Magnetic effect-triggered intelligent copper-releasing nanoformulation, preparation method thereof, and pharmaceutical application

Abstract

The present invention provides a magnetic effect-triggered intelligent copper-releasing nanoformulation, its preparation method, and pharmaceutical application. The preparation is ES@Fe3O4@SiO2-Cu. The preparation method includes adding FeCl3·6H2O and CH3COONa to ethylene glycol, hydrothermal reaction, washing, and magnetic separation; dispersing the iron oxide magnetic fluid in ammonia water and ultrasonication, adding an ethanol solution containing 20% ​​tetraethyl silicate and a copper nitrate mixture to continue the reaction, washing with ethanol and water, and magnetic separation. The Fe3O4@SiO2-Cu fluid is dispersed in an ammonium nitrate isopropanol solution. After magnetic separation, isopropanol and water are added and ultrasonicated, washed with ethanol and water, and the water is replaced with pure ethanol. The Fe3O4@SiO2-Cu is vacuum dried to obtain Fe3O4@SiO2-Cu, and ES is dispersed in water, and ultrasonic vibration is vigorously performed; the dialysis bag is placed overnight to collect the ES@Fe3O4@SiO2-Cu. The present invention has a strong penetration depth, is suitable for treating deep tumors, and has good heating efficiency under an alternating magnetic field.

Description

Technical Field

[0001] The present invention relates to the technical field of nano drug delivery, and in particular to a magnetic effect-triggered intelligent copper-releasing nano preparation, a preparation method thereof, and pharmaceutical applications. Background Art

[0002] Cuproptosis is a recently discovered copper-dependent immunogenic cell death pathway, and inducing cuproptosis is a promising anti-tumor therapeutic strategy. However, this copper-induced tumor cell death strategy faces two major challenges that need to be addressed: low copper ion concentrations within tumor cells and the short blood half-life of copper ion carrier drugs.

[0003] Therefore, constructing a copper-induced nano drug delivery system (NDDS) is the key to the clinical application of the copper-death tumor treatment strategy.

[0004] The following are examples of the current laboratory preparation steps for copper nanoparticles:

[0005] (1) Mix 5 mL of 0.5 mM HAuCl4 with 5 mL of 0.2 mM CTAB solution. Inject 0.6 mL of fresh 0.01 M sodium borohydride under vigorous stirring. After the color of the solution changes from yellow to brown, continue stirring for 2 min. Age the solution at room temperature for 30 min to obtain a seed solution. Prepare a growth solution by dissolving 1.8 g of CTAB and 0.22 g of 5-BrSA in 50 mL of warm water. After cooling the solution to 30°C, add 2.4 mL of 4 mM silver nitrate solution and stir gently. Keep the mixture at 30°C for 15 min, then add 50 mL of 1 mM HAuCl4 solution. After slowly stirring for 15 min, add 0.4 mL of 0.064 M ascorbic acid and stir vigorously for 30 s until colorless. Finally, inject 0.16 mL of the seed solution into the growth solution. Stir the mixture for 30 s and store at 30°C for 12 h.

[0006] (2) Prepare a 20% TEOS ethanol solution containing Cu(NO3)2, where the Si / Cu ratio is 30:1. The synthesized Au NRs were added to 30 mL of ammonium hydroxide (0.51 M) and stirred at 40°C. Subsequently, 90 μL of the Si / Cu ethanol solution was added to the solution three times. After gentle stirring for 2 days, the nanoparticles were collected by centrifugation (8000 rpm, 15 min).

[0007] (3) 10 mg of Au@MSN-Cu was dispersed in 10 mL of ethanol solution containing 2 mg / mL PEG-silane and gently stirred at 60 °C for 24 h. After cooling to room temperature, the Au@MSN-Cu / PEG was collected by centrifugation and washed three times with ethanol.

[0008] (4) Disperse 10 mg of Au@MSN-Cu / PEG in 2 mL of ethanol solution containing 40 mg of DSF (dicoflurane), ultrasonicate for 1 hour, and vigorously stir the mixture overnight (stirring overnight is 16 hours).

