Nanoparticles, methods of making and using the same

Abstract

The present application relates to a kind of nanoparticles and its preparation method and application, it is related to the field of nanotechnology. Nanoparticle is prepared by the reaction of 3'-(propane-2,2-sulfane) dipropionic acid with hydroxychloroquine, carrier, can inhibit neutrophil autophagy, realize the effect of efficient drug delivery, and the nanoparticle has good stability, biocompatibility, active oxygen response, can also respond to intracellular H2O2 in the case where chronic inflammation microenvironment, neutrophil is highly activated, intracellular ROS level increases, make drug break and play a role;Meanwhile, the nanoparticle improves the toxicity of hydroxychloroquine.

Description

Technical Field

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

[0002] One of the most common complications of diabetes is diabetic ulceration, which severely impairs limb function and quality of life, sometimes requiring amputation. Reports also indicate that diabetic patients with foot ulcers have a 10-year doubled risk of death. Traditional treatments for these complications involve controlling blood sugar levels while providing anti-infection, debridement, and circulation improvement. However, the uncertain pathogenesis of diabetic ulcers leads to a lack of targeted clinical treatment and limited efficacy. Persistent inflammation is one of the main reasons why chronic diabetic wounds are difficult to heal. Neutrophils in diabetic patients produce a large number of NETs, ​​forming NETosis, which delays wound healing. NET formation is mediated by autophagy. Based on previous research and understanding of the mechanism of NETosis, the most likely reasons for tissue damage and wound healing in diabetic patients under persistent chronic inflammation are: high neutrophil activation, increased ROS levels, and enhanced neutrophil autophagy-mediated NET formation. Therefore, researchers believe that by inhibiting autophagy, preventing NETosis, reducing NET formation, and inducing neutrophil apoptosis, the inflammatory microenvironment of the wound can be improved by regulating immune cells, which may promote wound repair and tissue regeneration and solve the problem of impaired wound healing in diabetic patients.

[0003] Currently, methods for inhibiting autophagy include siRNA-mediated silencing of autophagy-related genes (such as beclin 1 or ATG5) and cell therapy using autophagy inhibitors. In addition, studies have reported that some active ingredients in traditional Chinese medicine, such as andrographolide, triptolide, and quercetin, can alleviate RA by inducing neutrophil apoptosis through inhibiting autophagy-mediated NETosis. However, due to their short lifespan and high activation tendency, neutrophils are not suitable for autophagy gene silencing, and the above methods are not very effective in inhibiting neutrophil autophagy. Pharmacological inhibitors such as 3-methyladenine, chloroquine, and bafloxacin A1 should also be used with caution, as these inhibitors may have off-target effects in cells. Furthermore, active ingredients in traditional Chinese medicine may produce some unnecessary side effects. Therefore, selecting appropriate autophagy inhibition methods for neutrophils is necessary to achieve better results in improving disease symptoms. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a nanoparticle that can improve the toxicity of hydroxychloroquine, exhibits good biocompatibility and reactive oxygen species (ROS) response, inhibits neutrophil autophagy, promotes tissue regeneration and repair, and achieves efficient drug delivery.

[0005] To achieve the above objectives, the present invention provides a nanoparticle, the raw materials for which the nanoparticle is prepared include: 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia, solvent, acid-binding agent, esterification catalyst, and support;

[0006] The mass-to-volume ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the solvent is (0.001-0.02 g) / mL, the molar ratio of the acid-binding agent to the esterification catalyst is (0.5-2):2, the molar ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the esterification catalyst is 1:(1.1-1.3), and the weight ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the support is 1:(1-2).

[0007] The raw materials for preparing 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolic acid include: hydroxychloroquine and 3'-(propane-2,2-thion)dipropionic acid; the weight ratio of hydroxychloroquine to 3'-(propane-2,2-thion)dipropionic acid is (1-2):1.

