A nano-formulation with dual pH / ROS responsiveness, its preparation method and application

Abstract

This invention provides a pH / ROS dual-responsive nanomedicine carrier, the structure of which is cyclodextrin modified with cinnamaldehyde and 4-hydroxyphenylboronic acid pinacol ester. Lecithin, phospholipid-polyethylene glycol (DSPE-PEG), and phospholipid-polyethylene glycol-RGD peptide are used as the shell of the nanomedicine, and the core consists of glucocorticoids and the prepared pH / ROS dual-responsive drug carrier to form a nanoformulation. This invention designs a pH / ROS dual-responsive drug carrier based on the microenvironment of the disease site. Compared with single pH-responsive or single ROS-responsive drug carriers, this carrier has better microenvironment responsiveness and can more effectively degrade and release therapeutic molecules according to the microenvironment of the lesion site.

Description

Technical Field

[0001] This invention relates to the field of nanoparticle technology, and in particular to a nanoparticle with dual pH / ROS responsiveness, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis is a chronic, systemic autoimmune disease. Anti-inflammatory and immune-reducing drug therapy is the main treatment for rheumatoid arthritis, aside from surgical treatments such as joint replacement. It can alleviate patient suffering and prevent further damage to joint morphology and function.

[0003] Commonly used medications for treating rheumatoid arthritis include nonsteroidal anti-inflammatory drugs (NSAIDs) (such as celecoxib), disease-modifying antirheumatic drugs (DMARDs) (such as methotrexate and biologics), and glucocorticoids (such as dexamethasone and methylprednisolone). Taking glucocorticoids as an example, although they can control inflammation at the lesion site and alleviate patient suffering to some extent, their use is limited due to severe side effects. Traditional oral administration can cause serious adverse reactions such as osteoporosis, increased risk of infection, and even myocardial infarction. This is because maintaining a certain drug concentration at the lesion site requires keeping the drug concentration in the bloodstream at a high level, thus causing serious adverse reactions and severely limiting the use of glucocorticoids to "brief transitional treatment" for short-term, rapid symptom relief. Intra-articular administration, on the other hand, cannot solve the problems faced by drug therapy in clinical application due to inconvenience, poor patient compliance, and excessively long dosing intervals (at least 3-4 months between doses).

[0004] Therefore, developing nanomedicines that can maximize the efficacy of existing rheumatoid arthritis treatments while avoiding their adverse reactions is of great significance for the drug treatment of rheumatoid arthritis and has great clinical application prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a nano-formulation with dual pH / ROS responsiveness, its preparation method, and its application, which solves the problems of poor efficacy and numerous adverse reactions of existing rheumatoid arthritis treatment drugs.

[0006] In one aspect, the present invention provides a pH / ROS dual-responsive nanomedicine carrier, wherein the carrier is a cyclodextrin modified with cinnamaldehyde and 4-hydroxyphenylboronic acid pinacol ester, having the following general formula:

[0007]

[0008] Among them, when n=6, it is α-cyclodextrin; when n=7, it is β-cyclodextrin; and when n=8, it is γ-cyclodextrin.

[0009] In another aspect, the present invention provides a method for preparing a pH / ROS dual-responsive nanomedicine carrier, comprising the following steps:

[0010] (1) Cinnamaldehyde, cyclodextrin and activator 1 are dissolved, heated and then the reaction is terminated. The mixture is filtered, washed and dried to obtain cinnamaldehyde-modified cyclodextrin.

[0011] (2) The cinnamaldehyde-modified cyclodextrin was dissolved, and activator 2 and N,N'-carbonyldiimidazole-activated 4-hydroxyphenylboronic acid pinacol ester were added. The reaction was carried out at room temperature, water was added to precipitate, and the mixture was filtered to obtain a pH / ROS dual-responsive nanomedicine carrier.

[0012] Further, in step (1), the molar ratio of cinnamaldehyde, cyclodextrin and activator 1 is 2-4:1:2-4, and the activator 1 is p-toluenesulfonyl chloride. The heating temperature is 60-80℃ and the heating time is 1-2 days.

[0013] Further, in step (2), the molar ratio of cinnamaldehyde-modified cyclodextrin, N,N'-carbonyldiimidazole-activated 4-hydroxyphenylboronic acid pinacol ester and activator 2 is 1:1-3:1-3, wherein activator 2 is 4-dimethylaminopyridine, and the reaction time is 2-3 days.