[0009] However, the copper nanoparticles prepared in the prior art and the corresponding drug delivery methods have the following disadvantages:

[0010] 1. Gold (Au) nanoparticles are expensive and not suitable for industrial production and clinical transformation.

[0011] 2. Using near-infrared light to generate heat is not suitable for deep-seated tumors. Infrared light has difficulty penetrating fat layers and can only penetrate 4-5 cm below the skin, making it suitable for superficial tissue tumors and limiting its use. Furthermore, the intensity and duration of near-infrared light must be strictly controlled, otherwise it can easily cause skin damage.

[0012] 3. The existing thermal fields based on radio frequency, ultrasound, etc. as heat drivers are highly focused at one point. Heat needs to be conducted from the tissue to the drug delivery system. The heat conduction process will lead to overheating areas, suitable temperature areas and cold areas, making it difficult for the drug delivery system to release drugs evenly. Summary of the Invention

[0013] In view of this, the first purpose of the present invention is to provide a magnetic effect-triggered intelligent copper-releasing nanoformulation with iron oxide Fe3O4 as the core, which can be triggered by magnetic effect, has a strong penetration depth, is suitable for the treatment of deep tumors, induces uniform, rapid and controllable heat production, and reduces costs, making it suitable for industrial production and clinical transformation.

[0014] The second object of the present invention is to provide a method for preparing the above-mentioned magnetic effect-triggered intelligent copper-releasing nanoformulation, which has a simple production process and is convenient for on-site application.

[0015] The third object of the present invention is to provide the use of the above-mentioned magnetic effect-triggered intelligent copper-releasing nanoformulation in the preparation of drugs for treating tumors, which effectively promotes the uptake of nanomedicines by tumor cells through magnetic effect-triggered drug delivery.

[0016] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0017] The present invention provides a magnetically triggered smart copper-releasing nanoformulation, which is a copper compound ES@Fe3O4@SiO2-Cu. Specifically, the copper compound ES@Fe3O4@SiO2-Cu has iron oxide Fe3O4 as its core.

[0018] The present invention also provides a method for preparing a magnetic effect-triggered smart copper-releasing nanoformulation, which is used to prepare the magnetic effect-triggered smart copper-releasing nanoformulation as described above, comprising the following steps:

[0019] S1. Add equal parts by mass of FeCl3·6H2O and CH3COONa to ethylene glycol, add the mixture to a hydrothermal reactor, react at 195-205°C for 11-13 hours, wash, and magnetically separate to obtain iron oxide magnetic fluid Fe3O4;

[0020] S2, the iron oxide magnetic fluid Fe3O4 is dispersed in ammonia water, ultrasonically shaken, stirred, and added with molar concentration of Si 4+ :Cu 2+ The mixture of 20% tetraethyl silicate ethanol solution and copper nitrate in a ratio of 20:1 was continued to react for 47-49 hours; ethanol and water in a volume ratio of 1:1 were added for washing, and Fe3O4@SiO2-Cu fluid was obtained by magnetic separation;

[0021] S3, dispersing the Fe3O4@SiO2-Cu fluid in ammonium nitrate isopropanol solution and stirring; after magnetic separation, adding isopropanol water in a volume ratio of 1:1, ultrasonically vibrating for 25-35 minutes, adding ethanol water in a volume ratio of 1:1 for washing, and finally replacing the water with pure ethanol, vacuum drying to obtain Fe3O4@SiO2-Cu;

[0022] S4. Disperse the Fe3O4@SiO2-Cu and the carrier drug ilisimol ES in oxygen-free distilled water, and vibrate violently with ultrasound; dialyze overnight in a dialysis bag, and collect the final product ES@Fe3O4@SiO2-Cu nanoformulation.

[0023] Preferably, the stirring temperature for dispersing the iron oxide magnetic fluid Fe3O4 in the ammonia water in step S2 is 39-41°C.

