Responsive nano drug delivery system as well as preparation and application thereof
By developing a responsive nanopharmaceutical delivery system, using reactive oxygen response bonds to combine methoxy polyethylene glycol and silybin, and encapsulate near-infrared second-zone fluorescent dyes, the problem of early diagnosis and treatment of drug-induced liver injury is solved. , achieving efficient diagnostic and therapeutic effects.
Patent Information
- Application Number
- CN202411499568.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art is difficult to achieve the early accurate diagnosis and specific treatment of drug-induced liver injury, and the solubility and bioavailability of silybin are poor, resulting in slow therapeutic effect.
A responsive nano-drug delivery system was developed to chemically bind methoxy polyethylene glycol and silybin through reactive oxygen-responsive bonds, and self-assembled for the near-infrared two-zone fluorescent dyes through hydrophobic interactions. The system degrades responsively in the pathological microenvironment of the damaged liver, restoring fluorescent signals and releasing silybin.
It has achieved early accurate diagnosis and specific treatment of drug-induced liver injury, solved problems such as low drug loading rate, premature release, and blood circulation leakage, and improved the solubility and bioavailability of silybin.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of nanomedicine technology, and more specifically, to a responsive nano drug delivery system and its preparation and application. Background Art
[0002] Early diagnosis and timely treatment of drug-induced liver injury are crucial to prevent it from developing into chronic liver disease or even liver failure. At present, the clinical diagnostic methods for drug-induced liver injury are mainly liver function biomarkers and imaging diagnosis. However, these two diagnostic methods generally cannot achieve the purpose of early detection of damage, lack specificity, and have difficulty in real-time visualization of liver damage, which has certain limitations. The untimely early diagnosis of drug-induced liver injury is one of the main reasons for the difficulty in treatment. Near-infrared zone II fluorescence imaging has the advantages of high spatial resolution, high sensitivity, and high signal-to-noise ratio, which is more conducive to the early detection of the disease. The effective combination of near-infrared zone II fluorescence imaging and the pathological microenvironment of increased reactive oxygen in the drug-induced liver injury area can achieve early and accurate diagnosis of drug-induced liver injury.
[0003] Silybin, as a clinical liver protection drug, has a good effect on the repair of liver damage in patients with various chronic liver diseases and can effectively improve the prognosis of patients. However, due to the poor solubility and bioavailability of silybin, long-term oral administration is required, resulting in slow efficacy, limited to chronic liver disease, and unable to meet clinical needs. Nano drug delivery systems have the advantages of improving drug solubility, enhancing pharmacokinetics, and reducing toxic side effects on normal tissues, greatly increasing the possibility of silybin in treating drug-induced liver injury. However, traditional nano drug delivery systems using physical coatings have problems such as low drug loading rate, premature release, and blood circulation leakage, which are not convenient for specific treatment.
[0004] Therefore, it is urgent to develop a new nanodrug delivery system that can release the liver-protecting drug silybin through specific imaging of the pathological microenvironment of damaged liver, so as to solve the problems of untimely early diagnosis of drug-induced liver injury and the poor water solubility and short half-life of silybin, and realize the specific diagnosis and treatment of drug-induced liver injury. Summary of the invention
[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a responsive nano drug delivery system for diagnosis and treatment of liver injury, wherein the nano drug delivery system utilizes active oxygen responsive bond ketal bond chemistry to combine methoxypolyethylene glycol and silybin, and is self-assembled by encapsulating near-infrared zone II fluorescent dyes through hydrophobic interactions.
[0006] Preferably, the ketal bond is a dicarboxylated ketal bond; the methoxy polyethylene glycol is mainly methoxy polyethylene glycol amine; and the main molecular weights of the methoxy polyethylene glycol are 1000, 2000, and 5000.