[0014] In another aspect, the present invention provides a nanoformulation containing the above-mentioned pH / ROS dual-responsive nanomedicine carrier, wherein the pH / ROS dual-responsive nanomedicine carrier encapsulates a hydrophobic drug, and the outer layer of the pH / ROS dual-responsive nanomedicine carrier is coated with lecithin and distearylphosphatidylethanolamine-polyethylene glycol 2000, and is further coated with distearylphosphatidylethanolamine-polyethylene glycol 3400-RGD as a targeting unit.

[0015] Furthermore, the hydrophobic drug includes glucocorticoids, preferably methylprednisolone.

[0016] Furthermore, the nano-formulation has a particle size of approximately 160-180 nanometers, a polymerization dispersion index of less than 0.2, and a surface potential of -25 eV to -30 eV.

[0017] In another aspect, the present invention provides a method for preparing a nano-formulation containing a pH / ROS dual-responsive nanomedicine carrier, comprising the following steps:

[0018] (1) DSPE-PEG 2000 DSPE-PEG 3400-RGD and lecithin were dispersed in an organic solution, then added to water and ultrasonically dispersed, and heated to obtain solution 1;

[0019] (2) The pH / ROS dual-responsive nanomedicine carrier and the hydrophobic drug were dissolved in methanol and dimethyl sulfoxide to obtain solution 2;

[0020] (3) Add solution 2 to solution 1, stir, and self-assemble at room temperature to obtain nano-formulation.

[0021] Further, in step (1), DSPE-PEG 2000 DSPE-PEG 3400 - The mass ratio of RGD to lecithin is 1-2:1:1-2; in step (2), the mass ratio of pH / ROS dual-responsive nanomedicine carrier to hydrophobic drug is 5-10:1;

[0022] Preferably, the organic solution in step (1) is ethanol, the heating temperature is 50-65℃, and the time is 30 min;

[0023] Preferably, the self-assembly reaction time in step (3) is 1.5-3 hours.

[0024] The present invention also provides a pH / ROS dual-responsive nanomedicine carrier and the application of a nanoformulation containing a pH / ROS dual-responsive nanomedicine carrier in the preparation of a drug for treating rheumatoid arthritis.

[0025] The technical principle of this invention is as follows: The inventors previously determined that the inflammatory sites of rheumatoid arthritis contain high concentrations of reactive oxygen species (such as H2O2, O2). 2- Based on the high expression of hydroxylamine (H2O, etc.) and folic acid receptors, a reactive oxygen species-responsive dexamethasone nanoparticle formulation was prepared, which achieved good therapeutic effects on rheumatoid arthritis. Building on this, and considering the low pH and high reactive oxygen species concentrations at the lesion sites of rheumatoid arthritis, the applicant modified the structure of cyclodextrin using cinnamaldehyde and 4-hydroxyphenylboronic acid pinacol ester to prepare a pH / ROS dual-responsive drug carrier, finding it to achieve even better therapeutic effects. The inventors further modified the drug carrier with a targeting unit, RGD peptide, and found it to achieve even better targeting effects.

[0026] Specifically: The aldehyde group of cinnamaldehyde and the hydroxyl group of cyclodextrin can form an acetal. The acetal maintains good stability under neutral and alkaline conditions, but the bond breaks under acidic conditions. 4-Hydroxyphenylboronic acid pinacol ester is linked to cyclodextrin via a carbonate bond, maintaining good stability under normal physiological conditions, but the bond breaks rapidly under high H2O2 conditions. Since there is almost no hydrogen peroxide in the blood, the pH is neutral, allowing the nanomedicine to maintain good stability in blood circulation until it accumulates at the arthritis site. Upon reaching the arthritis site, the low pH and high concentration of hydrogen peroxide (H2O2) stimulate the carrier material to degrade, disrupting the nanomedicine structure and allowing for the controlled release of the anti-inflammatory drug glucocorticoid, achieving targeted therapy and reducing drug toxicity. Furthermore, the outer layer of the nanomedicine is modified with RGD peptides, which have a strong binding interaction with αvβ3 at the arthritis site, thus actively targeting the arthritis site, increasing the drug concentration at the arthritis site, reducing the distribution of the drug in other organs, and thereby reducing drug toxicity. Meanwhile, the hydroxyl groups of cyclodextrin are modified by cinnamaldehyde and pinacol ester of 4-hydroxyphenylboronic acid, which reduces water solubility and makes it lipophilic.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention designs a drug carrier with dual pH / ROS responsiveness based on the microenvironment of the disease site. Compared with a single pH responsive or a single ROS responsive drug carrier, the carrier has better microenvironment responsiveness and can be more controllably degraded and release therapeutic molecules according to the microenvironment of the lesion site.

