating the synthesis of the hyperbranched polymer obtained in Example 1 of the present invention.

[0056] Figure 2 The 1H NMR spectrum of dihydrolipoic acid prepared in Example 1 of this invention;

[0057] Figure 3 The 1H NMR spectrum of methyl thiocate prepared in Example 1 of this invention;

[0058] Figure 4 The 1H NMR spectrum of methyl dihydrolipoic acid obtained in Example 1 of this invention;

[0059] Figure 5The 1H NMR spectrum of methyl trimethylsilyl thioclate prepared in Example 1 of this invention;

[0060] Figure 6 The 1H NMR spectrum of diacetylacetic acid prepared in Example 1 of this invention;

[0061] Figure 7 The HBP prepared in Example 1 of this invention (LA) NPs and Bo-HBP (LA) Particle size distribution of NPs nanoparticles in aqueous medium;

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

[0063] Figure 9 The HBP prepared in Example 1 of this invention (LA) NPs and Bo-HBP (LA) Potentiogram of NPs nanoparticle formulation;

[0064] Figure 10 The 1H NMR spectra of the hyperbranched polymer prepared in Example 1 of this invention before and after the addition of hydrogen peroxide;

[0065] Figure 11 The Bo-HBP of Embodiment 1 of the present invention (LA) Graph of in vitro ABTS free radical scavenging experimental data of NPs nano-formulation;

[0066] Figure 12 The Bo-HBP of Embodiment 1 of the present invention (LA) Graph of in vitro hydroxyl radical scavenging experimental data for NPs nanoformulation;

[0067] Figure 13 The Bo-HBP of Embodiment 1 of the present invention (LA) In vivo therapeutic effect diagram of NPs nanoparticle formulation. Detailed Implementation

[0068] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0069] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0070] Example 1

[0071] A schematic flowchart of the preparation method of the hyperbranched polymer in this embodiment is shown below. Figure 1 As shown, the specific steps include:

[0072] (1) Lipoic 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 pale yellow solution. Under ice bath conditions, a small amount of sodium borohydride (1.90 g, 50.22 mmol, 2.03 eq) was added and stirred for 30 min. The mixture was then cooled to room temperature and stirred for another 30 min. The mixture was then placed in an ice bath again. 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 solution was purified by column chromatography to obtain a pale yellow oily liquid, dihydrolipoic acid. The NMR spectrum is shown in [reference needed]. Figure 2 The synthesis path is as follows:

[0073]

[0074] (2) Lipoic acid (5.00 g, 24.20 mmol, 1.00 eq) and 10 mL of anhydrous methanol (7.75 g, 242.50 mmol, 10.00 eq) were dissolved in 75 mL of dichloromethane. 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 complete, the solvent and unreacted methanol were removed by evaporation under reduced pressure. The mixture was redispersed in ethyl acetate. The organic phase was washed successively with 1 M hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride. The organic phase was separated, dried with anhydrous sodium sulfate, filtered to remove the solid, and purified by column chromatography to obtain the product methyl lipoate. The NMR spectrum is shown in [reference needed]. Figure 3 The synthesis path is as follows:

[0075]

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

[0077]

[0078] (4) Methyl dihydrolipoic acid (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 reacted for 5 min. Then, TMS-EBX (9.34 g, 26.98 mmol, 3.00 eq) was added and the reaction continued for another 5 min. Tetrahydrofuran was removed by rotary evaporation under reduced pressure. The product was dissolved in ethyl acetate, washed three times with a suitable amount of saturated NaCl solution, and the organic layer was collected. TLC column chromatography was used to purify the organic layer to obtain methyl trimethylsilyl lipoic acid (TMS-alk-DHLA). The NMR spectrum is shown in [reference needed]. Figure 5 The synthesis path is as follows:

[0079]

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

[0081]

[0082] (6) The dihydrolipoic acid (420.00 mg, 2.02 mmol, 4.00 eq) obtained in step (1), the diynyllipoic acid (129.21 mg, 504.00 μmol, 1.00 eq) obtained in step (5), and the photoinitiator DMPA (64.59 mg, 252.00 μmol, 0.50 eq) were added to a Shrek bottle. After three rounds of double deoxygenation, the bottle was placed under a 365 nm ultraviolet lamp for 3 hours. The solid hyperbranched polymer HBP was collected by sedimentation in ice-cold ether. (LA) The synthesis path is as follows:

[0083]

[0084] The obtained hyperbranched polymer is formulated into a nanoparticle, and the preparation method of the nanoparticle includes the following steps:

[0085] S1. Transfer HBP (LA)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 of DMSO. EDCI·HCl (40.83 mg, 0.213 mmol, 0.30 eq) was added dropwise under ice bath conditions. After 12 h of reaction, the mixture was dialyzed against DMSO for 4 h (molecular weight cutoff: 14 kDa), then against water for 12 h (molecular weight cutoff: 14 kDa). The mixture was then lyophilized to obtain solid Bo-HBP. (LA) The synthesis path is as follows:

[0086]

[0087] S2. Using the FNC nano-self-assembly platform, Bo-HBP dissolved in the organic phase DMSO was injected into channel 1 of a three-channel confined impingement jet reactor. (LA) A 20 mg / mL solution was prepared, and ultrapure water was injected into the other two channels. The solutions in all three channels were then thoroughly mixed at a constant preset flow rate using a CNC high-pressure injection pump to prepare uniform nanoparticles. These nanoparticles were then dialyzed in ultrapure water using a dialysis bag (molecular weight cutoff: 14 kDa) to remove the organic solvent DMSO, yielding the nanoparticle formulation (Bo-HBP). (LA) NPs); Similarly, the hyperbranched polymer HBP obtained in step (6) (LA) HBP nanoparticles were prepared using the FNC nano-self-assembly platform. (LA) NPs.

[0088] Example 2

[0089] The preparation method of the hyperbranched polymer in this embodiment includes the following steps:

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

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

[0092]

[0093] (2) Thioctamide (1.04 g, 5.00 mmol, 1.00 eq) was dispersed in 50 mL MeOH, and NaBH4 (0.38 g, 10.00 mmol, 2.00 eq) was added. After stirring at room temperature for 1 h, the reaction mixture was rotary evaporated to obtain a transparent solid dihydrothioctinamide. The synthetic route is as follows:

[0094]

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

[0096]

[0097] (4) Trimethylsilyl thioctinamide (1.00 g, 2.50 mmol, 1.00 eq) was dissolved in 10 mL of methanol. Under ice bath conditions, potassium carbonate (2.67 g, 20.01 mmol, 8.00 eq) was added and the reaction was carried out for 30 min. After removing the ice bath, the reaction was carried out at room temperature for 2 h. The reaction mixture was quenched with H2O. After rotary evaporation of methanol, the mixture was extracted with ethyl acetate, washed three times with saturated NaCl, dried over anhydrous sodium sulfate, and the mixed organic layer was concentrated under vacuum. The mixture was purified by TLC column chromatography to obtain diacetyl thioctinamide. The synthetic route is as follows:

[0098]

[0099] The dihydrolipoamide (100.00 mg, 0.48 mmol, 4.00 eq) obtained in step (2), the diacetylacetamide (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 to a Shrek bottle. After three rounds of double oxygen removal, the bottle was placed under a 365 nm UV lamp for 3 hours. The solid hyperbranched polymer HBP was collected by sedimentation in ice-cold ether. (LA-NH2) The synthesis path is as follows:

[0100]

[0101] Example 3

[0102] The preparation method of the hyperbranched polymer in this embodiment includes the following steps:

[0103] (1) Lipoic 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 pale yellow solution. Under ice bath conditions, a small amount of sodium borohydride (1.90 g, 50.22 mmol, 2.03 eq) was added and stirred for 30 min. The mixture was then cooled to room temperature and stirred for another 30 min. The mixture was then placed in an ice bath again, and the pH was adjusted to 1 with 2 M HCl. The mixture was extracted three times with chloroform (50 mL), and the organic layer was retained and dried over anhydrous sodium sulfate. The solution was purified by column chromatography to obtain a pale yellow oily liquid, dihydrolipoic acid. The synthetic route is as follows:

[0104]