[0008] During their research on neutrophils and inflammatory responses, the inventors discovered that hydroxychloroquine (HCQ), an FDA-approved drug, is currently the primary compound used in clinical trials aimed at treating tumors through autophagy inhibition. While numerous studies have been conducted on HCQ-based inhibition of neutrophil autophagy, achieving significant progress in alleviating inflammation, the side effects of long-term HCQ treatment persist, with retinopathy, neuromuscular, and cardiotoxicity being among the most serious and life-threatening side effects. Therefore, there is a need to improve HCQ to make it more effective and less toxic, and to identify the potential cellular and biological pathways specifically regulated by HCQ, thereby making it more suitable for inhibiting neutrophil autophagy. This has significant clinical implications for improving inflammatory diseases. Based on the above, the inventors proposed preparing the nanoparticles of this invention by reacting 3'-(propane-2,2-thionane)dipropionic acid (TK) with hydroxychloroquine (HCQ) and a support. These nanoparticles utilize the two carboxyl groups in the 3'-(propane-2,2-thionane)dipropionic acid structure to undergo a condensation reaction with the hydroxyl groups on hydroxychloroquine, preparing 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia. Then, 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia reacts with... The carrier undergoes a reaction to prepare nanoparticles that can inhibit neutrophil autophagy, achieving efficient drug delivery. Furthermore, by utilizing 3'-(propane-2,2-thionane)dipropionic acid (TK) as a ROS-responsive group, it not only connects the hydrophilic and hydrophobic portions of the nanoparticles, giving them good stability, biocompatibility, and reactive oxygen species responsiveness, but also allows them to respond to intracellular H2O2 in the context of a chronic inflammatory microenvironment, highly activated neutrophils, and increased intracellular ROS levels, causing drug fragmentation and exerting its effect. Moreover, because hydroxychloroquine has been chemically modified, these nanoparticles improve the toxicity of hydroxychloroquine.

[0009] In one embodiment, the 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiaza was prepared by dissolving hydroxychloroquine and 3'-(propane-2,2-thionane)dipropionic acid, mixing them, carrying out an esterification reaction, and then purifying them.

[0010] In one embodiment, the mass-to-volume ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the solvent is 0.01-0.02 g / mL, the molar ratio of the acid-binding agent to the esterification catalyst is (1-2):2, the molar ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the esterification catalyst is 1:1.2, and the weight ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the support is (1.5-2):1.

[0011] In one embodiment, the solvent comprises at least one of 1,4-dioxane, dichloromethane, and N,N-dimethylformamide; the acid-binding agent is triethylamine; the esterification catalyst comprises N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and the carrier is albumin.

[0012] The aforementioned albumin plays a role in maintaining plasma colloid osmotic pressure and has good water solubility. Using albumin as a carrier, it can bind to exogenous drugs, acting as a "ferry" for drugs to circulate in the bloodstream and targeting inflammatory-activated neutrophils. Simultaneously, albumin possesses good biocompatibility, non-immunogenicity, and biodegradability, making it suitable as a medium for drug regression and detection, thereby improving the toxicity of hydroxychloroquine (HCQ) and maximizing its efficacy. One of the two carboxyl groups in 3'-(propane-2,2-thionane)dipropionic acid (TK), one of which undergoes a condensation reaction with hydroxychloroquine (HCQ), and the remaining carboxyl group undergoes a condensation reaction with the amino group of albumin, thus linking hydroxychloroquine and albumin together. This achieves the binding of the drug component to the carrier, and the loading method differs from conventional physical encapsulation synthesis methods.

[0013] In one embodiment, the solvent comprises 1,4-dioxane.

[0014] The yield of the target product is higher when the solvent includes the above components.

[0015] The present invention also provides a method for preparing the nanoparticles, comprising the following steps:

[0016] Preparation of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolidinedioic acid: Hydroxychloroquine and 3'-(propane-2,2-thionane)dipropionic acid were dissolved, mixed, and subjected to esterification reaction, followed by purification.

[0017] Preparation of nanoparticles: 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia was dissolved in a solvent to obtain a 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia solution. The 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia solution was mixed with an esterification catalyst and an acid-binding agent, and reacted in the dark. A support was added, and the mixture was mixed to carry out a condensation reaction. After dialyzing and freeze-drying, nanoparticles were obtained by microfluidic method.

[0018] The above preparation method is simple, efficient, low-cost, requires minimal equipment, has mild preparation conditions, and a short cycle, making it suitable for mass production.