[0029] (2) The carrier has hydrophobic properties and can encapsulate hydrophobic drugs such as glucocorticoids through hydrophobic-hydrophobic interactions, forming core-shell nanomedicines under the stabilization of lecithin and phospholipid-polyethylene glycol-ligand.

[0030] (3) The nanomedicine has high dispersibility and small size, so it can be administered intravenously. After intravenous injection, the outer layer of the nanomedicine is modified with polyethylene glycol, which allows it to circulate in the body for a long time, thus reducing the number of administrations.

[0031] (4) The preparation method of this carrier is simple, completed in two steps, and can achieve large-scale preparation. The nanomedicines prepared using this carrier have the advantages of good dispersibility, good drug loading, strong targeting, good microenvironment responsiveness, and good in vivo anti-inflammatory effect. Attached Figure Description

[0032] Figure 1 This is a roadmap for preparing a pH / ROS dual-responsive drug carrier in Example 1 of the present invention.

[0033] Figure 2This is the 1H NMR spectrum of the pH / ROS dual-responsive drug carrier prepared in Example 1 of this invention.

[0034] Figure 3 This is a transmission electron microscope image of the nano-formulation prepared in Example 1 of the present invention.

[0035] Figure 4 This is a roadmap for preparing nano-formulations in Example 1 of the present invention.

[0036] Figure 5 This is an in vivo imaging image of the distribution of the nano-formulation in an arthritis model mouse in Example 5 of the present invention.

[0037] Figure 6 This describes the in vivo efficacy of free drug and nano-formulation in treating rheumatoid arthritis in mice in Example 6 of the present invention. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. All reagents used in the present invention are commercially available.

[0039] Example 1: Preparation method of nano-formulation

[0040] 1. Preparation of pH / ROS dual-responsive drug carriers

[0041] (1) Dissolve 3.34 mmol of cinnamaldehyde, 1.67 mmol of α-cyclodextrin, and 3.34 mmol of p-toluenesulfonyl chloride in 14 mL of dimethyl sulfoxide solution, stir at 60 °C for 2 days, and then stop the reaction. Add 140 mL of acetone to the reaction solution, place in a refrigerator overnight, filter, wash with 50 mL of acetone, and dry to obtain a white solid of cinnamaldehyde-modified cyclodextrin.

[0042] (2) Dissolve 0.6 mmol of cinnamaldehyde-modified cyclopaste in a dry dimethyl sulfoxide solution, then add 0.6 mmol of 4-dimethylaminopyridine and 0.6 mmol of CDI-activated 4-hydroxyphenylboronic acid pinacol ester. Stir the reaction at room temperature for 3 days, then add 40 mL of ultrapure water to the reaction flask. A solid precipitates out. Centrifuge the solid and transfer it to a dialysis bag. Dialyze the solid with distilled water for 2 days and freeze-dry to obtain a pH / ROS dual-responsive drug carrier.

[0043] The route diagram for preparing pH / ROS dual-responsive drug carriers is as follows: Figure 1 As shown. The compound's 1 HNMR such as Figure 2 As shown in the figure, characteristic peaks of the benzene rings of cinnamaldehyde and 4-hydroxyphenylboronic acid pinacol ester can be observed, indicating that both cinnamaldehyde and 4-hydroxyphenylboronic acid pinacol ester have been attached to the cyclodextrin, and the carrier has been successfully prepared.