[0105] (2) Diynyl lipoic acid (5.00 g, 24.20 mmol, 1.00 eq) and PEG 1k -OH (24.96 g, 24.20 mmol, 1.0 eq) was dissolved in 75 mL of dichloromethane solution. 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 complete, the dichloromethane solvent was removed by evaporation under reduced pressure. The mixture was redispersed in ethyl acetate. The organic phase was washed successively with 1 M hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride. The organic phase was separated, dried with anhydrous sodium sulfate, filtered to remove the solid, and purified by column chromatography to obtain the product, polyethylene glycol diacetyl lipoic acid. The synthetic route is as follows:

[0106]

[0107] (3) The dihydrolipoic acid (100.00 mg, 0.48 mmol, 4.00 eq) obtained in step (1), the diacetylacetic acid polyethylene glycol ester (152.35 mg, 0.12 mmol, 1.00 eq) obtained in step (2), and the photoinitiator DMPA (15.38 mg, 0.06 mmol, 0.50 eq) were added to a Shrek bottle. After three rounds of double deoxygenation, the bottle was placed under a 365 nm wavelength ultraviolet lamp for 3 hours. The solid hyperbranched polymer HBP was collected by sedimentation in ice-cold ether. (LA-PEG) The synthesis path is as follows:

[0108]

[0109] Nanoparticle formulation HBP prepared in Example 1 (LA) NPs and Bo-HBP (LA) The particle size distribution of NPs nanoparticles in water is shown in the figure below. Figure 7 As shown, from Figure 7 As can be seen, in an aquatic environment, the nano-formulation HBP (LA) The particle size of the NPs is approximately 29.7 nm, and the PDI is approximately 0.153; the nano-formulation Bo-HBP (LA) The particle size of NPs is approximately 35.88 nm, and the PDI is approximately 0.157.

[0110] Bo-HBP prepared in Example 1 at a concentration of 2 mg / mL (LA) The dispersion stability of NPs nanoparticles in both water and PBS (10 mM, pH 7.4) media is as follows: Figure 8 As shown, from Figure 8 It can be seen from this that 2 mg / mL of Bo-HBP (LA) During the 7-day monitoring period, the particle size and PDI of NPs did not change significantly in either PBS (10mM, pH 7.4) or water, indicating that Bo-HBP... (LA) NPs can be stably present in PBS (10 mM, pH 7.4) and water.

[0111] Figure 9 HBP prepared in Example 1 (LA) NPs and Bo-HBP (LA) Potentiogram of NPs nanoparticle formulations, from Figure 9 As can be seen from this, HBP (LA) The potential value of NPs is -33.56, Bo-HBP (LA)The potential of NPs is -27.50, indicating that the attached ice sheet structure shields some of the negative charges on the periphery of the nanoparticles, resulting in a slight increase in potential. Electronegativity makes the surface of the nanoparticles negatively charged, which helps to increase the electrostatic repulsion between particles, prevents particles from aggregating in the solution, and thus improves their dispersibility and stability.

[0112] The 1H NMR spectra of the hyperbranched polymer prepared in Example 1 before and after the addition of hydrogen peroxide are as follows: Figure 10 As shown, from Figure 10 As can be seen, the bonds of the thioacetate disappear, indicating that the hyperbranched polymer prepared in Example 1 of this invention can be effective under the H2O2 conditions.

[0113] Performance testing:

[0114] Figure 11 The Bo-HBP of Embodiment 1 of the present invention (LA) In vitro ABTS free radical scavenging experimental data of NPs nano-formulation; ABTS is oxidized to green ABTS· under the action of oxidant. + ABTS exhibits characteristic absorption peaks at 734 nm or 405 nm when antioxidants are present. + The inhibition of ABTS formation causes the reaction system to fade, and the absorbance at 405 nm decreases. Within a certain range, the change in absorbance is proportional to the degree of free radical scavenging. The degree of absorbance decrease reflects the sample's ability to scavenge ABTS free radicals. Figure 11 The test results show that the ability to scavenge ABTS free radicals increases with the increase of the concentration of the nano-formulation. When the polymer concentration reaches 2.0 mg / mL, the ability to scavenge ABTS free radicals is equivalent to that of 0.58 mM Trolox standard solution.

