A ROS-responsive prodrug and step-by-step targeted nanodrug delivery system and its preparation method and application

Abstract

The present invention discloses a ROS-responsive prodrug and a stepwise targeted nanodrug delivery system, as well as preparation methods and applications thereof. The ROS-responsive nanodrug has the structure shown in Formula I: #imgabs0#, and the ROS-responsive nanodrug delivery system comprises nanoparticles self-assembled using a thin film dispersion method using OARB, DSPE-PEG, DSPE-PEG-SHp, and DSPE-PEG-ANG as base materials. The ROS-responsive stepwise targeted delivery system provided by the present invention can achieve specific targeting of the blood-brain barrier and areas of brain damage, overcoming the difficulty of traditional drug delivery in achieving precise delivery and laying a technical foundation for the precise diagnosis and treatment of stroke and related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and in particular relates to a ROS-responsive prodrug and a step-by-step targeted nanomedicine delivery system, a preparation method thereof, and an application thereof in treating brain damage. Background Art

[0002] Research indicates that approximately 1.5 billion people worldwide suffer from central nervous system (CNS) disorders. CNS disorders, such as neurodegenerative diseases, including stroke, Alzheimer's disease, Parkinson's disease, spinal cord injury, and brain tumors, are highly detrimental and expensive to treat, placing a significant mental and financial burden on patients. Despite advances in drug discovery technology, the development of drugs for central nervous system (CNS) disorders remains challenging. Compared to most other areas of drug discovery, the failure rate of new drugs for major CNS diseases is high. Currently, a variety of treatment options are available, including surgery, deep brain stimulation, intravenous (IV), oral and topical formulations, and rehabilitation. However, traditional therapies have limitations. Drugs must enter the general circulation after crossing various physiological barriers, such as the blood-brain barrier (BBB), which is a fraction of the apparent blood volume of distribution. This results in only a small amount of drug reaching the brain, resulting in limited efficacy. In contrast, short-term and highly invasive surgical procedures and brain implants are considered unsafe treatments. Therefore, developing drugs with the ability to cross the BBB and enter the brain holds great promise for enhancing the treatment of CNS diseases.

[0003] An increasing number of studies have shown that danshensu is a potential drug for the treatment of central nervous system diseases. However, the inherent properties of DSS and the blood-brain barrier significantly limit its application in the brain. Therefore, alternative methods are needed to replace traditional drug delivery methods to effectively target DSS to the brain. Nanodelivery technology is considered an effective way to deliver drugs to the brain. Nano-strategies can improve drug concentration in the brain and enhance efficacy based on the disease microenvironment and / or binding to targeting ligands. Summary of the Invention

[0004] The present invention addresses the shortcomings of existing technologies and provides a ROS-responsive prodrug and a stepwise targeted nano-delivery system, as well as their preparation and application. The ROS-responsive stepwise targeted delivery system provided by the present invention can release drugs in the damaged brain area with good targeting, effectively reducing brain damage and improving brain nerve function. It can achieve specific targeting of the blood-brain barrier and the damaged brain area, overcoming the problem of traditional drug delivery that cannot achieve precise delivery, and laying a technical foundation for the precise diagnosis and treatment of stroke and related diseases.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a ROS-responsive nanoprodrug having the structure shown in Formula I:

[0007]

[0008] The second aspect of the present invention provides a method for preparing the above-mentioned ROS-responsive nanoprodrug, comprising:

[0009] 1) condensing oleic acid, ROS chain (2,2'-(propane-2,2-diylbis(sulfonamide))bis(ethane-1-amine)), EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and HOBT (1-hydroxybenzotriazole) in a solvent to obtain OAR ((Z)-N-(2-(2-((2-aminoethyl)thio)prop-2-yl)thio)ethyl)nonadeca-10-enamide); wherein the molar ratio of oleic acid, ROS chain, HOBT, and EDCI is 1-2:1-5:1-5:1-5;

[0010] 2) reacting the OAR obtained in step 1), bromoacetyl chloride, and triethylamine in a solvent to obtain OARB ((Z)-N-(2-((2-(2-bromoacetamido)ethyl)thio)prop-2-yl)thio)ethyl)nonadeca-10-enamide); wherein the molar ratio of the OAR, bromoacetyl chloride, and triethylamine is 1-2:1-5:1-5;

[0011] 3) directly condensing the OARB obtained in step 2) with danshensu to prepare a ROS-responsive nanoprodrug product OAD ((Z)-7,7-dimethyl-2,12-dioxo-6,8-dithio-3,11-diazatriazine-21-en-1-yl (R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropionate); wherein the molar ratio of the OARB to the danshensu is 1:1 to 5.