[0019] In one embodiment, the hydroxychloroquine is prepared by dissolving hydroxychloroquine sulfate in water, adding an alkaline solution to obtain a precipitate, extracting, removing water, allowing to stand, and rotary evaporating to obtain hydroxychloroquine.

[0020] In one embodiment, the alkaline solution is ammonia.

[0021] In one embodiment, the 3'-(propane-2,2-thion)dipropionic acid is prepared by mixing 3-mercaptopropionic acid with acetone, adding a catalyst, reacting, washing, and freeze-drying to obtain 3'-(propane-2,2-thion)dipropionic acid.

[0022] In one embodiment, the catalyst is trifluoroacetic acid.

[0023] In one embodiment, in the step of preparing 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolic acid, the esterification reaction is catalyzed by an EDC / DMAP system, and the esterification reaction time is 40-55 h; the purification is achieved by preparative chromatography.

[0024] In the step of preparing nanoparticles, the time for the light-protected reaction is 10-14 h, the temperature for the condensation reaction is 22-27 °C, and the time for the condensation reaction is 10-14 h.

[0025] During the research and development process, the inventors made various attempts to develop catalytic systems for the preparation of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazo, such as DCC / DMAP, EDC / DMAP, and NHS / EDC. Ultimately, the EDC / DMAP system was selected as the catalytic system, which resulted in a better reaction yield for 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazo.

[0026] The present invention also provides a pharmaceutical composition comprising the nanoparticles and pharmaceutically acceptable excipients.

[0027] The present invention also provides the use of the nanoparticles in the preparation of drugs for inhibiting neutrophil autophagy or for treating inflammation.

[0028] The drug prepared using the above-mentioned nanoparticles can inhibit neutrophil autophagy, improve inflammation, and has a good effect on promoting the healing of diabetic wounds.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] This invention discloses a nanoparticle, its preparation method, and its application. This nanoparticle can target inflammatory-activated neutrophils, inhibit autophagy, and achieve highly efficient drug delivery, allowing the drug components to better exert their therapeutic effects. It also exhibits good stability, biocompatibility, reactive oxygen species responsiveness, regular morphology, uniform size, and good dispersibility. In the context of a chronic inflammatory microenvironment, highly activated neutrophils, and increased intracellular ROS levels, it responds to intracellular H2O2, causing drug fragmentation and exerting its effect. Furthermore, it improves the toxicity of hydroxychloroquine, providing a reference for drug development for inflammation-related diseases. The preparation method is simple, efficient, low-cost, requires minimal equipment, has mild preparation conditions, and a short cycle time, offering the advantage of being suitable for mass production. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the synthetic route of hydroxychloroquine in Example 1;

[0032] Figure 2 The image shows the 1H NMR and 1C NMR spectra of hydroxychloroquine in Example 1.

[0033] Figure 3 This is a schematic diagram of the synthetic route for 3'-(propane-2,2-thionane)dipropionic acid (TK) in Example 1;

[0034] Figure 4 The 1H NMR spectrum and 1C NMR spectrum of 3'-(propane-2,2-thionane)dipropionic acid (TK) in Example 1;

[0035] Figure 5 This is a schematic diagram of the synthetic route for 17-((7-chloroquinoline)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia (HCQ-TK) in Example 1.

[0036] Figure 6 The 1H NMR spectrum and 1C NMR spectrum of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia (HCQ-TK) in Example 1;

[0037] Figure 7 This is a schematic diagram of the synthesis route of HCQ-TK-HSA NPs in Example 1;

[0038] Figure 8 The infrared absorption spectra of hydroxychloroquine (HCQ), 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolidinedioic acid (HCQ-TK), and nanoparticles (HCQ-TK-HSA NPs) in Example 1 are shown below. The three curves, from top to bottom, represent hydroxychloroquine (HCQ), 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolidinedioic acid (HCQ-TK), and nanoparticles (HCQ-TK-HSA NPs).

[0039] Figure 9 The particle size distribution diagram of the nanoparticles (HCQ-TK-HSA NPs) in the experimental example is shown. From left to right, they are HTH1, HTH2, HTH3, and HTH4.

