Nitric oxide driven nano motor composition and preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a nitric oxide-driven nano motor composition as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving cyclodextrin and Boc protected arginine in DMF, and adding EDC and DMAP; carrying out water bath reaction under a nitrogen condition; dialyzing, freeze-drying and separating to obtain a white product CD-Arg; the preparation method comprises the following steps: dissolving rapamycin and thioketal in anhydrous DMF, and adding EDC and DMAP; carrying out water bath reaction under a nitrogen condition; dialyzing, freeze-drying and separating to obtain a yellow product RAPA-TK; the preparation method comprises the following steps: dissolving CD-Arg and RAPA-TK in DMF, and then adding EDC and DMAP; carrying out water bath reaction under a nitrogen condition; dialyzing the product, and freeze-drying and separating to obtain the nano-motor composition. The nano motor provided by the invention has special chemical bonds sensitive and responsive to an inflammatory microenvironment, and is easy to break in the inflammatory environment, so that targeted drug release can be realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a nitric oxide driven nanomotor composition as well as a preparation method and application thereof. BACKGROUND

[0002] Nanotechnology is a new science and technology that utilizes the properties of nanoscale substances to manufacture nanomaterials and nanodevices with specific functions, and further studies the properties and practical applications thereof. Since the 20th century, nanotechnology has shown great application prospects in the fields of materials, chemical industry, medicine, environment, food and the like.

[0003] In the field of biomedical research, various nanodrugs and nanodrug carriers have been developed due to the continuous development of nanotechnology. A nanomotor is a nanoscale element with self-movement ability, which can convert various forms of energy including electrical energy, magnetic energy, optical energy, acoustic energy and chemical energy into mechanical energy for self-propulsion. An enzyme driven nanomotor can realize autonomous movement by the energy generated through enzymatic reaction in a specific chemical environment, and utilize the exposed active site to respond to specific chemical signals, thereby realizing chemotaxis to specific sites, which is particularly suitable for the field of biomedicine.

[0004] At present, the preparation of nanodrugs and nanodrug carriers in the prior art is relatively cumbersome, the yield is low, and the stability is poor, which affects the release of the drug. SUMMARY

[0005] In view of the above problems, the application provides a nitric oxide driven nanomotor composition as well as a preparation method and application thereof. The provided nanomotor has a special chemical bond that is sensitive to inflammatory microenvironment and is easy to break in an inflammatory environment, so that the drug can be released in the target inflammatory environment, the drug concentration in the target area is increased, the distribution of the drug in the non-target part is reduced, and the adverse reactions are reduced.

[0006] The application provides a nitric oxide driven nanomotor composition, which has the following structure: .

[0007] The application further provides a preparation method of the nitric oxide driven nanomotor composition, which comprises the following steps: S1, dissolving cyclodextrin and Boc protected arginine in anhydrous DMF, adding EDC and DMAP, and reacting under water bath in a nitrogen atmosphere; after dialysis, freeze-drying separation is performed to obtain a white product CD-Arg; S2, dissolving rapamycin and thioketal in anhydrous DMF, adding EDC and DMAP, and reacting under water bath in a nitrogen atmosphere; after dialysis, freeze-drying separation is performed to obtain a yellow product RAPA-TK; S3, dissolving CD-Arg and RAPA-TK in anhydrous DMF, then adding EDC and DMAP, and reacting in a water bath under nitrogen; the product is separated by dialysis and freeze-dried to obtain the nano motor composition CD-Arg-TK-RAPA.

[0008] Further, in steps S1 to S3, the temperature of the water bath reaction is 25-40℃, and the time is 12-30h.

[0009] Further, in step S1, the molar ratio of the cyclodextrin to the Boc-protected arginine is 1: (3.5-4.5); In step S2, the molar ratio of the rapamycin to the ketal is 1: (2.5-3.2); In step S3, the molar ratio of the CD-Arg to the RAPA-TK is 1: (1-1.2).

[0010] Further, the structure of the ketal is as follows: .

