A light-controlled nitric oxide delivery platform and its preparation method and application

Abstract

The present invention provides a light-controlled nitric oxide delivery platform and its preparation method and application, belonging to the field of optical nanomaterial technology. The nitric oxide donor microparticles provided by the present invention are self-assembled by components including energy ring nanoparticles, nitric oxide donor small molecules and amphiphilic polymers. The present invention uses Tm 3+ Doped NaYF4 nanoparticles, as energy ring nanoparticles, have a high refractive index (1.46), much higher than the refractive index of the dispersed system under use, making them easily captured by a near-infrared laser beam, enabling optical manipulation of the near-infrared laser beam. The light-controlled nitric oxide delivery platform provided by the present invention uses a near-infrared laser beam as a trigger to capture and manipulate the movement of nitric oxide donor particles, while simultaneously stimulating their release of nitric oxide. This platform has the advantage of spatiotemporally controllable nitric oxide delivery and can effectively isolate diseased cells, thereby achieving precise treatment of cardiovascular diseases such as thrombosis.

Description

Technical Field

[0001] The present invention relates to the technical field of optical nanomaterials, and in particular to a light-controlled nitric oxide delivery platform and a preparation method and application thereof. Background Art

[0002] Nitric oxide is an important gaseous signaling molecule that plays a key role in physiological processes such as neural communication, vasodilation, and cell apoptosis, and has great potential in the treatment of cardiovascular diseases, inflammation, and tumors. However, since nitric oxide is in gaseous form, it has a short half-life and diffusion distance. Currently, various nitric oxide donor drugs have been developed, but they are passively transported in vivo, and the complex in vivo environment may cause nitric oxide donor drugs to stick to tissues or even be cleared, resulting in low utilization of nitric oxide in the lesion. In addition, nitric oxide donor drugs usually release nitric oxide spontaneously, resulting in the generation of systemic side effects. Therefore, delivering nitric oxide in a spatiotemporally controllable manner remains the main challenge in the development of nitric oxide delivery platforms.

[0003] To address this problem, many stimuli-responsive nitric oxide donor microparticles have been developed. Endogenous stimuli include pH, reactive oxygen species, and enzymes, while exogenous triggers include magnetism, ultrasound, and X-rays. Under the action of stimuli, nitric oxide donor microparticles can release nitric oxide. However, these nitric oxide donor microparticles cannot move precisely to the target location, making it difficult to achieve spatiotemporally controlled nitric oxide delivery. Optical capture and manipulation technology has shown great potential in nitric oxide delivery. On the one hand, light can precisely guide the movement of microparticles, and on the other hand, light can trigger nitric oxide donor microparticles to produce nitric oxide. However, there are currently few reports on nitric oxide delivery methods based on optical manipulation technology. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a light-controlled nitric oxide delivery platform and its preparation method and application. The nitric oxide donor particles provided by the present invention can realize optical manipulation of near-infrared laser beams, and the constructed light-controlled nitric oxide delivery platform has the advantage of spatiotemporally controllable release of nitric oxide.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a nitric oxide donor microparticle, which is obtained by self-assembly of components including energy ring nanoparticles, nitric oxide donor small molecules and amphiphilic polymers;

[0007] The energy ring nanoparticles are Tm 3+ doped NaYF4 nanoparticles;

[0008] The nitric oxide donor small molecule is S-nitroso-N-acetyl-DL-penicillamine.

[0009] Preferably, the amphiphilic polymer is DSPE-PEG-NH2; the relative molecular weight of the DSPE-PEG-NH2 is 200 to 50,000.

[0010] Preferably, the energy ring nanoparticles have a Tm 3+ The doping amount is 0.1 to 5 wt%.

[0011] Preferably, the mass ratio of the amphiphilic polymer, the energy ring nanoparticles and the nitric oxide donor small molecule is 50-100:10:1;

[0012] The particle size of the nitric oxide donor particles is 1.2 to 1.6 μm.

[0013] The present invention provides a method for preparing the above-mentioned nitric oxide donor particles, comprising the following steps:

[0014] Ultrasonic mixing of energy ring nanoparticles, nitric oxide donor small molecules, amphiphilic polymers and organic solvents to obtain a mixed solution;

[0015] The organic solvent in the mixed solution is removed, and the remaining substance is mixed with water and dialyzed to obtain nitric oxide donor microparticles.

[0016] Preferably, the molecular weight cut-off of the dialysis is 100 kDa;

[0017] The dialysis is performed under centrifugal conditions, with a centrifugal speed of 3000-4000 rpm and a time of 3-5 minutes.