[0007] Another aspect of the present invention is to provide a method for preparing a responsive nano drug delivery system for diagnosis and treatment of liver injury. The specific steps of the preparation method are as follows: S1. dissolving ketal, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide and stirring, and protecting the carboxyl group with nitrogen to obtain solution I; S2. adding methoxypolyethylene glycol to the solution Ⅰ obtained in S1, stirring, dialyzing, and freeze-drying to obtain a lyophilized product Ⅰ; S3. The lyophilized product I obtained in S2 was dissolved in dimethyl sulfoxide with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, and stirred. The carboxyl groups were activated under nitrogen protection to obtain solution II. S4. Add silybin to the solution II obtained in S3, stir, dialyze, and freeze-dry to obtain a lyophilized product II; S5. The freeze-dried product II obtained in S4 and the near-infrared second-zone fluorescent dye are dissolved in acetonitrile, slowly added into rapidly stirred deionized water, and dialyzed and centrifuged to obtain a responsive nano drug delivery system.
[0008] Preferably, the molar ratio of ketal, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in S1 is 1:2:2, the stirring temperature is 28° C., and the stirring time is 2-4 h.
[0009] Preferably, the molar ratio of methoxypolyethylene glycol in S2 to ketal in solution I is 1:1.2, the stirring time is 12-24 hours, the molecular weight cutoff of the dialysis bag is 1000DA, and the dialysis time is 24 hours.
[0010] Preferably, the molar ratio of the freeze-dried product I, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in S3 is 1:2:2, the stirring temperature is 28°C, and the stirring time is 2-4h.
[0011] Preferably, the molar ratio of silybin to the lyophilized product I in S4 is 1.2:1, the stirring time is 12-24 hours, the molecular weight cut-off of the dialysis bag is 1000DA, and the dialysis time is 24 hours.
[0012] Preferably, the mass ratio of the freeze-dried product II and the near-infrared second zone fluorescent dye in S5 is 50:1, the ratio of acetonitrile to deionized water is 1:10-20, the stirring time is 6-10h, the dialysis bag molecular weight cutoff is 3500DA, the dialysis time is 24h, the centrifugation uses an ultrafiltration centrifuge tube 10KD, the speed is 4000r / min, and the time is 10min.
[0013] Preferably, the near-infrared second region fluorescent dye in S5 is IR-1061.
[0014] Another aspect of the present invention is to provide an application of a responsive nano drug delivery system for diagnosis and treatment of liver injury, and the application of the responsive nano drug delivery system in preparing drugs for diagnosis and treatment of drug-induced liver injury.
[0015] The present invention provides a responsive nano drug delivery system for diagnosis and treatment of drug-induced liver injury. The nano drug delivery system uses active oxygen responsive bond ketal bond to chemically combine methoxy polyethylene glycol and silybin, and is self-assembled by encapsulating near-infrared zone II fluorescent dye through hydrophobic interaction. The introduction of polyethylene glycol effectively improves the water solubility and biocompatibility of the liver protection drug silybin. At the same time, due to the characteristics of drug-induced liver injury microenvironment that can produce a large amount of active oxygen, the nano drug delivery system responsively degrades at the site of injury, restores the quenched near-infrared zone II fluorescence, releases silybin to exert anti-inflammatory and antioxidant effects, effectively identifies damaged livers, solves the problems of low drug loading rate, premature release, blood circulation leakage, etc. in the nano drug delivery system using physical coating, and realizes the specific diagnosis and treatment of drug-induced liver injury. The preparation method is simple and easy, and the effect is obvious.
[0016] Additional aspects and advantages of the invention will become apparent from the following description, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 This is the ultraviolet absorption spectrum of the synthetic responsive nano drug delivery system of Example 1 of the present invention; Figure 2 This is a near-infrared second-zone fluorescence imaging image of the synthetic responsive nano drug delivery system of Example 1 of the present invention; Figure 3 This is a cytotoxicity diagram of the synthetic responsive nano drug delivery system of Example 1 of the present invention; Figure 4 This is a cell fluorescence spectrum of the synthetic responsive nano drug delivery system of Example 1 of the present invention; Figure 5 This is a fluorescence imaging diagram of the synthetic responsive nano drug delivery system in Example 1 of the present invention in the in vivo acetaminophen-induced liver injury; Figure 6 This is a diagram showing the therapeutic effect of the synthetic responsive nano drug delivery system in Example 1 of the present invention on acetaminophen-induced liver damage in vivo. DETAILED DESCRIPTION
[0018] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.