[0044] 2. Preparation methods of nano-formulations

[0045] 6 mg DSPE-PEG 2000 6 mg DSPE-PEG 3400 -RGD and 6 mg of lecithin were ultrasonically dispersed in 0.4 mL of anhydrous ethanol, followed by the addition of 7 mL of water and continued ultrasonic dispersion. The mixture was then stirred at 65°C for 30 minutes. Separately, 50 mg of the pH / ROS dual-responsive drug carrier prepared in step 1 and 10 mg of methylprednisolone were dissolved in 0.4 mL of methanol and 0.2 mL of dimethyl sulfoxide solution. This solution was added dropwise to the above dispersion, and the mixture was rapidly stirred at 800 rpm for 3 minutes. The mixture was then allowed to self-assemble at room temperature for 2 hours. After centrifugation and washing, the nanoformulation was obtained. A non-targeted nanoformulation was set up as a control group. The non-targeted nanoformulation, without RGD modification, could not actively target the arthritis site. The morphology of the nanoformulation was observed using transmission electron microscopy. Figure 3 As shown, both the non-targeted and targeted nanoformulations are spherical with a particle size of approximately 160-180 nm, a polymerization dispersion index below 0.2, and a surface potential of -25 to -30 eV, making them suitable for in vivo application. The synthetic route for the nanoformulations is as follows: Figure 4 As shown.

[0046] Example 2: Preparation method of nano-formulation

[0047] 1. Preparation of pH / ROS dual-responsive drug carriers

[0048] (1) Dissolve 5.01 mmol of cinnamaldehyde, 1.67 mmol of β-cyclodextrin, and 5.01 mmol of p-toluenesulfonyl chloride in 20 mL of dimethyl sulfoxide solution, stir at 65 °C for 1.5 days, and then stop the reaction. Add 200 mL of acetone to the reaction solution, place in a refrigerator overnight, filter, wash with 70 mL of acetone, and dry to obtain a white solid of cinnamaldehyde-modified cyclodextrin.

[0049] (2) 0.6 mmol of cinnamaldehyde-modified cyclopaste was dissolved in a dry dimethyl sulfoxide solution, and then 1.2 mmol of 4-dimethylaminopyridine and 1.2 mmol of CDI-activated 4-hydroxyphenylboronic acid pinacol ester were added. The mixture was stirred at room temperature for 2.5 days. Then, 50 mL of ultrapure water was added to the reaction flask, and a solid precipitated out. The solid was centrifuged and transferred to a dialysis bag. The solid was dialyzed with distilled water for 2 days and then lyophilized to obtain a pH / ROS dual-responsive drug carrier.

[0050] 2. Preparation methods of nano-formulations

[0051] 12 mg of DSPE-PEG 2000 6 mg DSPE-PEG 3400-RGD and 12 mg of lecithin were ultrasonically dispersed in 0.4 mL of anhydrous ethanol, and then 7 mL of water was added for further ultrasonic dispersion. The mixture was then stirred at 65 °C for 30 min. Separately, 50 mg of the pH / ROS dual-responsive drug carrier prepared in step 1 and 10 mg of methylprednisolone were weighed and dissolved in 0.4 mL of methanol and 0.2 mL of dimethyl sulfoxide solution. This solution was added dropwise to the above dispersion phase, and the mixture was rapidly stirred at 800 rpm for 3 min. The mixture was then allowed to self-assemble at room temperature for 2 hours. After centrifugation and washing, the nano-formulation was obtained.

[0052] Example 3: Preparation method of nano-formulation

[0053] 1. Preparation of pH / ROS dual-responsive drug carriers

[0054] (1) Dissolve 6.68 mmol of cinnamaldehyde, 1.67 mmol of γ-cyclodextrin, and 6.68 mmol of p-toluenesulfonyl chloride in 26 mL of dimethyl sulfoxide solution, stir at 80 °C for 1 day, and then stop the reaction. Add 250 mL of acetone to the reaction solution, place in a refrigerator overnight, filter, wash with 80 mL of acetone, and dry to obtain a white solid of cinnamaldehyde-modified cyclodextrin.

[0055] (2) Dissolve 0.6 mmol of cinnamaldehyde-modified cyclopaste in a dry dimethyl sulfoxide solution, then add 1.8 mmol of 4-dimethylaminopyridine and 1.8 mmol of CDI-activated 4-hydroxyphenylboronic acid pinacol ester, stir at room temperature for 2 days, then add 70 mL of ultrapure water to the reaction flask, a solid precipitates out, centrifuge, transfer the solid to a dialysis bag, dialyze with distilled water for 2 days, freeze dry, and obtain a pH / ROS dual-responsive drug carrier.