[0012] Furthermore, in the step 1), the molar ratio of oleic acid, ROS chain, HOBT and EDCI is 1-1.2:1-3:1.2-2:1.2-2.

[0013] Furthermore, the condensation reaction in step 1) is carried out at room temperature, and the reaction time is 24 to 48 hours.

[0014] Furthermore, in the step 2), the molar ratio of OAR, bromoacetyl chloride and triethylamine is 1-1.2:1.5-2:1-1.5.

[0015] Furthermore, the reaction temperature in step 2) is room temperature, and the reaction time is 4 h to 10 h.

[0016] Furthermore, the condensation reaction temperature in step 3) is room temperature, and the reaction time is 24 hours to 48 hours.

[0017] The third aspect of the present invention provides a ROS-responsive nanodrug delivery system comprising the above-mentioned nano prodrug OAD, comprising: the nano drug delivery system is based on OARB, DSPE-PEG (phospholipid-polyethylene glycol), DSPE-PEG-SHp (phospholipid-polyethylene glycol-center homing peptide) and DSPE-PEG-ANG (phospholipid-polyethylene glycol-angiotensin II), and is self-assembled into nanoparticles using a thin film dispersion method.

[0018] A fourth aspect of the present invention provides a method for preparing the above-mentioned ROS-responsive nanodrug delivery system, comprising:

[0019] OARB, DSPE-PEG, DSPE-PEG-SHp and DSPE-PEG-ANG were mixed in a solvent, the solvent was removed by rotary evaporation, and then hydrated in a shaker. The hydrated system was filtered to obtain a ROS-responsive nanodrug delivery system.

[0020] Furthermore, the molar ratio of OARB, DSPE-PEG, DSPE-PEG-SHp and DSPE-PEG-ANG is 1-5:2-10:1-10:1-10.

[0021] Furthermore, the preparation of the DSPE-PEG-SHp comprises:

[0022] Mal-PEG-DSPE and SHp ​​were dissolved in DMF and phosphate buffer, respectively. Under nitrogen protection, the SHp solution was added to the Mal-PEG-DSPE solution. The SHp peptide was covalently coupled to the maleimide group of Mal-PEG-DDPE through the thiol of cysteine. The DSPE-PEG-SHp copolymer was dialyzed for 24 to 48 hours using a membrane with a molecular weight cutoff of 3.5 to 10 kDa.

[0023] Furthermore, the preparation of the DSPE-PEG-ANG comprises:

[0024] DSPE-PEG-Mal and Angiopep-2 at a molar ratio of 1:3 were dissolved in PBS buffer and reacted at room temperature for 24 to 48 hours. The DSPE-PEG-ANG copolymer was dialyzed with deionized water for 48 hours to remove free ANG.

[0025] Furthermore, the rotary evaporation temperature in the step is 37-45° C., and the rotation speed is 80-120 r / min.

[0026] Furthermore, the volume of pure water used in the hydration treatment is 4-10 mL, the temperature is 37-45° C., the time is 1-2 h, and the rotation speed is 100-200 r / min.

[0027] Furthermore, the filtration uses a 220nm filter membrane.

[0028] Another aspect of the present invention also provides the use of the above-mentioned ROS-responsive nano-prodrug and ROS-responsive nano-drug delivery system in the preparation of drugs for treating brain injury.

[0029] Furthermore, the brain injury includes stroke, Alzheimer's disease, traumatic brain injury, glioma, etc.

[0030] The ROS-responsive nanodrug delivery system of the present invention can bind to receptors on brain endothelial cells, helping the entire system to cross the blood-brain barrier and enter the brain. Under the action of DSPE-PEG-SHp, the entire system is brought to the area of ​​brain damage. Under the action of ROS, the ROS response chain breaks, causing the nanoparticles to disintegrate.

[0031] The disintegration of nanoparticles can not only clear the excessive ROS in the damaged environment and inhibit oxidative stress, but also release the drug danshensu, which acts on microglia, regulating their transformation to the M2 anti-inflammatory phenotype and inhibiting the production of inflammatory factors.