[0019] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.
[0020] Embodiment 1 S1. Dissolve 34.5 mg of ketal, 52.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 31.5 mg of N-hydroxysuccinimide in 4 ml of dimethyl sulfoxide and stir for 2 h. Activate the carboxyl group under nitrogen protection to obtain solution I. S2. 225 mg of methoxy polyethylene glycol with a molecular weight of 2000 was added to the solution Ⅰ prepared in S1 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product Ⅰ; S3. 115 mg of S2 was dissolved in 4 ml of dimethyl sulfoxide with lyophilized product Ⅰ, 19.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.2 mg of 4-dimethylaminopyridine, and stirred for 2 h. The carboxyl groups were activated under nitrogen protection to obtain solution Ⅱ. S4. 28.8 mg of silybin was added to the solution II obtained in S3 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product II; S5. 10 mg of S4 to obtain freeze-dried product II and 0.2 mg of near-infrared zone II fluorescent dye IR-1061 were dissolved in 1 ml of acetonitrile, slowly added into 10 ml of rapidly stirred deionized water and stirred for 6 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 3500 Da, and the product was separated for 10 min using a 10KD ultrafiltration centrifuge tube at 4000 r / min to obtain a responsive nano drug delivery system; The prepared responsive nano drug delivery system solution is administered by mouse tail vein injection to detect the diagnosis and treatment effects of the responsive nano drug delivery system.
[0021] Figure 1 This is the ultraviolet absorption spectrum of the responsive nano drug delivery system synthesized in Example 1 of the present invention. As can be seen from the figure, the synthesized responsive nano micelles have a characteristic absorption peak of silybin at 288nm, and a characteristic absorption peak of the near-infrared second region dye IR-1061 at 770-1000nm.
[0022] Figure 2This is a near-infrared second-zone fluorescence imaging image of the synthetic responsive nano drug delivery system of Example 1 of the present invention. As can be seen from the figure, as the concentration of tert-butyl hydroperoxide increases and the time prolongs, the fluorescence signal of the nano drug delivery system solution gradually increases, indicating that the nano drug delivery system can be degraded in the presence of active oxygen and restore the quenched near-infrared second-zone fluorescence.
[0023] Figure 3 This is a cytotoxicity graph of the synthetic responsive nano drug delivery system of Example 1 of the present invention. As can be seen from the graph, even if the concentration of the nano drug delivery system reaches 200 μg / mL, the cell survival rate is still above 90%.
[0024] Figure 4 This is a cell fluorescence spectrum of the synthetic responsive nano drug delivery system of Example 1 of the present invention. As can be seen from the figure, as time goes by, the nano drug delivery system recovers fluorescence under the condition of the presence of cellular active oxygen.
[0025] Figure 5 This is a fluorescence imaging diagram of the synthetic responsive nano drug delivery system in Example 1 of the present invention in the in vivo acetaminophen-induced liver injury. As can be seen from the figure, compared with the control group, the liver fluorescence intensity of mice with acetaminophen-induced liver injury after tail vein administration is more obvious.
[0026] Figure 6 This is a diagram showing the therapeutic effect of the synthetic responsive nano drug delivery system in Example 1 of the present invention on acetaminophen-induced liver injury in vivo. As shown in the figure, compared with the acetaminophen-induced liver injury group, the range of the injured lesions was significantly reduced and the inflammatory cell infiltration was reduced after the nano drug delivery system was injected into the tail vein of mice.
[0027] Based on the above results, it is shown that this responsive nanodrug delivery system can specifically restore the quenched near-infrared second-zone fluorescence at the site of liver damage, release silybin, and thus achieve effective diagnosis and treatment of drug-induced liver injury.