[0011] Further, the synthesis process of step S1 is as follows: Formula I; The synthesis process of step S2 is as follows: Formula II; The synthesis process of step S3 is as follows: Formula III.

[0012] The application also provides a use of the above-mentioned nitric oxide-driven nano motor composition or the nano motor composition obtained by the preparation method in any of the above in the preparation of an atherosclerosis-relieving drug.

[0013] Further, the preparation of the atherosclerosis-relieving drug comprises: self-assembling the drug active ingredient and the nitric oxide-driven nano motor composition into nanoparticles in a solution to encapsulate the drug active ingredient, thereby obtaining the atherosclerosis-relieving drug.

[0014] The application has the following beneficial effects: The nitric oxide-driven nano motor composition provided by the application has a special chemical bond sensitive to an inflammatory microenvironment, which is easy to break in an inflammatory environment, thereby achieving targeted drug release, increasing the drug concentration in the target area, reducing the distribution in non-targeted parts, reducing adverse reactions, and enabling the lipid-lowering drug to self-assemble with the nano motor into nanoparticles in a solution to achieve drug encapsulation, thereby significantly improving the stability of the drug, and the preparation method of the drug-loaded nano motor has a simple process flow and high product stability. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Transmission electron microscopy of drug-loaded nanomotor in the embodiment of the present application.

[0016] Figure 2 Particle size distribution of drug-loaded nanomotor in the embodiment of the present application.

[0017] Figure 3 NMR hydrogen spectrum of drug-loaded nanomotor under the action of hydrogen peroxide in the embodiment of the present application.

[0018] Figure 4 Brownian motion trajectory and velocity distribution of solvent water of drug-loaded nanomotor in the embodiment of the present application.

[0019] Figure 5 Motion trajectory and velocity distribution of drug-loaded nanomotor in aqueous solution in the embodiment of the present application.

[0020] Figure 6 Laser confocal image of nitric oxide produced by co-incubation of drug-loaded nanomotor and control sample with endothelial cells and macrophages in the embodiment of the present application. DETAILED DESCRIPTION

[0021] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0022] The present application provides a nitric oxide-driven nanomotor composition, and the synthesis structure formula is shown in general formula I to III: Formula I.

[0023] As shown in the above structure formula, wherein: Synthesis of CD-Arg: Cyclodextrin (113.5 mg, 0.1 mmol) and Boc-protected arginine (200 mg, 0.4 mmol) were dissolved in 30 mL of anhydrous DMF, and EDC (115 mg, 0.6 mmol, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and DMAP (10 mg, 0.08 mmol, 4-dimethylaminopyridine) were added; under the condition of nitrogen, 30°C water bath for 24h; the product was separated by dialysis and freeze-dried to obtain white product CD-Arg, with a yield of 79%, and the chemical formula of CD-Arg is C 64 H 110 N8O41 .

[0024] As shown in formula II: Formula II; As shown in the above structural formula, wherein: Synthesis of RAPA-TK: rapamycin (200 mg, 0.2 mmol) and ketal (200 mg, 0.57 mmol) were dissolved in 30 mL of anhydrous DMF, and EDC (50 mg, 0.25 mmol) and DMAP (10 mg, 0.08 mmol) were added; under nitrogen, 30°C water bath for 24h; the product was separated by dialysis and freeze-dried to obtain a yellow product RAPA-TK with a yield of 69%, and the chemical formula of RAPA-TK was C 70 H 95 NO 16 S2.

[0025] As shown in formula III: Formula III; As shown in the above formula, wherein: Synthesis of CD-Arg-TK-RAPA: CD-Arg (164.6 mg, 0.1 mmol) and RAPA-TK (152.2 mg, 0.1 mmol) were dissolved in 50 mL of anhydrous DMF, and then EDC (50 mg, 0.25 mmol) and DMAP (10 mg, 0.08 mmol) were added; under nitrogen, 30°C water bath for 24h; the product was separated by dialysis and freeze-dried to obtain a yellow product CD-Arg-TK-RAPA with a yield of 49%, and the chemical formula of CD-Arg-TK-RAPA was C 134 H 203 N9O 56 S2.