[0032] Compared to existing technologies, this invention uses a chemical method to transform the highly water-soluble small molecule drug danshensu into an amphiphilic prodrug responsive to ROS. The prodrug is then self-assembled into responsive nanoparticles using DSPE-PEG, DSPE-PEG-Shp, and DSPE-PEG-ANG for the treatment of brain diseases. The well-prepared nanoparticles not only prolong the circulation time of danshensu in the body but also impart targeting capabilities, enabling them to effectively reach the site of injury and release the drug at a specific location.

[0033] The ROS-responsive nanoparticle drug delivery system provided by the present invention includes a modified peptide DSPE-PEG-ANG targeting the blood-brain barrier to help the entire nanoparticle cross the blood-brain barrier, DSPE-PEG that prolongs circulation in the body to increase the drug's circulation time in the body, a modified peptide DSPE-PEG-SHp that targets the brain injury area to deliver the entire nanoparticle to the injured area, and a self-produced prodrug OAD with excellent biocompatibility that can self-assemble into nanoparticles. The nanoparticle delivery system provided by the present invention can effectively scavenge ROS, inhibit the production of inflammatory factors, reduce cerebral infarction, and improve neurological function.

[0034] The TOAD NPs nanoparticle drug delivery system constructed in this invention can effectively release drugs through step-by-step targeting and ROS response, achieving a synergistic ROS response and anti-inflammatory therapeutic effect, resulting in a highly effective reduction of cerebral infarction. The construction of this nanoplatform provides a new strategy for the treatment of brain diseases through a ROS-responsive step-by-step targeted nanoparticle delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 For DSPE-PEG-SHp and DSPE-PEG-ANG 1 H NMR spectrum; (A) is DSPE-PEG-SHp, (B) is DSPE-PEG-ANG;

[0036] Figure 2 TEM images; (A) is OAD NPs, (B) is TOAD NPs (scale bar = 100 nm);

[0037] Figure 3 are water, particle size and zeta potential; where (A) is the particle size of OAD NPs and TOAD NPs, and (B) is the potential of OAD NPs and TOAD NPs;

[0038] Figure 4 is the drug release curve of TOAD NPs under different pH conditions;

[0039] Figure 5 Results of cell uptake and brain endothelial cell targeting; (A) is the uptake experiment of HBMEC, (B) is the result of TOAD NPs targeting brain endothelial cells detected by flow cytometry, and (C) is the fluorescence quantification result of Figure B;

[0040] Figure 6 is the cell survival rate after cells were incubated with different preparations;

[0041] Figure 7 The results of TOAD NPs system clearing intracellular ROS; (A) is the fluorescence microscopy image of ROS detection using DCFH DA as a ROS probe, and (B) is the fluorescence intensity statistics of DCF;

[0042] Figure 8In vivo evaluation of cerebral ischemia-reperfusion injury. (A) shows the protective effect of different preparations against ischemia-reperfusion injury after 7 days of treatment, (B) shows the protective effect of different preparations against ischemia-reperfusion injury after 14 days of treatment, (C) shows the quantitative graph of the protective effect of different preparations against ischemia-reperfusion injury after 7 days of treatment, and (D) shows the quantitative graph of the protective effect of different preparations against ischemia-reperfusion injury after 14 days of treatment. Note: *P≤0.05, **P≤0.01. N=4, mean±SD.

[0043] Figure 9 The figures show the inhibitory effects of different preparations on brain inflammation in vivo; (A) shows the regulatory effect on M1 microglia, (B) shows the regulatory effect on M2 microglia, (C) shows the fluorescence intensity statistics of CD86, and (D) shows the fluorescence intensity statistics of CD206. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0045] The reagents used in the following examples can all be obtained through commercial sources.

[0046] Example 1: Preparation of ROS-responsive prodrugs

[0047] (1) Synthesis of intermediate product OAR

[0048] Oleic acid (200 mg, 0.7 mmol) was weighed and dissolved in DMF, HOBT (143 mg, 1.1 mmol) and EDCI (203 mg, 1.1 mmol) were added, and the mixture was reacted at room temperature for 30 min. TEA (214.5 mg, 2.1 mmol) and ROS linker (137 mg, 0.7 mmol) were added and the mixture was reacted at room temperature for 24 h. The mixture was purified by column chromatography (petroleum ether: ethyl acetate = 10:1 to 1:1 v / v) to obtain compound OAR, the structure of which is shown in FIG. 1 H NMR identification; 1H NMR(500MHz, CDCl3)δ0.84-0.88(m,3H),1.23-1.35(m,20H),1.59-1.62(m,8H),1.97-2.05(m,4H),2.14-2.17(m,2H), 2.76-2.87(m,4H),2.87(s,2H),2.93-2.96(m,2H),3.43-3.46(q,J=6.5Hz,2H),5.27-5.39(m,2H),6.23-6.24(m,1H).