[0018] The present invention provides a light-controlled nitric oxide delivery platform, comprising a trigger and the above-mentioned nitric oxide donor particles;

[0019] The trigger is a near-infrared laser beam.

[0020] Preferably, the wavelength of the near-infrared laser beam is 700-1700 nm.

[0021] The present invention provides a method for preparing the above-mentioned light-controlled nitric oxide delivery platform, comprising the following steps:

[0022] A near-infrared laser beam is assembled with nitric oxide donor microparticles to obtain a light-controlled nitric oxide delivery platform;

[0023] The assembly method is scanning optical tweezers assembly.

[0024] The present invention provides the use of the above-mentioned light-controlled nitric oxide delivery platform in the preparation of cardiovascular disease drugs or cardiovascular disease medical devices.

[0025] The present invention provides a nitric oxide donor microparticle, which is obtained by self-assembly of components including energy ring nanoparticles, nitric oxide donor small molecules and amphiphilic polymers; the energy ring nanoparticles are Tm 3+ Doped NaYF4 nanoparticles (abbreviated as ELNP); the nitric oxide donor small molecule is S-nitroso-N-acetyl-DL-penicillamine (abbreviated as SNAP). The present invention uses Tm 3+ The doped NaYF4 nanoparticles serve as energy ring nanoparticles with a high refractive index (1.46), which is much higher than the refractive index of the dispersed system under the use environment. They are easily captured by near-infrared laser beams, thus realizing optical manipulation of near-infrared laser beams. Under the excitation of near-infrared light, the energy ring nanoparticles generate ultraviolet light and blue light, among which ultraviolet light can excite the nitric oxide donor small molecule S-nitroso-N-acetyl-DL-penicillamine to release nitric oxide. The amphiphilic polymers in the particles play the role of connecting the energy ring nanoparticles with the nitric oxide donor small molecules.

[0026] The light-controlled nitric oxide delivery platform constructed by the present invention comprises a near-infrared laser beam trigger and the aforementioned nitric oxide donor microparticles. The light-controlled nitric oxide delivery platform provided by the present invention uses a laser beam as a trigger to capture and manipulate the movement of the nitric oxide donor microparticles, while simultaneously stimulating their release of nitric oxide. This platform offers the advantages of spatiotemporally controllable nitric oxide delivery and can effectively isolate diseased cells, thereby enabling precise treatment of cardiovascular diseases such as thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the structure of nitric oxide donor particles and the mechanism of nitric oxide release;

[0028] Figure 2 is the hydration kinetic diameter diagram of nitric oxide donor microparticles;

[0029] Figure 3 Imaging of the precise movement of the light-controlled nitric oxide delivery platform;

[0030] Figure 4 This is a diagram showing the effect of light-controlled nitric oxide delivery platform inducing deactivation of activated platelets;

[0031] Figure 5 This figure shows the therapeutic effect of the light-controlled nitric oxide delivery platform on thrombotic zebrafish lesions. DETAILED DESCRIPTION

[0032] The present invention provides a nitric oxide donor microparticle, which is obtained by self-assembly of components including energy ring nanoparticles, nitric oxide donor small molecules and amphiphilic polymers;

[0033] The energy ring nanoparticles are Tm 3+ doped NaYF4 nanoparticles;

[0034] The nitric oxide donor small molecule is S-nitroso-N-acetyl-DL-penicillamine.

[0035] In the present invention, the nitric oxide donor microparticles have energy ring nanoparticles as the core, and the energy ring nanoparticles and the nitric oxide donor small molecules are connected by amphiphilic polymers.

[0036] In the present invention, the energy ring nanoparticles are Tm 3+ Doped NaYF4 nanoparticles, the Tm 3+ Tm in doped NaYF4 nanoparticles 3+ The doping amount is preferably 0.1 to 5 wt %, more preferably 0.5 to 4 wt %, and further preferably 1 to 3 wt %.

[0037] In the present invention, the Tm 3+ The method for preparing doped NaYF4 nanoparticles preferably comprises the following steps:

[0038] The aqueous solution of yttrium acetate and thulium acetate is heated and mixed with oleic acid and octadecene to obtain a lanthanide oleic acid complex;

[0039] The lanthanide oleic acid complex is mixed with a methanol solution of ammonium fluoride and sodium hydroxide, and after removing the methanol, heat treatment is performed to obtain Tm 3+ Doped NaYF4 nanoparticles.