[0115] Figure 12 The Bo-HBP of Embodiment 1 of the present invention (LA) In vitro hydroxyl radical scavenging experimental data of NPs nanoparticles; the hydroxyl radical scavenging experiment is a commonly used method to determine the scavenging ability of a substance against hydroxyl radicals. The experimental principle is to utilize the Fenton reaction to generate hydroxyl radicals, then add an analyte with hydroxyl radical scavenging function to reduce the generation of hydroxyl radicals, thereby correspondingly reducing the amount of colored compounds formed. Figure 12 The test results show that when the concentration of the nano-formulation reaches 1.5 mg / mL, the hydroxyl radical scavenging rate is close to 100%.

[0116] Figure 13 The Bo-HBP of Embodiment 1 of the present invention (LA)The image shows the in vivo therapeutic effect of NPs nanoparticles. TTC (Tunica Tecta Catalase) staining is a method used to identify stroke. It uses TTC (triphenyltetrazolium chloride), a redox indicator, to detect the active enzyme catalase in tissue. Under normal conditions, catalase is present in brain tissue, which reduces the dye TTC, making the tissue appear red or orange. During a stroke, cells in the damaged brain tissue lose activity, and catalase levels decrease, thus failing to reduce TTC, resulting in the damaged tissue appearing white or grayish-white after staining. The experimental method was as follows: Immediately after reperfusion following 1 hour of ischemia, α-lipoic acid (LA) and edaravone (Eda) were administered as controls. When the nanoparticle injection dose reached 20 mg / kg, the patient was sacrificed after 24 hours, and the brain was collected and placed in a special sectioning mold. The brain was cut into 2 mm thick coronal sections, immediately stained with 1% TTC at 37°C for 15 min, and then fixed with 4% paraformaldehyde. Non-ischemic areas are shown in red, and infarcted areas are shown in white. (Through...) Figure 13 The test results show that TTC treated with the nano-formulation of this invention had almost no infarct area, indicating that the mice received effective treatment.

[0117] The hyperbranched polymers of Examples 2 and 3 have similar backbone structures and responsive groups to those of Example 1. It is expected that if they are formulated into nanoparticles, these nanoparticles, when stimulated by ROS, can release the active pharmaceutical ingredient of thioctic acid in response, similar to the nanoparticles of Example 1, thereby playing an antioxidant role and a therapeutic role in ischemic stroke.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nano-formulation, characterized in that, The nano-formulation includes hyperbranched polymers and borneol; The nano-formulation is obtained by polycondensation of hyperbranched polymer and borneol under the action of a condensing agent, followed by self-assembly. The molar ratio of the hyperbranched polymer to borneol is (1.00-5.00):(0.20-1.00); The hyperbranched polymer is obtained by free radical polymerization of compound A and compound B via mercapto-alkynyl click chemiluminescence; the structure of compound A is shown in Formula I, and the structure of compound B is shown in Formula II. R1 and R2 are each independently -OH.

2. The nano-formulation according to claim 1, characterized in that, The preparation method of the hyperbranched polymer includes the following steps: mixing compound A and compound B with a photoinitiator, and irradiating with ultraviolet light to obtain the hyperbranched polymer.

3. The nano-formulation 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 nano-formulation according to claim 2, characterized in that, The molar ratio of compound A, compound B, and photoinitiator is (4.00-20.00): (1.00-5.00): (0.50-2.50).

5. The nano-formulation according to claim 2, characterized in that, The preparation method of compound A includes the following steps: reducing raw material A under the action of a reducing agent to obtain compound A; the structure of raw material A is shown in formula A: In formula A, R1 is -OH.

6. The nano-formulation according to claim 2, characterized in that, The preparation method of compound B includes the following steps: (1) The esterification reaction of lipoic acid yields lipoic acid ester; (2) The thioctic acid ester obtained in step (1) is reduced under the action of a reducing agent to obtain dihydrothioctic acid ester; (3) The dihydrothioctic acid ester obtained in step (2) is reacted with an alkynyl group-containing compound to undergo a thiol alkynylation reaction, followed by hydrolysis to obtain the compound B.

7. A method for preparing the nano-formulation according to claim 1, characterized in that, The process includes the following steps: polycondensing hyperbranched polymers and borneol under the action of a condensing agent, followed by self-assembly to obtain the nano-formulation.

8. The method for preparing nano-formulations according to claim 7, characterized in that, The molar ratio of the hyperbranched polymer to borneol is (1.00-5.00):(0.20-1.00).

9. The use of the nano-formulation according to claim 1 in the preparation of drugs for treating ischemic stroke.

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