[0040] Figure 10 The figure shows the stability results of the nanoparticles (HCQ-TK-HSA NPs) in plasma in the experimental example;

[0041] Figure 11 The image shows the drug release curves of the nanoparticles (HCQ-TK-HSA NPs) in the experimental example, which release HCQ in response to different concentrations of H2O2.

[0042] Figure 12 This is a diagram showing the results of the biocompatibility verification experiment in the experimental example;

[0043] Figure 13 This is a diagram showing the wound treatment effect in the experimental case;

[0044] Figure 14 This is a quantitative graph of wound closure rate in the experimental case;

[0045] Figure 15This is a diagram showing the H&E staining results in the experimental example;

[0046] Figure 16 This is a quantitative graph of epidermal regeneration in the experimental example;

[0047] Figure 17 This is a quantitative graph of granulation tissue thickening in the experimental example;

[0048] Figure 18 This is a diagram showing the results of Masson staining in the experimental example;

[0049] Figure 19 This is a quantitative graph of collagen deposition in the experimental example;

[0050] Figure 20 This is a graph showing the fluorescence expression results of CD31.

[0051] Figure 21 A quantitative map of the area of ​​CD31-positive regions. Detailed Implementation

[0052] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0054] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.

[0055] definition:

[0056] EDC: refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0057] DMAP: refers to 4-dimethylaminopyridine.

[0058] NHC: refers to N-hydroxysuccinimide.

[0059] Example 1

[0060] A type of nanoparticle.

[0061] The preparation method of the nanoparticles is as follows.

[0062] I. Preparation of hydroxychloroquine (HCQ).

[0063] Dissolve 5g of hydroxychloroquine sulfate in deionized water, slowly add an appropriate amount of ammonia (alkaline solution), and react at room temperature for 20 minutes until no white turbidity forms. Then extract three times with dichloromethane. After extraction, add anhydrous sodium sulfate to remove residual water and let stand overnight. Evaporate to dryness under vacuum to obtain hydroxychloroquine, which will be used in subsequent experiments.

[0064] The above synthesis path is as follows Figure 1 As shown, the proton and carbon NMR spectra of hydroxychloroquine are as follows: Figure 2 As shown.

[0065] II. Preparation of 3'-(propane-2,2-thionane)dipropionic acid (TK).

[0066] 10 g of 3-mercaptopropionic acid was added to a round-bottom flask containing 2.6 g of acetone, followed by the addition of 40 μL of trifluoroacetic acid (catalyst). The mixture was reacted at 0 °C for 6 hours, then washed with ice water and n-hexane, respectively, repeating the process three times. After freeze-drying, a white solid product was obtained for subsequent experiments.

[0067] The above synthesis path is as follows Figure 3 As shown, the 1H NMR spectrum and 1C NMR spectrum of 3'-(propane-2,2-thionane)dipropionic acid (TK) are as follows. Figure 4 As shown.

[0068] III. Preparation of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia (HCQ-TK).

[0069] First, 4.1 g of 3'-(propane-2,2-thion)dipropionic acid (TK) and 5.5 g of hydroxychloroquine (HCQ) were dissolved in dichloromethane in a round-bottom flask containing dichloromethane. The esterification reaction was carried out for 2 days using an EDC / DMAP system as a catalyst, causing the carboxyl group of 3'-(propane-2,2-thion)dipropionic acid (TK) to condense with the hydroxyl group of hydroxychloroquine (HCQ). The resulting product was purified by preparative chromatography.

[0070] The specific method for preparing the above chromatogram is as follows:

[0071] Purification was performed using an HPLC column. HPLC sample processing involved dissolving the product thoroughly in methanol solution, then diluting it with a mobile phase (0.045 mol / L potassium dihydrogen phosphate solution (pH = 3.0): acetonitrile = (85:15)) to achieve a product concentration of approximately 100 mg / L. The solution was then filtered through a 0.45 μm aqueous filter membrane for HPLC analysis. Quantification was performed using the area normalization method, yielding a white product for subsequent experiments.