[0040] In the present invention, an aqueous solution of yttrium acetic acid and thulium acetic acid is heated and mixed with oleic acid and octadecene to obtain a lanthanide oleic acid complex. In the present invention, the concentration of the aqueous solution of yttrium acetic acid and thulium acetic acid is preferably 0.2 mmol / L. In the present invention, the volume ratio of oleic acid to octadecene is preferably 2:3, and the volume ratio of oleic acid to the aqueous solution of yttrium acetic acid and thulium acetic acid is preferably 2:1.

[0041] In the present invention, the temperature of the heating and mixing is preferably 160° C., and the time is preferably 40 minutes. After obtaining the lanthanide oleic acid complex, the present invention preferably cools it to 50° C. at room temperature.

[0042] The present invention mixes the lanthanide oleic acid complex with a methanol solution of ammonium fluoride and sodium hydroxide, removes the methanol, and then performs heat treatment to obtain Tm 3+ Doped NaYF4 nanoparticles. In the present invention, in the methanol solution of ammonium fluoride and sodium hydroxide, the usage ratio of ammonium fluoride, sodium hydroxide and methanol is preferably 1.5 mmol: 1 mmol: 5 mL.

[0043] In the present invention, the heat treatment is carried out under inert gas protection conditions; the temperature of the heat treatment is preferably 290° C., and the time is preferably 1 hour.

[0044] In the present invention, after the heat treatment, the heat-treated product is preferably cooled to room temperature, anhydrous ethanol is added to precipitate a solid, and the solid is centrifuged and washed to obtain Tm 3+ Doped NaYF4 nanoparticles.

[0045] In the present invention, the detergent used in the washing is preferably ethanol, and the number of washing times is preferably 3 times.

[0046] In the present invention, the Tm 3+ The particle size of the doped NaYF4 nanoparticles is preferably 40 to 60 nm.

[0047] In the present invention, the amphiphilic polymer is preferably DSPE-PEG-NH2, whose Chinese name is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (polyethylene glycol) amine; the relative molecular weight of the DSPE-PEG-NH2 is preferably 200 to 50,000, more preferably 500 to 40,000, and further preferably 10,000 to 30,000.

[0048] In the present invention, the mass ratio of the amphiphilic polymer, the energy ring nanoparticles and the nitric oxide donor small molecule is preferably 50 to 100:10:1, more preferably 60 to 80:10:1.

[0049] In the present invention, the particle size of the nitric oxide donor microparticles is preferably 1.2 to 1.6 μm, more preferably 1.4 μm.

[0050] The present invention provides a method for preparing the above-mentioned nitric oxide donor particles, comprising the following steps:

[0051] Ultrasonic mixing of energy ring nanoparticles, nitric oxide donor small molecules, amphiphilic polymers and organic solvents to obtain a mixed solution;

[0052] The organic solvent in the mixed solution is removed, and the remaining substance is mixed with water and dialyzed to obtain nitric oxide donor microparticles.

[0053] The present invention ultrasonically mixes energy ring nanoparticles, nitric oxide donor small molecules, amphiphilic polymers and an organic solvent to obtain a mixed solution. In the present invention, the organic solvent is preferably dichloromethane or chloroform.

[0054] In the present invention, the energy ring nanoparticles are preferably provided in the form of a cyclohexane dispersion, and the concentration of the cyclohexane dispersion of the energy ring nanoparticles is preferably 20 mg / mL; the nitric oxide donor small molecule is preferably provided in the form of an acetone dispersion, and the concentration of the acetone dispersion of the nitric oxide donor small molecule is preferably 5 mg / mL.

[0055] In the present invention, the power of the ultrasonic mixing is preferably 20 to 50 kHz, more preferably 30 to 40 kHz, and the time is preferably 3 to 5 minutes, more preferably 4 minutes.

[0056] After obtaining the mixed solution, the present invention removes the organic solvent from the mixed solution, mixes the remaining substance with water, and performs dialysis to obtain nitric oxide donor particles. In the present invention, the method of removing the organic solvent is preferably natural drying.

[0057] In the present invention, the method of mixing the remaining substance with water is preferably ultrasonic mixing to obtain a uniformly dispersed particle solution. In the present invention, the power of the ultrasonic mixing is preferably 20 to 50 kHz, more preferably 30 to 40 kHz, and the time is preferably 3 to 5 minutes, more preferably 4 minutes.