[0024] Preferably, the stirring speed of the iron oxide magnetic fluid Fe3O4 dispersed in the ammonia water in step S2 is 95-105 rpm / min.

[0025] Preferably, the method of adding the ethanol solution containing 20% ​​tetraethyl silicate and the copper nitrate mixture in step S2 is: adding one-third of the ethanol solution containing 20% ​​tetraethyl silicate and the copper nitrate mixture every 30 minutes, for a total of 3 times.

[0026] Preferably, the stirring time of the Fe3O4@SiO2-Cu fluid dispersed in the ammonium nitrate isopropanol solution in step S3 is 7-9 hours.

[0027] Preferably, the Fe3O4@SiO2-Cu and the carrier drug ilisimol ES in step S4 are dispersed in oxygen-free distilled water and subjected to intense ultrasonic oscillation for 18-22 minutes.

[0028] The present invention also provides the use of the magnetic effect-triggered intelligent copper-releasing nanoformulation described above in the preparation of drugs for treating tumors.

[0029] The intelligent copper-releasing nanoformulation prepared by the present invention is triggered by the magnetic effect. The magnetic field is evenly distributed, and the induced heat generation is uniform, rapid and controllable. By controlling the magnetic field intensity, the core iron oxide Fe3O4 can be quickly heated to 50°C within 10 minutes and maintained at 50°C±0.2°C for up to 60 minutes.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Compared with the existing near-infrared photothermal-driven copper nanoparticles, the magnetic effect-triggered intelligent copper-releasing nanoformulation provided by the present invention has a strong penetration depth of the magnetic field and few side effects, and is suitable for the treatment of deep tumors. It overcomes the limitations of the existing technologies based on radio frequency, ultrasound, etc. as thermal drives, and has the advantages of uniform, rapid and controllable induced heat production. It is an ideal thermal drive solution for temperature-sensitive intelligent drug delivery systems. It releases copper ions and ilisimol drugs through the self-induced heating of the core iron oxide Fe3O4 under an alternating magnetic field. It has a wider range of indications and higher safety. It is not limited by the size, location and irregular shape of the tumor. The magnetic field can penetrate the human body evenly without obvious side effects, allowing the core of the drug delivery system to directly generate heat, causing the silicon shell to disintegrate, minimizing damage to adjacent cancer tissues. The core iron oxide Fe3O4 nanoparticles are simpler to prepare and have higher economic benefits than gold nanoparticles. The heating efficiency under an alternating magnetic field is better than infrared light, and the heating rate to 50°C is increased by 17.6%. DETAILED DESCRIPTION

[0032] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0033] Example 1

[0034] Prepare oxygen-free distilled water;

[0035] 2.4 g FeCl3·6h2O and 2.4 g CH3COONa were added to 30 mL ethylene glycol and placed in a hydrothermal reactor. The mixture was reacted at 200°C for 12 hours. The iron oxide magnetic fluid Fe3O4 was obtained by washing and magnetic separation.

[0036] Prepare a mixture of 20% tetraethyl silicate in ethanol and copper nitrate, with a molar concentration of Si 4+ :Cu 2+ is 20:1;

[0037] The iron oxide magnetic fluid Fe3O4 was dispersed in ammonia water, ultrasonically shaken, and stirred at 40°C at a stirring speed of 100 rpm / min. 2 mL of the ethanol solution containing 20% ​​tetraethyl silicate and copper nitrate was added every 30 minutes for a total of 3 times, and the reaction was continued for 48 hours. The mixture was washed with ethanol and water in a volume ratio of 1:1 for 3 times, and magnetic separation was performed to obtain Fe3O4@SiO2-Cu fluid.

[0038] The Fe3O4@SiO2-Cu fluid was dispersed in 60 mL of ammonium nitrate isopropanol solution and stirred at 35°C for 7 h. After magnetic separation, isopropanol-water was added in a volume ratio of 1:1. After ultrasonic vibration for 30 minutes, ethanol-water was added in a volume ratio of 1:1 for multiple washings. Finally, the water was replaced with 20 mL of pure ethanol and vacuum dried to obtain Fe3O4@SiO2-Cu.