[0049] (2) Synthesis of intermediate product OARB

[0050] OAR (20.0 g, 43.7 mmol), bromoacetyl chloride (8.2 g, 52.2 mmol) and triethylamine (5.3 g, 52.4 mmol) were weighed and further reacted in THF to obtain OARB, and the mixture was stirred at room temperature under nitrogen for 6 hours until the reaction was completed; after extraction with dichloromethane, the organic layer was dried with Na2SO4 and condensed in vacuo to obtain a crude product; further purification was performed using a chromatographic column (dichloromethane: methanol = 50:1 to 20:1, v / v) to obtain compound OARB, the structure of which is shown in FIG. 1 H NMR identification; 1 HNMR(500MHz, CDCl3)δ0.87-0.90(m,3H),1.29-1.36(m,20H),1.62-1.63(m,8H),1.97-2.07(m,4H),2.17-2.20(m,2H),2 .76-2.83(m,4H),3.45-3.49(m,2H),3.50-3.55(m,2H),3.90-3,91(m,2H),5.30-5.41(m,2H),5.95(s,1H),7.04(s,1H).

[0051] (3) Synthesis of ROS prodrug OAD

[0052] Compound OARB (10 g, 40.82 mmol) was dissolved in N,N-dimethylformamide, and Danshensu DSS (10 g, 45.5 mmol) was added to the solution under magnetic stirring, and the reaction was carried out at room temperature for 24 h until the reaction was completed; the solvent was removed by vacuum rotary evaporation at 70°C, and ethanol was added to the mixture to obtain a crude product; column chromatography purification (dichloromethane: methanol = 50:1-20:1, v / v) was performed to obtain a light yellow oily substance with a structure of 1 Identification by H NMR. 1H NMR(500MHz, CDCl3)δ0.87-0.90(m,3H),1.25-1.36(m,20H),1.57(s,6H),1.60-1.6 3(m,2H),1.96-2.07(m,4H),2.19-2.24(m,2H),2.66-2.72(m,2H),2.72-2.81(m,2H ),2.93-2.96(m,1H),3.02-3.05(m,1H),3.35-3.47(m,4H),4.49-4.51(m,2H),4.89 -4.61(m,2H),5.31-5.41(m,2H),6.31(s,2H),6.61-6.62(m,1H),6.80-6.84(m,2H).

[0053] Example 2: Preparation of ROS-responsive nanodrug delivery system

[0054] (1) Preparation of DSPE-PEG-SHp

[0055] Maleimide-PEG-DSPE (Mal-PEG-DPE, MW of 2000Da, 4mg) and SHp ​​(10mg) were dissolved in N,N-dimethylformamide (DMF) and phosphate buffer (0.2M, pH 7.4), respectively. Under nitrogen protection, the SHp solution was added to the Mal-PEG-DSPE solution, and the SHp peptide was covalently coupled to the maleimide group of Mal-PEG-DDPE via the thiol of cysteine. The DSPE-PEG-SHp copolymer was dialyzed against deionized water using a dialysis bag (MWCO of 3.5kDa). Finally, the obtained DSPE-PEG-SHp was freeze-dried and filtered. 1 Identification by H NMR ( Figure 1 A);

[0056] (2) Preparation of DSPE-PEG-ANG

[0057] DSPE-PEG-Mal and Angiopep-2 at a molar ratio of 1:3 were dissolved in PBS buffer (pH 7.4) and reacted at room temperature for 12 to 24 h. The product DSPE-PEG-ANG was isolated by dialysis against deionized water (MWCO 35 kDa) for 48 h to remove free ANG, and then freeze-dried and filtered. 1 Identification by H-NMR ( Figure 1 B);

[0058] (3) Preparation of targeted nanodelivery system

[0059] TOAD NPs were prepared by a thin film hydration method. OAD, DSPE-PEG2k (Xi'an Ruixi Biotechnology Co., Ltd.), DSPE-PEG-SHp, and DSPE-PEG-ANG were dissolved in DCM and MeOH (v:v = 3:2) at a mass ratio of 1:4:1:1. The solution was rotary evaporated at 37°C and 120 r / min to form a thin film, which was then dried under vacuum overnight to remove any residual organic solvent. The formed film was then hydrated with 5 mL of ddH2O at 40°C for 2 h to achieve complete equilibrium. The film was then filtered through a 0.22 μm membrane to obtain ROS-responsive TOAD NPs.