[0072] The conditions for the above liquid chromatography analysis were as follows: Column: Hypersil GOLD™, C18, 250 mm * 4.6 mm, 5 μm; Detector: DAD 3000 (UV detector); Mobile phase: 0.045 mol / L potassium dihydrogen phosphate solution (pH = 3.0): acetonitrile = (85:15); Flow rate: 1.0 mL / min; Detection wavelengths: 210 nm, 254 nm, 329 nm, 343 nm; Column temperature: 30 ℃; Injection volume: 10 μL; Run time: 15 min.

[0073] The above synthesis path is as follows Figure 5 As shown, the 1H NMR and 1C NMR spectra of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia (HCQ-TK) are as follows: Figure 6 As shown.

[0074] IV. Preparation of nanoparticles.

[0075] 300 mg of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazoline (HCQ-TK) was dissolved in 20 ml of 1,4-dioxane (solvent), and then dissolved in an NHS / EDC (esterification catalyst) solution to achieve a molar ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazoline (HCQ-TK) to NHS / EDC (esterification catalyst) of 1:1.2. Triethylamine (acid-binding agent) was added, with a molar ratio of triethylamine (acid-binding agent) to NHS / EDC (esterification catalyst) of 1:2. The reaction was carried out in the dark at room temperature for 12 hours, in the dark, to obtain a mixed solution.

[0076] Then, 200 mg of HSA (human serum albumin) was dissolved in 40 ml of deionized water to obtain an HSA solution. This HSA solution was then mixed with the mixture obtained in the previous step and reacted at room temperature (22-27℃) for 12 h to obtain the product HSA-TK-HCQ. The HSA-TK-HCQ product was then removed, dialyzed in deionized water, and freeze-dried. Nanoparticles were obtained using a microfluidic method.

[0077] The microfluidic method described above is as follows: Using the product HSA-TK-HCQ, an ethanol solution of 10.0 mg / mL HSA-TK-HCQ is accurately prepared using ethanol as the solvent. A 12.5% ​​sodium chloride solution is also prepared. The solutions are placed in a water bath and heated to a constant temperature of 40°C. A certain volume of deionized water is then placed in a 50 mL centrifuge tube and heated to a constant temperature of 60°C. Then, 1.0 mL and 10.0 mL syringes are fixed to a microinjection pump, with the 1.0 mL syringe connected to inlet 2 of the MF chip and the 10.0 mL syringe connected to inlets 1 and 3 (inlets 1 and 3 are used to introduce the aqueous phase, and inlet 2 is used to introduce the organic phase). A 10.0 mg / mL HSA-TK-HCQ ethanol solution, a 12.5% ​​NaCl solution, and HSA were mixed in a volume ratio of 2:1:1 and placed in a 1.0 mL syringe. Deionized water at 60°C was placed in a 10.0 mL syringe. The flow rate of the intermediate organic phase (HSA-TK-HCQ ethanol solution, 12.5% ​​NaCl solution, and HSA) was adjusted sequentially to 15, 20, 30, and 40 μL / min. The flow rate of the aqueous phase (60°C deionized water) was initially controlled at 100 μL / min and gradually increased according to the flow rate ratio. After mixing thoroughly in the channels of an inverted W-shaped passive mixing chip, the mixture was connected to a connecting tube and then rapidly cooled using an ice-water bath to obtain a nanoparticle (HCQ-TK-HSA NPs) solution. The freshly prepared nanoparticle (HCQ-TK-HSA NPs) solution was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 50 kDa and centrifuged at 3200 × g for 10 min to obtain a concentrated solution and filtrate of nanoparticle (HCQ-TK-HSA NPs). The solution was then frozen in a refrigerator and quickly placed in a freeze dryer for drying before being removed.

[0078] The above synthesis path is as follows Figure 7 As shown, the infrared absorption spectra of hydroxychloroquine (HCQ), 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolidinedioic acid (HCQ-TK), and nanoparticles (HCQ-TK-HSA NPs) are as follows. Figure 8 As shown.