[0028] Embodiment 2 S1. Dissolve 34.5 mg of ketal, 52.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 31.5 mg of N-hydroxysuccinimide in 4 ml of dimethyl sulfoxide and stir for 2 h. Activate the carboxyl group under nitrogen protection to obtain solution I. S2. 562.5 mg of methoxy polyethylene glycol with a molecular weight of 5000 was added to the solution I prepared in S1 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product I; S3. Dissolve 288 mg of S2 to obtain the lyophilized product I, 19.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1.2 mg of 4-dimethylaminopyridine in 4 ml of dimethyl sulfoxide and stir for 2 h. Activate the carboxyl group under nitrogen protection to obtain solution II. S4. 28.8 mg of silybin was added to the solution II obtained in S3 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product II; S5. Dissolve 10 mg of S4 to obtain freeze-dried product II and 0.2 mg of near-infrared zone II fluorescent dye IR-1061 in 1 ml of acetonitrile, slowly add into 15 ml of rapidly stirred deionized water and stir for 10 hours, dialyze for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and separate the product with a 10KD ultrafiltration centrifuge tube at 4000 r / min for 10 minutes to obtain a responsive nano drug delivery system.
[0029] Embodiment 3 S1. Dissolve 34.5 mg of ketal, 52.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 31.5 mg of N-hydroxysuccinimide in 4 ml of dimethyl sulfoxide and stir for 2 h. Activate the carboxyl group under nitrogen protection to obtain solution I. S2. 113 mg of 1000 molecular weight methoxypolyethylene glycol was added to the solution Ⅰ prepared in S1 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product Ⅰ; S3. Dissolve 58 mg of S2 to obtain the lyophilized product I, 19.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1.2 mg of 4-dimethylaminopyridine in 4 ml of dimethyl sulfoxide and stir for 2 h. Activate the carboxyl group under nitrogen protection to obtain solution II. S4. 28.8 mg of silybin was added to the solution II obtained in S3 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product II; S5. Dissolve 10 mg of S4 to obtain freeze-dried product II and 0.2 mg of near-infrared zone II fluorescent dye IR-1061 in 1 ml of acetonitrile, slowly add into 20 ml of rapidly stirred deionized water and stir for 10 hours, dialyze for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and separate the product with a 10KD ultrafiltration centrifuge tube at 4000 r / min for 10 minutes to obtain a responsive nano drug delivery system.
[0030] Embodiment 4 S1. Dissolve 34.5 mg of ketal, 52.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 31.5 mg of N-hydroxysuccinimide in 4 ml of dimethyl sulfoxide and stir for 2 h. Activate the carboxyl group under nitrogen protection to obtain solution I. S2. 225 mg of methoxy polyethylene glycol with a molecular weight of 2000 was added to the solution Ⅰ prepared in S1 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product Ⅰ; S3. 115 mg of S2 was dissolved in 4 ml of dimethyl sulfoxide with lyophilized product Ⅰ, 19.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.2 mg of 4-dimethylaminopyridine, and stirred for 2 h. The carboxyl groups were activated under nitrogen protection to obtain solution Ⅱ. S4. 28.8 mg of silybin was added to the solution II obtained in S3 and stirred for 24 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product II; S5. Dissolve 10 mg of S4 to obtain freeze-dried product II and 0.2 mg of near-infrared zone II fluorescent dye IR-1061 in 1 ml of acetonitrile, slowly add into 20 ml of rapidly stirred deionized water and stir for 10 hours, dialyze for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and separate the product with a 10KD ultrafiltration centrifuge tube at 4000 r / min for 10 minutes to obtain a responsive nano drug delivery system.
[0031] Embodiment 5 S1. Dissolve 34.5 mg of ketal, 52.5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 31.5 mg of N-hydroxysuccinimide in 4 ml of dimethyl sulfoxide and stir for 4 h. Activate the carboxyl group under nitrogen protection to obtain solution I. S2. 225 mg of methoxy polyethylene glycol with a molecular weight of 2000 was added to the solution Ⅰ prepared in S1 and stirred for 12 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product Ⅰ; S3. 115 mg of S2 obtained by lyophilization of product Ⅰ and 19.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.2 mg of 4-dimethylaminopyridine were dissolved in 4 ml of dimethyl sulfoxide and stirred for 4 h, and the carboxyl group was activated under nitrogen protection to obtain solution Ⅱ; S4. 28.8 mg of silybin was added to the solution II obtained in S3 and stirred for 12 h, dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da, and freeze-dried to obtain a lyophilized product II; S5. Dissolve 10 mg of S4 to obtain freeze-dried product II and 0.2 mg of near-infrared zone II fluorescent dye IR-1061 in 1 ml of acetonitrile, slowly add into 10 ml of rapidly stirred deionized water and stir for 6 hours, dialyze for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and separate the product with a 10KD ultrafiltration centrifuge tube at 4000 r / min for 10 minutes to obtain a responsive nano drug delivery system.