[0056] 2. Preparation methods of nano-formulations

[0057] 6 mg DSPE-PEG 2000 6 mg DSPE-PEG 3400 -RGD and 6 mg of lecithin were ultrasonically dispersed in 0.4 mL of anhydrous ethanol, and then 7 mL of water was added for further ultrasonic dispersion. The mixture was then stirred at 60 °C for 30 min. Separately, 50 mg of the pH / ROS dual-responsive drug carrier prepared in step 1 and 5 mg of methylprednisolone were weighed and dissolved in 0.4 mL of methanol and 0.2 mL of dimethyl sulfoxide solution. This solution was added dropwise to the above dispersion phase, and the mixture was stirred rapidly at 800 rpm for 3 min. The mixture was then allowed to self-assemble at room temperature for 3 hours. After centrifugation and washing, the nano-formulation was obtained.

[0058] Example 4: Preparation method of nano-formulation

[0059] 1. Preparation of pH / ROS dual-responsive drug carriers

[0060] (1) Dissolve 4.175 mmol of cinnamaldehyde, 1.67 mmol of α-cyclodextrin, and 4.175 mmol of p-toluenesulfonyl chloride in 17 mL of dimethyl sulfoxide solution, stir at 70 °C for 2 days, and then stop the reaction. Add 160 mL of acetone to the reaction solution, place in a refrigerator overnight, filter, wash with 60 mL of acetone, and dry to obtain a white solid of cinnamaldehyde-modified cyclodextrin.

[0061] (2) Dissolve 0.6 mmol of cinnamaldehyde-modified cyclopaste in a dry dimethyl sulfoxide solution, then add 0.9 mmol of 4-dimethylaminopyridine and 0.9 mmol of CDI-activated 4-hydroxyphenylboronic acid pinacol ester, stir the reaction at room temperature for 0.5 days, then add 45 mL of ultrapure water to the reaction flask, a solid precipitates out, centrifuge, transfer the solid to a dialysis bag, dialyze with distilled water for 2 days, freeze dry, and obtain a pH / ROS dual-responsive drug carrier.

[0062] 2. Preparation methods of nano-formulations

[0063] 9 mg of DSPE-PEG 2000 9 mg DSPE-PEG 3400 -RGD and 9 mg of lecithin were ultrasonically dispersed in 0.6 mL of anhydrous ethanol, and then 7 mL of water was added for further ultrasonic dispersion. The mixture was stirred at 50 °C for 30 min. Separately, 50 mg of the pH / ROS dual-responsive drug carrier prepared in step 1 and 10 mg of methylprednisolone were weighed and dissolved in 0.4 mL of methanol and 0.2 mL of dimethyl sulfoxide solution. This solution was added dropwise to the above dispersion phase, and the mixture was stirred rapidly at 800 rpm for 3 min. The mixture was then self-assembled at room temperature for 1.5 h, centrifuged, and washed to obtain the nano-formulation.

[0064] A standard working curve for methylprednisolone was prepared using high-performance liquid chromatography (HPLC). The nano-formulations obtained by centrifugation in Examples 1-4 were redispersed in 200 μL of deionized water. 20 μL of the dispersed nano-formulation was freeze-dried at low temperature, weighed, and dissolved in 1 mL of a 1:1 mixture of methanol and acetonitrile. The drug content of methylprednisolone was determined by HPLC, and its encapsulation efficiency and drug loading were calculated. The results are shown in Table 1. Wherein:

[0065]

[0066]

[0067] Table 1 Encapsulation efficiency and drug loading of methylprednisolone nanoformulations

[0068] Example 1 Example 2 Example 3 Example 4 Encapsulation efficiency (%) 86.1 81.5 89.6 83.8 Drug loading (%) 9.2 8.9 6.5 8.3