[0079] Because long-term administration of hydroxychloroquine alone can cause side effects such as retinopathy, neuromuscular and cardiotoxicity, which can lead to very serious and even life-threatening side effects, the inventors propose to improve hydroxychloroquine (HCQ) using the chemical method of this embodiment to make it more effective and less toxic, and to identify the potential cellular and biological pathways specifically regulated by HCQ, thereby making it more suitable for inhibiting neutrophils and improving the inflammatory microenvironment.

[0080] Experimental Example

[0081] The performance of the nanoparticles (HCQ-TK-HSA NPs) prepared in Example 1 was verified.

[0082] I. The nanoparticles (HCQ-TK-HSA NPs) prepared in Example 1 were subjected to particle size and zeta potential tests, and the results are as follows: Figure 9 And as shown in the table below. Wherein, Figure 9 The image shows the particle size distribution of the nanoparticles (HCQ-TK-HSA NPs). The table below shows the particle size zeta potentials of the nanoparticles (HCQ-TK-HSA NPs), with particle sizes of approximately 125.4 nm, 252.5 nm, 376.3 nm, and 512.3 nm.

[0083]

[0084] II. To evaluate the stability of the nanoparticles (HCQ-TK-HSA NPs) in plasma, the inventors used 10% FBS to simulate a plasma environment and then examined the particle size change. The results are as follows: Figure 10 As shown, the nanoparticles (HCQ-TK-HSA NPs) with a particle size of 376.3 nm exhibit little fluctuation in particle size in 10% FBS, demonstrating good stability.

[0085] III. Experiment on ROS-responsive release behavior of nanoparticles (HCQ-TK-HSA NPs).

[0086] Experimental procedure: The release behavior of hydroxychloroquine from the nanoparticles (HCQ-TK-HSANPs) synthesized in Example 1 under different concentrations of ROS was studied by dialysis at 37℃. The results are as follows: Figure 11 As shown in the figure. In contrast, almost no release occurred in PBS alone. Compared to a high concentration of 500 μM H2O2, HCQ-TK-HSA NPs released slowly in PBS containing 100 μM and 200 μM H2O2, while at 500 μM, the cumulative release rate reached 90% after 10 hours. This indicates that the nanoparticles (HCQ-TK-HSA NPs) can release drugs on demand under conditions of high ROS expression, and exhibit high stability. Even in the presence of low concentrations of reactive oxygen species, the chemical bonds are not easily broken, effectively protecting the drug from degradation and increasing its potential for clinical application.

[0087] IV. Verification experiment on the good biocompatibility of nanoparticles (HCQ-TK-HSA NPs).

[0088] The inventors used CCK-8 to detect its toxicity to normal cells. The specific procedure was as follows: 3T3 cells in logarithmic growth phase were seeded into 96-well plates at 6000 cells / well. After 24 hours, different concentrations (5, 10, 15, 20, 25, 50, 100 μg / mL) of HCQ and HCQ-TK-HSA NPs were added. After co-incubation for 24 hours, the cells were carefully washed three times with sterile PBS, and then treated with 100 μL of solution (CCK-8 solution: fresh medium = 1:10). The cells were then incubated again at 37°C for 2 hours. Cell viability was then assessed by measuring absorbance at 450 nm using a microplate reader. The cell viability was calculated using the following formula:

[0089] Cell viability (%) = [(Ax-Ab) / (Ac-Ab)] × 100

[0090] Where Ax is the absorbance of the sample (a CCK-8 solution containing cells and HCQ-TK-HSA NPs); Ac is the absorbance of the control group (a CCK-8 solution containing cells but without HCQ-TK-HSA NPs); and Ab is the absorbance of the blank (a CCK-8 solution without cells or HCQ-TK-HSA NPs).

[0091] The results are as follows Figure 12 As shown in the figure, compared with HCQ alone, HCQ-TK-HSA NPs had almost no cytotoxic effect on normal fibroblast (3T3) cells. At 25 μg / mL, the HCQ alone group already showed cytotoxicity, with a cell survival rate of only 50%, and all cells died at 50 μg / mL. In contrast, the HCQ-TK-HSA NPs group maintained a cell survival rate as high as 90% even at 100 μg / mL. This indicates that modification of albumin nanoparticles can improve and mitigate the toxicity of HCQ itself, giving the nanoparticles good biocompatibility.