[0039] 100 mg of the Fe3O4@SiO2-Cu and 4 mg of the carrier drug ilisimol ES were dispersed in oxygen-free distilled water and subjected to vigorous ultrasonic shaking for 20 minutes; the dialysis bag was dialyzed overnight, and the final product ES@Fe3O4@SiO2-Cu nanoformulation was collected.

[0040] Example 2

[0041] Prepare oxygen-free distilled water;

[0042] 2.4 g FeCl3·6h2O and 2.4 g CH3COONa were added to 30 mL ethylene glycol and placed in a hydrothermal reactor. The reaction was carried out at 205°C for 11 hours. The iron oxide magnetic fluid Fe3O4 was obtained by washing and magnetic separation.

[0043] Prepare a mixture of 20% tetraethyl silicate in ethanol and copper nitrate, with a molar concentration of Si 4+ :Cu 2+ is 20:1;

[0044] The iron oxide magnetic fluid Fe3O4 was dispersed in ammonia water, ultrasonically shaken, and stirred at 39°C at a stirring speed of 95 rpm. 2 mL of the ethanol solution containing 20% ​​tetraethyl silicate and copper nitrate was added every 30 minutes for a total of 3 times, and the reaction was continued for 47 hours. The solution was washed with ethanol and water in a volume ratio of 1:1 for 3 times, and magnetic separation was performed to obtain the Fe3O4@SiO2-Cu fluid.

[0045] The Fe3O4@SiO2-Cu fluid was dispersed in 60 mL of ammonium nitrate isopropanol solution and stirred at 35°C for 8 h. After magnetic separation, isopropanol-water was added in a volume ratio of 1:1. After ultrasonication for 30 minutes, the mixture was washed several times with ethanol-water in a volume ratio of 1:1. Finally, the water was replaced with 20 mL of pure ethanol and vacuum dried to obtain Fe3O4@SiO2-Cu.

[0046] 100 mg of the Fe3O4@SiO2-Cu and 4 mg of the carrier drug ilisimol ES were dispersed in oxygen-free distilled water and subjected to vigorous ultrasonic shaking for 18 minutes; the dialysis bag was dialyzed overnight, and the final product ES@Fe3O4@SiO2-Cu nanoformulation was collected.

[0047] Example 3

[0048] Prepare oxygen-free distilled water;

[0049] 2.4 g FeCl3·6h2O and 2.4 g CH3COONa were added to 30 mL ethylene glycol and placed in a hydrothermal reactor. The reaction was carried out at 205°C for 13 hours. The iron oxide magnetic fluid Fe3O4 was obtained by washing and magnetic separation.

[0050] Prepare a mixture of 20% tetraethyl silicate in ethanol and copper nitrate, with a molar concentration of Si 4+ :Cu 2+ is 20:1;

[0051] The iron oxide magnetic fluid Fe3O4 was dispersed in ammonia water, ultrasonically shaken, and stirred at 41°C at a stirring speed of 100 rpm / min. 2 mL of the ethanol solution containing 20% ​​tetraethyl silicate and copper nitrate was added every 30 minutes for a total of 3 times, and the reaction was continued for 49 hours. The mixture was washed with ethanol and water in a volume ratio of 1:1 for 3 times, and magnetic separation was performed to obtain Fe3O4@SiO2-Cu fluid.

[0052] The Fe3O4@SiO2-Cu fluid was dispersed in 60 mL of ammonium nitrate isopropanol solution and stirred at 35°C for 9 h. After magnetic separation, isopropanol-water was added in a volume ratio of 1:1. After ultrasonication for 35 minutes, ethanol-water was added in a volume ratio of 1:1 for multiple washings. Finally, the water was replaced with 20 mL of pure ethanol and vacuum dried to obtain Fe3O4@SiO2-Cu.