[0058] In the present invention, the molecular weight cut-off for dialysis is preferably 100 kDa; and the present invention preferably uses an ultrafiltration centrifuge tube for dialysis. In the present invention, the dialysis is preferably performed under centrifugal conditions, with the centrifugal speed preferably being 3000-4000 rpm, more preferably 3500-4000 rpm, and the time preferably being 3-5 minutes, more preferably 4 minutes.

[0059] After the dialysis, the present invention preferably filters the obtained dialysis product, and the present invention preferably uses a polyethersulfone filter membrane with a pore size of 0.22 μm for the filtration.

[0060] The present invention provides a light-controlled nitric oxide delivery platform, comprising a trigger and the above-mentioned nitric oxide donor particles; the trigger is a near-infrared laser beam.

[0061] In the present invention, the wavelength of the near-infrared laser beam is preferably 700 to 1700 nm, more preferably 1064 to 1500 nm.

[0062] The present invention provides a method for preparing the above-mentioned light-controlled nitric oxide delivery platform, comprising the following steps:

[0063] A near-infrared laser beam is assembled with nitric oxide donor particles to obtain a light-controlled nitric oxide delivery platform; the assembly method is scanning optical tweezers assembly.

[0064] In the present invention, the assembly is preferably carried out in an aqueous dispersion system.

[0065] The present invention provides the use of the above-mentioned light-controlled nitric oxide delivery platform in the preparation of cardiovascular disease drugs or cardiovascular disease medical devices. In the present invention, the cardiovascular disease is preferably thrombosis.

[0066] In the present invention, the structural diagram of the nitric oxide donor particles and the mechanism diagram of nitric oxide release are as follows: Figure 1 shown.

[0067] The light-controlled nitric oxide delivery platform, preparation method and application thereof provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] (1) Preparation of energy ring nanoparticles:

[0070] A reaction flask was added with 4 mL of oleic acid and 6 mL of 1-octadecene. Then, 2 mL of a 0.2 mmol / L aqueous solution of yttrium acetate and thulium acetate was added. The mixture was heated at 160°C for 40 minutes to form the lanthanide oleic acid complex. The mixture was then cooled to 50°C at room temperature. A methanol solution containing 5 mL of 1.5 mmol of ammonium fluoride and 1 mmol of sodium hydroxide was added to the mixture and stirred for 30 minutes. The methanol was then removed, and the solution was heated at 290°C under inert gas for 1 hour. After the reaction, the mixture was cooled to room temperature, and 5 mL of anhydrous ethanol was added to precipitate the product. The resulting crude product was centrifuged and washed three times with 5 mL of ethanol to yield 80 mg of nanoparticles.

[0071] (2) Preparation of energy ring nanoparticle solution:

[0072] The energy ring nanoparticles were mixed evenly with 4 mL of cyclohexane to obtain a solution of energy ring nanoparticles.

[0073] (3) Preparation of Nitric Oxide Donor Microparticles:

[0074] A 20 mg / mL solution of energy ring nanoparticles (25 μL), 5 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (polyethylene glycol)amine, 10 μL of a 5 mg / mL solution of S-nitroso-N-acetyl-DL-penicillamine in acetone, and 1 mL of dichloromethane were mixed and ultrasonically dispersed for 3 minutes to obtain a mixed solution. The mixed solution, including dichloromethane, n-hexane, and acetone, was dried using a nitrogen stream to obtain a dry solid film. 1 mL of ultrapure water was added, and the solution containing the solid film was ultrasonically dispersed for 3 minutes to obtain a uniformly dispersed microparticle solution. The microparticle solution was then centrifuged at 4000 rpm for 10 minutes using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. The supernatant was removed, and the crude product obtained by centrifugation was filtered through a 0.22 μm polyethersulfone filter membrane to obtain a solution of nitric oxide donor microparticles with a uniform particle size.

[0075] The hydration kinetic diameter of the obtained nitric oxide donor microparticles is shown in FIG. Figure 2 As shown, it can be seen that the particle size of the obtained nitric oxide donor microparticles is about 1.4 μm.

[0076] Example 2

[0077] The nitric oxide donor microparticle solution obtained in Example 1 was diluted 10-fold with ultrapure water to obtain a 1 mg / mL microparticle solution. 100 μL of the diluted microparticle solution was then placed on a glass slide. The slide with the microparticle solution was then placed on the three-dimensional displacement stage of a scanning optical tweezers system (Aresis Tweez250si). A 1064 nm laser beam was then used to irradiate the microparticles on the slide, capturing and manipulating the microparticles in the solution, stimulating their nitric oxide production while precisely guiding their trajectory.