[0026] In addition, the application also provides the use of the above-mentioned nitric oxide-driven nanomotor composition in the preparation of a medicine for relieving atherosclerosis.

[0027] Example 1 A preparation method of a nitric oxide-driven nanomotor composition is as follows: Synthesis of CD-Arg: cyclodextrin (113.5 mg, 0.1 mmol) and Boc-protected arginine (200 mg, 0.4 mmol) were dissolved in 30 mL of anhydrous DMF, and EDC (115 mg, 0.6 mmol) and DMAP (10 mg, 0.08 mmol) were added; under nitrogen, 30°C water bath for 24h; the product was separated by dialysis and freeze-dried to obtain a white product CD-Arg with a yield of 79%, and the chemical formula of CD-Arg was C 64 H110 N8O 41 . 1 H NMR (600 MHz, DMSO-D6) δ 4.80 – 5.10 (m, 2H), 3.55 –3.85 (m, 2H), 3.45 – 3.65 (m, 2H), 3.25 – 3.45 (m, 2H). 2.82 (m, 4H). 2.58(m, 4H). 1.42 (s, 18H).

[0028] Synthesis of RAPA-TK: Rapamycin (200 mg, 0.2 mmol) and the ketal (200 mg, 0.57 mmol) were dissolved in 30 mL of anhydrous DMF, and EDC (50 mg, 0.25 mmol) and DMAP (10 mg, 0.08 mmol) were added; the mixture was stirred at 30 °C under nitrogen for 24 h; the product was isolated by dialysis and freeze-drying to obtain a yellow product, RAPA-TK, in a yield of 69%, and the chemical formula of RAPA-TK was C 70 H 95 NO 16 S2. 1 H NMR (600 MHz, DMSO-D6) δ 7.45 –7.75 (s, 1H). 6.15 – 6.45 (d,2H). 5.70 – 5.90 (m, 1H). 3.35 – 3.65 (m, 3H). 2.82 (m, 4H). 2.58 (m, 4H).1.59 (s, 6H). 0.8 – 0.9 (d, 9H).

[0029] Synthesis of CD-Arg-TK-RAPA: CD-Arg (164.6 mg, 0.1 mmol) and RAPA-TK (152.2 mg, 0.1 mmol) were dissolved in 50 mL of anhydrous DMF, and then EDC (50 mg, 0.25 mmol) and DMAP (10 mg, 0.08 mmol) were added; the mixture was stirred at 30 °C under nitrogen for 24 h; the product was isolated by dialysis and freeze-drying to obtain a yellow product, CD-Arg-TK-RAPA, in a yield of 49%, and the chemical formula of CD-Arg-TK-RAPA was C 134 H 203 N9O 56 S2. 1H NMR (600 MHz, DMSO-D6) δ 7.45 - 7.75 (s, 1H). 6.15 - 6.45 (d, 2H). 5.70 - 5.90 (m, 1H). 4.80 - 5.10 (m, 2H). 3.55 - 3.85 (m, 2H), 3.45 - 3.65 (m, 2H). 3.35 - 3.65 (m, 3H). 3.25 - 3.45 (m, 2H). 2.82 (m, 4H). 2.58 (m, 4H). 1.59 (s, 6H). 1.42 (s, 18H). 0.8 - 0.9 (d, 9H).