[0060] (4) Preparation of non-targeted nanodelivery systems

[0061] OAD NPs were prepared by thin film hydration method. OAD and DSPE-PEG2k were dissolved in DCM and MeOH (v:v = 3:2) in a mass ratio of 1:4. The solution was rotary evaporated at 37°C and 120 r / min to form a thin film, which was then dried under vacuum overnight to remove any residual organic solvent. The formed film was then hydrated with 5 mL of ddH2O at 40°C for 2 h to achieve complete equilibrium. The film was then filtered through a 0.22 μm membrane to obtain ROS-responsive OAD NPs.

[0062] Example 3: Characterization of the physicochemical properties of nanoparticles

[0063] (1) Transmission electron microscopy (TEM)

[0064] The OAD NPs and TOAD NPs solutions prepared above were added dropwise onto the super carbon film. After the solvent evaporated, the film was stained in 2% phosphotungstic acid for 5 min and then evaporated. The morphology was observed and images were collected under TEM at an accelerating voltage of 100 kV. The results are shown in Figure 2. Figure 2 As shown in the results, it can be seen that the prepared nanoparticles have good dispersion and the particle size of OAD NPs is about 10nm ( Figure 2 A), TOAD NPs particle size is about 12 nm ( Figure 2 B).

[0065] (2) Determination of particle size and zeta potential

[0066] The particle size and Zeta potential of OAD NPs and TOAD NPs solutions were measured using a DLS analyzer. A small amount of sample was dispersed in ultrapure water and ultrasonically dispersed before measurement at a temperature of 25°C. Figure 3 The results show that the average number of nanoparticles is 10nm, which is consistent with Figure 2 The transmission electron microscopy results are consistent with those of .

[0067] Example 4: Determination of drug loading and drug release in vitro

[0068] (1) Drug loading determination

[0069] High-performance liquid chromatography (HPLC) was used to determine the drug loading of OAD NPs and TOAD NPs. The prepared TOAD NPs were dissolved in methanol and centrifuged to remove the long lipid chains. The peak areas of the supernatant and precipitate were measured by HPLC to calculate the drug loading. The HPLC conditions were: absorption wavelength of 280 nm; mobile phase of methanol:0.05% glacial acetic acid in water = 90:10; flow rate of 1 mL / min. The OAD loading, i.e., the DSS loading, in the OAD and TOAD NPs was determined. The drug loading rate of TOAD NPs was 3.8%, and the encapsulation efficiency was 95.3%.

[0070] (2) Drug release

[0071] The release profiles of DSS from TOAD NPs were measured at pH 6 and pH 6.2 in PBS. TOAD NPs were dispersed in PBS, pH 6, and pH 6.2 solutions. The samples were placed in dialysis bags and tested at 37°C in a constant temperature shaker. 500 μL of the release solution was removed at 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours, and 500 μL of the corresponding solution was added simultaneously. The DSS concentration in the release solution was determined by HPLC, and the cumulative release rates of the two drugs were calculated.

[0072] The results showed that the release amount of nanoparticles was relatively small under physiological conditions, but it could be released quickly under low pH conditions and completely released within 48 hours, indicating that it could be effectively released in the environment of brain damage ( Figure 4 ).

[0073] Example 5: Cellular Uptake and In Vitro Blood-Brain Barrier Targeting

[0074] (1) Cellular uptake

[0075] Nanocarriers were fluorescently labeled with FITC to measure OAD NPs and TOAD NPs for cellular uptake measurements. HBMEC cells were seeded in confocal laser culture dishes and incubated overnight. OAD NPs and TOAD NPs (10 μg / mL) were added to each well and incubated for different times (0.5 h, 1 h, 2 h, 4 h). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and visualized under confocal laser scanning microscope (CLSM).

[0076] The results showed that the targeted nanoparticles TOAD NPs were partially taken up by cells at 2 hours. As time went on, the amount of nanoparticles taken up by cells gradually increased and was completely taken up at 4 hours. However, after co-incubation with cells, the non-targeted nanoparticles OAD NPs were taken up by cells in a small amount at 2 hours, partially taken up at 4 hours, and not completely taken up until 8 hours. This shows that the addition of targeting peptides can significantly increase the uptake of nanoparticles by cells ( Figure 5 A).