[0032] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may also have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A responsive nano drug delivery system, characterized in that: The nano drug delivery system utilizes active oxygen responsive ketal bond chemistry to combine methoxypolyethylene glycol and silybin, and is self-assembled by encapsulating near-infrared zone II fluorescent dye through hydrophobic interaction.
2. A responsive nano drug delivery system according to claim 1, characterized in that: The ketal bond is a dicarboxylated ketal bond; the methoxy polyethylene glycol is mainly methoxy polyethylene glycol amine; the main molecular weights of the methoxy polyethylene glycol are 1000, 2000 and 5000.
3. The method for preparing a responsive nano drug delivery system according to claim 1, characterized in that: The specific steps of the preparation method are as follows: S1. dissolving ketal, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide and stirring, and protecting with nitrogen to activate the carboxyl group to obtain solution I; S2. adding methoxypolyethylene glycol to the solution Ⅰ obtained in S1, stirring, dialyzing, and freeze-drying to obtain a lyophilized product Ⅰ; S3. The lyophilized product I obtained in S2 was dissolved in dimethyl sulfoxide with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, and stirred. The carboxyl groups were activated under nitrogen protection to obtain solution II. S4. Add silybin to the solution II obtained in S3, stir, dialyze, and freeze-dry to obtain a lyophilized product II; S5. The freeze-dried product II obtained in S4 and the near-infrared second-zone fluorescent dye are dissolved in acetonitrile, slowly added into rapidly stirred deionized water, and dialyzed and centrifuged to obtain a responsive nano drug delivery system.
4. The method for preparing a responsive nano drug delivery system according to claim 3, characterized in that: The molar ratio of ketal, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in S1 is 1:2:2, the stirring temperature is 28° C., and the stirring time is 2-4 h.
5. The method for preparing a responsive nano drug delivery system according to claim 3, characterized in that: The molar ratio of the methoxy polyethylene glycol in S2 to the ketal in solution I is 1:1.2, the stirring time is 12-24 hours, the molecular weight cutoff of the dialysis bag is 1000DA, and the dialysis time is 24 hours.
6. The method for preparing a responsive nano drug delivery system according to claim 3, characterized in that: The molar ratio of the freeze-dried product I, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in S3 is 1:2:2, the stirring temperature is 28° C., and the stirring time is 2-4 h.
7. The method for preparing a responsive nano drug delivery system according to claim 3, characterized in that: The molar ratio of silybin to the lyophilized product I in S4 is 1.2:1, the stirring time is 12-24 hours, the molecular weight cutoff of the dialysis bag is 1000DA, and the dialysis time is 24 hours.
8. The method for preparing a responsive nano drug delivery system according to claim 3, characterized in that: The mass ratio of the freeze-dried product II and the near-infrared second zone fluorescent dye in the S5 is 50:1, the ratio of acetonitrile to deionized water is 1:10-20, the stirring time is 6-10 hours, the molecular weight cutoff of the dialysis bag is 3500DA, the dialysis time is 24 hours, the centrifugation uses an ultrafiltration centrifuge tube 10KD, the speed is 4000r / min, and the time is 10 minutes.
9. The method for preparing a responsive nano drug delivery system according to claim 3 or 8, characterized in that: The near-infrared second-region fluorescent dye in the S5 is IR-1061.
10. The use of a responsive nano drug delivery system according to claim 1, characterized in that: The responsive nano drug delivery system is used to prepare drugs for diagnosing and treating drug-induced liver injury.