[0069] Example 5: Evaluation of the in vivo targeting effect of nano-formulation

[0070] A mouse model of rheumatoid arthritis was established. The nano-formulations and non-targeted nano-formulations prepared in Example 1 were labeled with the fluorescent probe Cy5. The free drug was covalently linked to Cy5. Model mice were administered Cy5-labeled free drug, Cy5-labeled non-targeted nano-formulations, and Cy5-labeled targeted nano-formulations, respectively. The control group consisted of normal mice administered an equal volume of physiological saline. Figure 5 As shown, non-targeted nano-formulations accumulate significantly more at the arthritis site than free drugs. This is because non-targeted nano-formulations can passively target and accumulate at the arthritis site through enhancement and retention effects, thus accumulating more at the site than free drugs. Conversely, targeted nano-formulations accumulate significantly more at the arthritis site than non-targeted nano-formulations. This is because the targeted nano-formulations are modified with RGD peptides, which can specifically bind to αvβ3, which is highly expressed at the arthritis site, thus actively targeting the arthritis site and accumulating there. These results demonstrate that the pH / ROS dual-responsive nano-formulations prepared in this invention can actively target the arthritis site.

[0071] Example 6: Validation of in vivo therapeutic activity for rheumatoid arthritis

[0072] A mouse model of rheumatoid arthritis was established to evaluate the in vivo anti-inflammatory activity of the nanoformulation. Figure 6 As shown, the toes of the model mice showed significant swelling compared to normal mice, indicating successful model establishment. Treatment with the blank nano-formulation (without methylprednisolone) did not significantly reduce toe swelling, demonstrating the poor anti-inflammatory effect of the blank nano-formulation. Treatment with the free methylprednisolone group and the non-targeted nano-formulation group alleviated toe swelling to some extent, demonstrating that both free methylprednisolone and the non-targeted nano-formulation (Example 1) had some therapeutic effect. However, after treatment with the targeted nano-formulation (Example 1), the swelling of the toes was significantly reduced, approaching that of the normal group, indicating that the targeted nano-formulation had the best therapeutic effect. Small animal CT results showed that the model mice's toes suffered severe erosion due to inflammation, but the bone erosion was alleviated after treatment with the free drug and the non-targeted nano-formulation. After treatment with the targeted nano-formulation, the mouse joints became smooth, approaching that of the normal group, indicating that the targeted nano-formulation had a good anti-inflammatory effect.

[0073] Comparative Example 1: Preparation method of nano-formulation

[0074] Similar to Example 1, the difference is that step 1 only includes step (1) and does not include step (2), thus obtaining cinnamaldehyde-modified cyclodextrin. Meanwhile, nano-formulations are prepared according to the method in step 2.

[0075] Preparation method of comparative 2 nano-formulation

[0076] Similar to Example 1, the difference is that step 1 does not include step (1), but only step (2) is included, that is, CDI-activated 4-hydroxyphenylboronic acid pinacol ester modified cyclodextrin is obtained. At the same time, nano-formulations are prepared according to the method in step 2.

[0077] The effects were tested using the experimental method described in Example 6. The results showed that the anti-inflammatory effects of the nano-formulation prepared in Comparative Example 1 and Comparative Example 2 were significantly less than those of the nano-formulation prepared in Example 1. This may be because cinnamaldehyde has a certain anti-arthritis effect, and the two have a mutually promoting and synergistic effect in anti-inflammatory efficacy.

[0078] Therefore, the drug carrier with dual pH / ROS responsiveness prepared in this invention is significantly better than the drug carriers with only pH responsiveness or only ROS responsiveness.

[0079] 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 it. 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 spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A pH / ROS dual-responsive nanomedicine carrier, characterized in that, The carrier is a cyclodextrin modified with cinnamaldehyde and 4-hydroxyphenylboronic acid pinacol ester, which has the following general formula: Among them, when n=6, it is α-cyclodextrin; when n=7, it is β-cyclodextrin; and when n=8, it is γ-cyclodextrin. It is prepared by the following method: (1) Cinnamaldehyde, cyclodextrin and p-toluenesulfonyl chloride were dissolved, heated, and then the reaction was terminated. The mixture was filtered, washed and dried to obtain cinnamaldehyde-modified cyclodextrin. (2) The cinnamaldehyde-modified cyclodextrin was dissolved, and 4-dimethylaminopyridine and N,N'-carbonyldiimidazole-activated 4-hydroxyphenylboronic acid pinacol ester were added. The reaction was carried out at room temperature, water was added to precipitate, and the mixture was filtered to obtain a pH / ROS dual-responsive nanomedicine carrier.