[0077] (2) In vitro blood-brain barrier targeting

[0078] The targeting ability of TOAD NPs was determined using the trans-well method. Cells were seeded at a density of 2×105 cells / well in the upper insert of a 12-well transwell plate (polyester film, 0.4m, Corning, USA) and cultured for one week. Transendothelial electrical resistance (TEER) was used to test a cell resistance meter (Millicell ERS2, USA), and TEER exceeding 300ohm·cm2 was used for measurement. Then, 2×105cell / well were seeded in the lower chamber and incubated for 24h. Then, OAD NPs and TOAD NPs solutions were added to the upper chamber and cultured for another 24h. The fluorescence intensity of SH-SY5Y cells in the bottom chamber was detected using CytoFLEX.

[0079] The results showed that when non-targeted nanoparticles OAD NPs and targeted nanoparticles TOAD NPs were incubated with cells for the same time, the targeted nanoparticles TOAD could quickly reach the lower chamber and be taken up by nerve cells. These data indicate that the targeting peptide can bind to the receptors of brain endothelial cells and help the nanoparticles cross the blood-brain barrier ( Figure 5 B).

[0080] Example 6: In vitro cell activity experiment

[0081] SH-SY5Y cells were cultured at a rate of 1 × 10 5 Cells were seeded at a density of 10 cells / well in a 96-well plate and incubated overnight. To establish a cell model of ROS damage, SH-SY5Y cells were incubated with 200 μM tert-butyl hydroperoxide (t-BHP) for 2 hours and then replaced with normal culture medium. They were then treated with OAD and TOAD, respectively. Injured SH-SY5Y cells without any treatment served as a positive control, and uninjured SH-SY5Y cells without any treatment served as a negative control. Cell activity was then detected using CCK8.

[0082] The results showed that the cell viability of cells stimulated by t-BHP decreased significantly, while the cell viability recovered to a certain extent after the action of OAD and TOAD NPs, and the effect was more obvious after the action of TOAD NPs ( Figure 6 ).

[0083] Example 7: Intracellular ROS scavenging

[0084] SH-SY5Y cells were cultured at 5×10 5 The cells were seeded in 6-well plates at a density of 10 cells / well and incubated overnight. To establish a cell model of ROS damage, SH-SY5Y cells were incubated with 200 μM tert-butyl hydroperoxide (t-BHP) for 2 hours and then replaced with normal culture medium. They were then treated with OAD and TOAD NPs, respectively. Injured SH-SY5Y cells without any treatment served as a positive control, and uninjured SH-SY5Y cells without any treatment served as a negative control. To detect the ROS scavenging ability, the SH-SY5Y cells treated each time were stained with DCFH-DA (Abcam, Britain) for 20 minutes for further visualization and photography using an inverted fluorescence microscope (Leica, Germany).

[0085] The results showed that in cells stimulated by t-BHP, the fluorescence of ROS in the cells increased significantly, and the fluorescence in the cells decreased after the action of OAD. The ROS level in the cells decreased significantly after the action of TOAD NPs, indicating that targeted nanoparticles can reduce the ROS in cells and alleviate the oxidative environment in cells ( Figure 7 ).

[0086] Example 8: TOAD NPs reduce cerebral infarction

[0087] (1) Construction of rat MCAO / R model

[0088] A rat MCAO / R model was established using suture occlusion. Briefly, male Sprague-Dawley rats were anesthetized with isoflurane via a pneumatic anesthesia machine. The left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. A nylon suture was then inserted from the ECA into the ICA to block the blood supply to the middle cerebral artery. The suture was removed after 2 hours to allow reperfusion. In the sham-operated group, only the arteries were isolated without suture insertion.

[0089] (2) Animal grouping and drug administration

[0090] Rats undergoing MCAO / R were randomly divided into four groups: model, OAD, OAD NPs, and TOAD NPs. A sham-operated group served as the control group. Rats were injected with PBS or OAD, OAD NPs, or TOAD NPs (based on a uniform conversion of 5 mg / kg of ORD) via the tail vein for 14 days. Twelve hours after the last injection, behavioral testing was performed on days 7, 14, and 21. Rats were then sacrificed, and brain tissue was harvested for subsequent staining.