[0092] V. Experimental verification of the wound healing effect of nanoparticles (HCQ-TK-HSA NPs).

[0093] 1. To evaluate the effect of nanoparticles in Example 1 on the healing of diabetic wounds, the inventors photographed and recorded the changes in wound area of ​​different treatment groups at different time points. The specific procedures for the treatment groups were as follows: Mice with successfully established diabetic wounds were randomly divided into four groups of six mice each: Control group, HSA group, HCQ group, and HCQ-TK-HSA NPs group. The drugs were injected into the wounds, and the mice were placed in a warm environment until recovery. Wound healing changes were photographed and recorded at 3, 6, 9, and 12 days, using orange cards as a reference. The wound area was quantified using ImageJ software.

[0094] The results are as follows Figure 13 , Figure 14 As shown, HCQ-TK-HSA NPs significantly promoted wound healing, achieving a healing rate of approximately 90% by day 12, compared to only about 70% in the hydroxychloroquine (HCQ) group. The control group and HSA group only achieved 40-50% healing.

[0095] 2. Detection of epidermal length and granulation tissue thickness: Epidermal length and granulation tissue thickness reflect the tissue regeneration after wound injury. H&E staining was used to analyze the wound tissue of each group after 12 days of treatment. Figure 15 , 16 As shown in Figure 17, compared with the control group, the hydroxychloroquine (HCQ) group and the HCQ-TK-HSA NPs group significantly promoted epidermal regeneration and granulation tissue thickening, with the HCQ-TK-HSA NPs group showing a more significant increase. The HSA group showed almost the same effect as the control group, with no therapeutic effect. This indicates that albumin only acts as a target for neutrophils in the wound, while the drug-loaded albumin HCQ-TK-HSA can improve the regenerative capacity of damaged tissue. Masson staining stains collagen in tissues, reflecting the maturity of regenerated tissue; the depth of blue represents the degree of maturity. Figure 18 , Figure 19 As shown, significant collagen deposition was observed in the hydroxychloroquine (HCQ) group and the HCQ-TK-HSA NPs group, with the HCQ-TK-HSA NPs group exhibiting a more abundant blue staining area, suggesting that the damaged tissue had recovered and matured. In contrast, the control group and the HSA group still showed a large number of red muscle fibers, indicating that the damaged tissue had not yet healed.

[0096] 3. Detection of CD31 expression: CD31 is a marker of angiogenesis. CD31 expression was detected by immunofluorescence staining of paraffin sections. Dewaxed and rehydrated tissue sections were heat-treated in a 100°C boiling water bath for 15 min in citrate buffer (10 mM, pH 6.0) for antigen retrieval. The sections were washed three times with PBS, 5 min each time. The sections were then blocked with PBS containing 10% goat serum at room temperature for 2 h. To examine wound angiogenesis, rabbit anti-mouse CD31 polyclonal antibody (1:4000) was used as the primary antibody and incubated overnight at 4°C. The sections were washed three times with PBS, 5 min each time. Goat anti-rabbit IgG (Alexa Fluor-561) antibody was added as the secondary antibody and incubated at room temperature in the dark for 1 h. The sections were washed three times with PBS and mounted with mounting medium containing DAPI to quench fluorescence. The slides were observed and photographed under a fluorescence microscope.

[0097] from Figure 20 , Figure 21The study found that CD31 fluorescence expression was not significant in the control group and HSA group, while the CD31 positive area increased in the HCQ-TK-HSANPs group. HCQ treatment also had a positive effect on increasing the CD31 positive area, but the effect was not as good as the HCQ-TK-HSA NPs treatment group. The results indicate that HCQ-TK-HSA NPs treatment can promote wound angiogenesis.

[0098] The nanoparticles of this invention are reactive oxygen species (ROS) responsive and neutrophil-targeting nanoparticles in an inflammatory environment. These nanoparticles utilize albumin as a nanocarrier and thioacetate as a carrier linker, connecting hydroxychloroquine to form a nanostructure. These nanoparticles exhibit good biocompatibility and ROS responsiveness, while also targeting neutrophils in an inflammatory environment to alleviate inflammation. The preparation method for these nanoparticles is simple, efficient, and requires mild conditions and a short preparation cycle, which is also beneficial for wound healing and the treatment of inflammatory diseases.