[0053] 100 mg of the Fe3O4@SiO2-Cu and 4 mg of the carrier drug ilisimol ES were dispersed in oxygen-free distilled water and subjected to vigorous ultrasonic shaking for 22 minutes; the dialysis bag was dialyzed overnight, and the final product ES@Fe3O4@SiO2-Cu nanoformulation was collected.

[0054] Example 4

[0055] Other conditions are the same as those in Example 1, except that 2.0 g of FeCl 3 ·6h 2 O and 2.0 g of CH 3 COONa are used.

[0056] Example 5

[0057] Other conditions are the same as those in Example 1, except that 2.8 g of FeCl 3 ·6h 2 O and 2.8 g of CH 3 COONa are used.

[0058] Comparative Example 1

[0059] Different from Example 1, the ethanol solution containing 20% ​​tetraethyl silicate and the copper nitrate mixture d molar concentration Si 4+ :Cu 2+ Changed to 19:1.

[0060] Comparative Example 2

[0061] Different from Example 1, FeCl3·6h2O and CH3COONa are used in different mass fractions, namely 2.2g FeCl3·6h2O and 2.6g CH3COONa.

[0062] Comparative Example 3

[0063] Different from Example 1, the stirring speed of the iron oxide magnetic fluid Fe3O4 dispersed in the ammonia water is changed to 90 rpm / min.

[0064] Comparative Example 4

[0065] Different from Example 1, half of the mixed solution of 20% tetraethyl silicate in ethanol and copper nitrate was added every 30 minutes for a total of 2 times.

[0066] Test results

[0067] The copper nanoparticle preparations prepared in the above examples and comparative examples were tested for the drug delivery heating rate. The test data are shown in Table 1 below:

[0068] Table 1

[0069]

[0070] The data in Table 1 show that, through a reasonable preparation process, the magnetic effect-triggered intelligent copper-releasing nanoformulation of the present invention has a drug delivery temperature increase rate that is superior to that of Comparative Examples 1 to 4. Furthermore, the process parameters used in Example 1 of the present invention produce the most excellent effect.

[0071] For the comparison results of Comparative Example 1 and Example 1, when the ethanol solution containing 20% ​​tetraethyl silicate and the copper nitrate mixture d molar concentration Si 4+ :Cu 2+ When it is changed to 19:1, the coating effect of the SiO2-Cu complex is weakened, which has a certain impact on the chemical stability of the iron oxide core, thereby reducing the drug delivery heating efficiency.

[0072] As for the comparison results of Comparative Example 2 and Example 1, when FeCl3·6h2O and CH3COONa have different masses, the product of the inner core iron oxide is reduced, which affects the effect of the magnetic effect and thus reduces the drug delivery heating efficiency.

[0073] For the comparison results of Comparative Example 3 and Example 1, when the stirring speed of the iron oxide magnetic fluid Fe3O4 dispersed in ammonia water is changed to 90 rpm / min, the effect of the dispersion is reduced, affecting the effect of subsequent magnetic separation, thereby reducing the drug delivery heating efficiency.

[0074] For the comparison results of Comparative Example 4 and Example 1, when half of the ethanol solution containing 20% ​​tetraethyl silicate and copper nitrate mixture is added every 30 minutes, the number of additions is reduced, the reaction efficiency between the components is reduced, and the effect of subsequent magnetic separation is affected, thereby reducing the drug delivery heating efficiency.

[0075] Comparing Example 1 with Example 2 and Example 3 in Table 1, it can be seen that the drug delivery heating efficiency is best when the intermediate values ​​of the process parameters such as reaction temperature, stirring speed, and reaction time are used.

[0076] In summary, the magnetic effect-triggered intelligent copper-releasing nanoformulation prepared in the embodiment of the present invention has a strong magnetic field penetration depth and few side effects, is suitable for the treatment of deep tumors, and has the advantages of uniform, rapid and controllable induced heat production. It is an ideal thermal driving solution for temperature-sensitive intelligent drug delivery systems. The self-induced heating of the core iron oxide Fe3O4 under the alternating magnetic field releases copper ions and ilisimol drugs, with a wider range of indications and higher safety. It is not limited by the size, location and irregular shape of the tumor. The magnetic field can penetrate the human body evenly without obvious side effects, allowing the core of the drug delivery system to directly generate heat, causing the silicon shell to disintegrate, minimizing damage to adjacent cancer tissues. The core iron oxide Fe3O4 nanoparticles are simpler to prepare and more economical than gold nanoparticles. The heating efficiency under the alternating magnetic field is better than that of infrared light, and the heating rate to 50°C is increased by 17.6%, effectively solving the limitations of the existing technology based on radio frequency, ultrasound, etc. as thermal driving.