[0091] TTC results showed that after MCAO, the rats had obvious infarction in their brains. After 7 days of administration, the free drug did not show any therapeutic effect, the non-targeted nanoparticles had a certain therapeutic effect, and the targeted nanoparticles could significantly reduce cerebral infarction. After 14 days of administration, the free drug showed a certain therapeutic effect, and the non-targeted nanoparticles could also reduce cerebral infarction, but the targeted nanoparticles had the most significant effect. This shows that nanoparticles can increase cell uptake to a certain extent and enter the brain more easily than free drugs. After the addition of the targeting peptide, the content of targeted nanoparticles in the brain was further increased under the action of the targeting peptide. Therefore, the effect of the targeted nanoparticles was more obvious ( Figure 8 ).

[0092] Further immunofluorescence staining of brain tissue revealed that after MCAO, the content of inflammatory glial cells (CD86) in brain tissue increased significantly, and anti-inflammatory phenotype microglia (CD206) were almost invisible. After drug treatment, the free drug group, non-targeted nanoparticle group, and targeted nanoparticle group were able to promote the transformation of microglia to an anti-inflammatory phenotype, among which the targeted nanoparticles had the most significant effect, indicating that targeted nanoparticles can improve the inflammatory environment of cells ( Figure 9 ).

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A ROS-responsive nanoprodrug having the structure shown in Formula I: (Formula I).

2. The method for preparing a ROS-responsive nanoprodrug according to claim 1, comprising: 1) condensing oleic acid, a ROS chain as shown in Formula II, EDCI, and HOBT in a solvent to obtain an OAR as shown in Formula III; wherein the molar ratio of oleic acid, ROS chain, HOBT, and EDCI is 1-2:1-5:1-5:1-5; (Formula II) (Formula III) 2) reacting the OAR obtained in step 1), bromoacetyl chloride, and triethylamine in a solvent to obtain OARB as shown in Formula IV; wherein the molar ratio of the OAR, bromoacetyl chloride, and triethylamine is 1-2:1-5:1-5; (Formula IV) 3) directly condensing the OARB obtained in step 2) with danshensu to prepare a ROS-responsive nanoprodrug product, OAD, as shown in Formula I; wherein the molar ratio of OARB to danshensu is 1:1-5; (Formula I).

3. The preparation method according to claim 2, wherein: The molar ratio of oleic acid, ROS chain, HOBT and EDCI in step 1) is 1-1.2:1-3:1.2-2:1.2-2; the condensation reaction temperature is room temperature, and the reaction time is 24h-48h.

4. The preparation method according to claim 2, wherein: The molar ratio of OAR, bromoacetyl chloride and triethylamine in step 2) is 1-1.2:1.5-2:1-1.5; the reaction temperature in step 2) is room temperature, and the reaction time is 4h-10h.

5. The preparation method according to claim 2, wherein: The condensation reaction temperature in step 3) is room temperature, and the reaction time is 24h~48h.

6. A ROS-responsive nanodrug delivery system comprising the nano prodrug OAD according to claim 1, comprising: The nano drug delivery system uses OAD, DSPE-PEG, DSPE-PEG-SHp and DSPE-PEG-ANG as basic materials and adopts a thin film dispersion method to self-assemble into nanoparticles.

7. The method for preparing the ROS-responsive nano drug delivery system according to claim 6, comprising: OAD, DSPE-PEG, DSPE-PEG-SHp and DSPE-PEG-ANG were mixed in a solvent, the solvent was removed by rotary evaporation, and then hydrated in a shaker. The hydrated system was filtered to obtain a ROS-responsive nanodrug delivery system.

8. The method for preparing the ROS-responsive nano drug delivery system according to claim 7, wherein: The molar ratio of OAD, DSPE-PEG, DSPE-PEG-SHp and DSPE-PEG-ANG is 1-5:2-10:1-10:1-10.

9. The method for preparing the ROS-responsive nano drug delivery system according to claim 7, wherein: The volume of pure water used in the hydration treatment is 4-10 mL, the temperature is 37-45° C., the time is 1-2 h, and the rotation speed is 100-200 r / min.

10. Use of the ROS-responsive nano prodrug according to claim 1 and the ROS-responsive nano drug delivery system according to claim 6 in the preparation of drugs for treating brain diseases.

11. The use according to claim 10, characterized in that: The brain diseases include stroke, Alzheimer's disease, traumatic brain injury or glioma.

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