[0099] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0100] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A nanoparticle, characterized in that, The raw materials for preparing the nanoparticles include: 17-((7-chloroquinoline)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia, solvent, acid-binding agent, esterification catalyst, and support; The mass-to-volume ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the solvent is (0.001-0.02 g) / mL, the molar ratio of the acid-binding agent to the esterification catalyst is (0.5-2):2, the molar ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the esterification catalyst is 1:(1.1-1.3), and the weight ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the support is (1.5-2):

1. The raw materials for preparing 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolic acid include: hydroxychloroquine and 3'-(propane-2,2-thion)dipropionic acid; the weight ratio of hydroxychloroquine to 3'-(propane-2,2-thion)dipropionic acid is (1-2):

1. The solvent comprises at least one of 1,4-dioxane, dichloromethane, and N,N-dimethylformamide; the acid-binding agent is triethylamine; the esterification catalyst comprises N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the carrier is human serum albumin; The method for preparing the nanoparticles includes the following steps: Preparation of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolidinedioic acid: Hydroxychloroquine and 3'-(propane-2,2-thionane)dipropionic acid were dissolved, mixed, and subjected to esterification, followed by purification; the esterification reaction was catalyzed by an EDC / DMAP system, and the esterification reaction time was 40-55 h; the purification was achieved by preparative chromatography. Preparation of nanoparticles: 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia was dissolved in a solvent to obtain a 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia solution. The 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia solution was mixed with an esterification catalyst and an acid-binding agent, and reacted in the dark. A support was added, and the mixture was mixed to carry out a condensation reaction. The mixture was dialyzed, freeze-dried, and nanoparticles were obtained by microfluidic method. The time of the light-protected reaction was 10-14 h, the temperature of the condensation reaction was 22-27 °C, and the time of the condensation reaction was 10-14 h.

2. The nanoparticles according to claim 1, characterized in that, The mass-to-volume ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the solvent is 0.01-0.02 g / mL, the molar ratio of the acid-binding agent to the esterification catalyst is (1-2):2, and the molar ratio of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia to the esterification catalyst is 1:1.

2.

3. The method for preparing nanoparticles according to any one of claims 1-2, characterized in that, Includes the following steps: Preparation of 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithiazolidinedioic acid: Hydroxychloroquine and 3'-(propane-2,2-thionane)dipropionic acid were dissolved, mixed, and subjected to esterification, followed by purification; the esterification reaction was catalyzed by an EDC / DMAP system, and the esterification reaction time was 40-55 h; the purification was achieved by preparative chromatography. Preparation of nanoparticles: 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia was dissolved in a solvent to obtain a 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia solution. The 17-((7-chloroquine)amino)-13-ethyl-5,5-dimethyl-9-oxo-10-oxo-4,6-dithia solution was mixed with an esterification catalyst and an acid-binding agent, and reacted in the dark. A support was added, and the mixture was mixed to carry out a condensation reaction. The mixture was dialyzed, freeze-dried, and nanoparticles were obtained by microfluidic method. The time of the light-protected reaction was 10-14 h, the temperature of the condensation reaction was 22-27 °C, and the time of the condensation reaction was 10-14 h.

4. The preparation method according to claim 3, characterized in that, The hydroxychloroquine was prepared by the following method: hydroxychloroquine sulfate was dissolved in water, an alkaline solution was added, a precipitate was obtained, the precipitate was extracted, water was removed, the precipitate was allowed to stand, and the precipitate was obtained by rotary evaporation.

5. The preparation method according to claim 3, characterized in that, The 3'-(propane-2,2-thion)dipropionic acid was prepared by the following method: 3-mercaptopropionic acid was mixed with acetone, a catalyst was added, the mixture was reacted, washed, and freeze-dried to obtain 3'-(propane-2,2-thion)dipropionic acid.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nanoparticles as described in any one of claims 1-2 and pharmaceutically acceptable excipients.

7. The use of the nanoparticles according to any one of claims 1-2 in the preparation of a drug for healing diabetic wounds.

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