[0077] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. It is therefore intended that the appended claims include all such changes and modifications that fall within the scope of the present invention.

Claims

1. A magnetic effect-triggered intelligent copper-releasing nanoformulation, characterized in that: The intelligent copper-releasing nanoformulation is a copper compound ES@Fe3O4@SiO2-Cu, wherein the copper compound ES@Fe3O4@SiO2-Cu has iron oxide Fe3O4 as a core; The preparation method of the magnetic effect-triggered intelligent copper-releasing nanoformulation comprises the following steps: S1. Add equal parts by mass of FeCl3·6H2O and CH3COONa to ethylene glycol, add the mixture to a hydrothermal reactor, react at 195-205°C for 11-13 hours, wash, and magnetically separate to obtain iron oxide magnetic fluid Fe3O4; S2, the iron oxide magnetic fluid Fe3O4 is dispersed in ammonia water, ultrasonically shaken, stirred, and added with molar concentration of Si 4+ :Cu 2+ The mixture of 20% tetraethyl silicate ethanol solution and copper nitrate in a ratio of 20:1 was continued to react for 47-49 hours; ethanol and water in a volume ratio of 1:1 were added for washing, and Fe3O4@SiO2-Cu fluid was obtained by magnetic separation; S3, dispersing the Fe3O4@SiO2-Cu fluid in ammonium nitrate isopropanol solution and stirring; after magnetic separation, adding isopropanol water in a volume ratio of 1:1, ultrasonically vibrating for 25-35 minutes, adding ethanol water in a volume ratio of 1:1 for washing, and finally replacing the water with pure ethanol, vacuum drying to obtain Fe3O4@SiO2-Cu; S4. Disperse the Fe3O4@SiO2-Cu and the carrier drug ilisimol ES in oxygen-free distilled water, and vibrate violently with ultrasound; dialyze overnight in a dialysis bag, and collect the final product ES@Fe3O4@SiO2-Cu nanoformulation.

2. The magnetic effect-triggered intelligent copper-releasing nanoformulation according to claim 1, characterized in that: The stirring temperature of the iron oxide magnetic fluid Fe3O4 dispersed in the ammonia water in step S2 is 39-41°C.

3. The magnetic effect-triggered intelligent copper-releasing nanoformulation according to claim 1, characterized in that: The stirring speed of the iron oxide magnetic fluid Fe3O4 dispersed in the ammonia water in step S2 is 95-105 rpm / min.

4. The magnetic effect-triggered intelligent copper-releasing nanoformulation according to claim 1, characterized in that: The method for adding the mixed solution of 20% tetraethyl silicate in ethanol and copper nitrate in step S2 is as follows: adding one-third of the mixed solution of 20% tetraethyl silicate in ethanol and copper nitrate every 30 minutes, for a total of 3 times.

5. The magnetic effect-triggered smart copper-releasing nanoformulation according to claim 1, characterized in that: The stirring time of the Fe3O4@SiO2-Cu fluid dispersed in the ammonium nitrate isopropanol solution in step S3 is 7-9 hours.

6. The magnetic effect-triggered smart copper-releasing nanoformulation according to claim 1, characterized in that: The Fe3O4@SiO2-Cu and the carrier drug ilisimol ES in step S4 are dispersed in oxygen-free distilled water and the ultrasonic vibration time is 18-22 minutes.

7. Use of the magnetic effect-triggered intelligent copper-releasing nanoformulation according to claim 1 in the preparation of a drug for treating tumors.

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