[0030] Figure 1 The transmission electron microscope image of the drug-loaded nanomotor in the embodiment of the application is shown, and it can be seen that the morphology is uniformly dispersed spherical particles; Figure 2 The particle size distribution diagram of the drug-loaded nanomotor in the embodiment of the application is shown, and the average particle size is 129.1 nm. Figure 3 The responsiveness of the application in the inflammatory microenvironment is shown, and the drug can be smoothly broken down. Figure 4 and Figure 5 The aqueous solvent molecular Brownian motion and the motion trajectory and speed distribution of the drug-loaded nanomotor show that the drug-loaded nanomotor can be driven by nitric oxide, and the driving speed is 6 times the Brownian motion speed. Figure 6 (LPS, LPS+CDA, LPS+CDATR respectively refer to: lipopolysaccharide, lipopolysaccharide+CD-Arg, lipopolysaccharide+CD-Arg-TK-RAPA. CD-Arg-TK-RAPA is the drug-loaded nanomotor.) Further verify that the drug-loaded nanomotor has the strongest green fluorescence in the inflammatory macrophage, and the highest amount of nitric oxide is generated.

[0031] The nanomotor provided in the embodiment has a special chemical bond sensitive to reactive oxygen in the inflammatory microenvironment, which is easy to break down in the presence of reactive oxygen, thereby achieving targeted release of drugs, increasing the drug concentration in the target area, reducing its distribution in non-targeted parts, and reducing adverse reactions. At the same time, the arginine modified on the nanomotor generates nitric oxide under the action of reactive oxygen and inducible nitric oxide synthase, which regulates vasodilation and provides gas driving force.

[0032] Although the application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that the technical solutions recorded in the foregoing embodiments can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application.

Claims

1. A nitric oxide-driven nanomotor synthesis, characterized in that, The structure is shown in the following formula: 。 2. A method for preparing a nitric oxide-driven nanomotor synthesis, characterized in that, The method comprises the following steps: S1, dissolve the cyclodextrin and Boc-protected arginine in anhydrous DMF, add EDC and DMAP; react in a water bath under nitrogen; after dialysis, freeze-drying separation to obtain white product CD-Arg; S2, dissolve rapamycin and thioketone in anhydrous DMF, add EDC and DMAP; react in a water bath under nitrogen; after dialysis, freeze-drying separation to obtain yellow product RAPA-TK; S3, dissolve CD-Arg and RAPA-TK in anhydrous DMF, then add EDC and DMAP; react in a water bath under nitrogen; the product is separated by dialysis and freeze-drying to obtain nanomotor synthesis CD-Arg-TK-RAPA.

3. The method of claim 2, wherein the nitric oxide-driven nanomotor composition is prepared by the steps of: (a) providing a nitric oxide source; (b) providing a nanomotor; and (c) mixing the nitric oxide source and the nanomotor. In steps S1 to S3, the temperature of the water bath reaction is 25-40℃, and the time is 12-30h.

4. The method of claim 2, wherein the nitric oxide-driven nanomotor composition is prepared by the steps of: (a) mixing a nitric oxide donor and a nitric oxide scavenger; (b) adding a nanomotor to the mixture of step (a); and (c) adding a stabilizer to the mixture of step (b). In step S1, the molar ratio of the cyclodextrin to the Boc-protected arginine is 1: (3.5-4.5); In step S2, the molar ratio of the rapamycin to the thioketone is 1: (2.5-3.2); In step S3, the molar ratio of the CD-Arg to the RAPA-TK is 1: (1-1.2).

5. The method for preparing the nitric oxide-driven nanomotor synthesis according to claim 2, characterized in that, The structure of the thioketone is shown in the following formula: 。 6. The preparation method of the nitric oxide-driven nanomotor synthesis of any one of claims 2-5, wherein, The synthesis process of step S1 is shown in the following formula: Formula I; The synthesis process of step S2 is shown in the following formula: Formula II; The synthesis process of step S3 is shown in the following formula: Formula III.

7. Use of the nitric oxide-driven nanomotor synthesis of claim 1 or the nanomotor synthesis obtained by the preparation method of any one of claims 2-5 in the preparation of an atherosclerosis-relieving drug.

8. Use according to claim 7, characterized in that, The preparation of the atherosclerosis-relieving drug comprises: self-assembling the drug active ingredient and the nitric oxide-driven nanomotor synthesis into nanoparticles in a solution to encapsulate the drug active ingredient, thereby obtaining the atherosclerosis-relieving drug.

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