[0078] Figure 3 The motion diagram of the light-controlled nitric oxide delivery platform described in multiple embodiments 1 is shown in FIG. Figure 3 It can be seen that the light-controlled nitric oxide delivery platform provided by the present invention can accurately control the movement trajectory of nitric oxide donor particles.

[0079] Application Example 1

[0080] The microparticle solution obtained in Example 1 was added to an imaging dish incubated with cells and diluted 100 times to obtain a microparticle solution with a concentration of 0.1 mg / mL. The imaging dish was placed on the three-dimensional displacement platform of a scanning optical tweezers system (Aresis Tweez 250si), and a laser beam with a wavelength of 1064 nm was used to irradiate the microparticle solution in the imaging dish to capture and manipulate the microparticles in the solution, while imaging the fluorescence generated by the microparticles. The light-controlled nitric oxide delivery platform was manipulated to the vicinity of the activated platelets to deliver nitric oxide thereto, thereby completing the deactivation of the activated platelets, as shown in FIG. Figure 4As shown, by observing the morphological changes of platelets before and after treatment, it is proved that the nitric oxide delivery platform can deliver nitric oxide in a spatiotemporally controlled manner to complete the deactivation of activated platelets.

[0081] Application Example 2

[0082] Two-day-old zebrafish larvae were incubated with 1 μmmol / L phenylhydrazine solution for 12 h to establish a zebrafish thrombosis disease model.

[0083] The microparticle solution obtained in Example 1 was diluted 10-fold with phosphate buffer to obtain a microparticle solution with a concentration of 1 mg / mL. 10 nL of the diluted microparticle solution was injected into the vein of the thrombus-bearing zebrafish using a microinjection device. The microparticles were captured by laser and manipulated to the thrombus lesion. Figure 5 As shown, the light-controlled nitric oxide delivery platform can release nitric oxide at the lesion site, inducing cells at the thrombus site to detach from the blood vessel wall, and then use optical tweezers to remove the cells detached from the blood vessel wall, thereby completing the removal of the thrombus adhered to the blood vessel.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nitric oxide donor microparticle, which is self-assembled from components including energy ring nanoparticles, a nitric oxide donor small molecule, and an amphiphilic polymer; The energy ring nanoparticles are Tm 3+ doped NaYF4 nanoparticles; The nitric oxide donor small molecule is S-nitroso-N-acetyl-DL-penicillamine; The amphiphilic polymer is DSPE-PEG-NH2; the relative molecular weight of the DSPE-PEG-NH2 is 200 to 50,000; Tm in the energy ring nanoparticles 3+ The doping amount is 0.1 to 5 wt%; The mass ratio of the amphiphilic polymer, the energy ring nanoparticles and the nitric oxide donor small molecule is 50-100:10:1; The particle size of the nitric oxide donor particles is 1.2 to 1.6 μm.

2. The method for preparing the nitric oxide donor microparticles according to claim 1, comprising the following steps: Ultrasonic mixing of energy ring nanoparticles, nitric oxide donor small molecules, amphiphilic polymers and organic solvents to obtain a mixed solution; The organic solvent in the mixed solution is removed, and the remaining substance is mixed with water and dialyzed to obtain nitric oxide donor microparticles.

3. The preparation method according to claim 2, characterized in that The molecular weight cut-off of the dialysis is 100 kDa; The dialysis is performed under centrifugal conditions, with a centrifugal speed of 3000-4000 rpm and a time of 3-5 minutes.

4. A light-controlled nitric oxide delivery platform comprising a trigger and the nitric oxide donor microparticles according to claim 1 or the nitric oxide donor microparticles prepared by the preparation method according to claim 2 or 3; The trigger is a near-infrared laser beam.

5. The light-controlled nitric oxide delivery platform according to claim 4, characterized in that The wavelength of the near-infrared laser beam is 700-1700 nm.

6. The method for preparing the light-controlled nitric oxide delivery platform according to claim 4 or 5, comprising the following steps: A near-infrared laser beam is assembled with nitric oxide donor microparticles to obtain a light-controlled nitric oxide delivery platform; The assembly method is scanning optical tweezers assembly.

7. Use of the light-controlled nitric oxide delivery platform according to claim 4 or 5, or the light-controlled nitric oxide delivery platform prepared by the preparation method according to claim 6, in the preparation of drugs or medical devices for treating cardiovascular diseases; The cardiovascular disease is thrombosis.

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