2. The method for preparing a pH / ROS dual-responsive nanomedicine carrier according to claim 1, comprising the following steps: (1) Cinnamaldehyde, cyclodextrin and p-toluenesulfonyl chloride were dissolved, heated, and then the reaction was terminated. The mixture was filtered, washed and dried to obtain cinnamaldehyde-modified cyclodextrin. (2) The cinnamaldehyde-modified cyclodextrin was dissolved, and 4-dimethylaminopyridine and N,N'-carbonyldiimidazole-activated 4-hydroxyphenylboronic acid pinacol ester were added. The reaction was carried out at room temperature, water was added to precipitate, and the mixture was filtered to obtain a pH / ROS dual-responsive nanomedicine carrier.

3. The method for preparing a pH / ROS dual-responsive nanomedicine carrier as described in claim 2, characterized in that, In step (1), the molar ratio of cinnamaldehyde, cyclodextrin and p-toluenesulfonyl chloride is 2-4:1:2-4, the heating temperature is 60-80℃, and the heating time is 1-2 days.

4. The method for preparing a pH / ROS dual-responsive nanomedicine carrier as described in claim 2, characterized in that, In step (2), the molar ratio of cinnamaldehyde-modified cyclodextrin, N,N'-carbonyldiimidazole-activated 4-hydroxyphenylboronic acid pinacol ester and 4-dimethylaminopyridine is 1:1-3:1-3, and the reaction time is 2-3 days.

5. A nanoformulation containing the pH / ROS dual-responsive nanomedicine carrier of claim 1, characterized in that, The pH / ROS dual-responsive nanomedicine carrier encapsulates a hydrophobic drug, including glucocorticoids. The outer layer of the pH / ROS dual-responsive nanomedicine carrier is coated with lecithin and distearate phosphatidylethanolamine-polyethylene glycol 2000, and is further coated with distearate phosphatidylethanolamine-polyethylene glycol 3400-RGD as a targeting unit.

6. The nanoformulation containing a pH / ROS dual-responsive nanomedicine carrier as described in claim 5, characterized in that, The glucocorticoid in question is methylprednisolone.

7. The nanoformulation containing a pH / ROS dual-responsive nanomedicine carrier as described in claim 5, characterized in that, The nanoparticles have a particle size of approximately 160-180 nanometers, a polymerization dispersion index of less than 0.2, and a surface potential of -25 eV to -30 eV.

8. A method for preparing a nano-formulation containing a pH / ROS dual-responsive nanomedicine carrier as described in any one of claims 5-7, characterized in that, Includes the following steps: (1) Disteazyle phosphatidylethanolamine-polyethylene glycol 2000, disteazyle phosphatidylethanolamine-polyethylene glycol 3400-RGD and lecithin were added to an organic solution and dispersed. Then, they were added to water and dispersed by ultrasonication and heated to obtain solution 1. (2) Dissolve the pH / ROS dual-responsive nanomedicine carrier and hydrophobic drug described in claim 1 in methanol and dimethyl sulfoxide to obtain solution 2; (3) Add solution 2 to solution 1, stir, and self-assemble at room temperature to obtain nano-formulation.

9. A method for preparing nano-formulations containing pH / ROS dual-responsive nanomedicine carriers as described in claim 8, characterized in that, In step (1), the mass ratio of distearylphosphatidylethanolamine-polyethylene glycol 2000, distearylphosphatidylethanolamine-polyethylene glycol 3400-RGD and lecithin is 1-2:1:1-2; in step (2), the mass ratio of pH / ROS dual-responsive nanomedicine carrier and hydrophobic drug is 5-10:

1.

10. A method for preparing a nano-formulation containing a pH / ROS dual-responsive nanomedicine carrier as described in claim 8, characterized in that, The organic solution mentioned in step (1) is ethanol, and the heating temperature is 50-65℃ for 30 min.

11. A method for preparing nano-formulations containing pH / ROS dual-responsive nanomedicine carriers as described in claim 8, characterized in that, The self-assembly reaction time in step (3) is 1.5-3 hours.

12. The use of the nanoformulation containing a pH / ROS dual-responsive nanomedicine carrier as described in claim 6 in the preparation of a drug for treating rheumatoid arthritis.

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