A preparation method of oxygen-producing nanoliposomes

Abstract

A preparation method of oxygen-producing nanoliposomes. In a non-polar organic solvent, soybean lecithin and a pectin solution are added. Sodium percarbonate solution and calcium hydroxide solution react at low temperature to generate peroxide, and the calcium hydroxide solution and the pectin solution can also undergo a cross-linking reaction to coat a calcium pectate gel film outside the peroxide. It is made into oxygen-producing liposomes by vortex ultrasonic mixing in lecithin-non-polar organic solvent, reverse evaporation, and then adding water and vortex ultrasonic mixing. The prepared oxygen-producing nanoliposomes have a high oxygen production amount, can be stored for a long time under freezing conditions, have high performance stability, and the effective oxygen release time at room temperature is as high as 55h to 60h.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of liposomes, and specifically relates to a method for preparing oxygen-producing nano-liposomes. Background Art

[0002] Liposomes refer to ultra-micro spherical drug carrier preparations prepared by encapsulating drugs in the film formed by a lipid bilayer. Since their structure is similar to biological membranes, they can encapsulate water-soluble and lipophilic drugs, reduce the drug dosage, lower toxicity, delay release, change the drug distribution in the body, and have a targeted therapeutic effect, improving the drug efficacy.

[0003] Cancer treatment is an unsolved problem today and a major challenge that the medical community has been facing. Drug treatment is an important means of cancer treatment. Although traditional anti-cancer drugs have achieved certain curative effects, their side effects such as allergic reactions, suppression of the bone marrow hematopoietic system, and hair loss have greatly limited their clinical applications and become one of the difficult problems in clinical treatment. How to improve the curative effect of cancer treatment is an urgent problem in modern medicine. Hypoxia is an important feature of the solid tumor microenvironment. In a hypoxic environment, cancer cells promote the metastasis of cancer cells by changing their metabolic patterns and inhibit the anti-tumor immune response of the body. The hypoxic environment in the core of tumors causes resistance. Anti-cancer therapies are expected to be an effective way to improve the curative effect of malignant solid tumors by improving the oxygen partial pressure or increasing the reactive oxygen species concentration of hypoxic tumors.

[0004] The currently prepared oxygen-producing nano-liposomes have poor safety performance and low stability, and still cannot meet the requirements for improving the curative effect of malignant solid tumors, and need to be further improved. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing oxygen-producing nano-liposomes with high oxygen production, stable performance, and controllable and sustained oxygen release.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing oxygen-producing nano-liposomes, comprising the following steps:

[0007] (1) Add soybean lecithin to a non-polar organic solvent, vortex, and mix under ultrasonic waves for 4 min to 6 min to obtain a mixed solution. The addition amount of soybean lecithin in each milliliter of non-polar organic solvent is 10 mg to 40 mg;

[0008] (2) Add a pectin solution with a concentration of 20 mg / ml to 30 mg / ml to the mixed solution, stir for 35 min to 45 min, and mix under ultrasonic waves for 18 min to 25 min to obtain a suspension. The volume ratio of the pectin solution to the non-polar organic solvent in step (1) is 0.8 to 1.2:100;

[0009] (3) Add a sodium percarbonate solution with a concentration of 10 mg / ml to 20 mg / ml to the suspension, vortex, and ultrasonically mix for 18 min to 25 min at 10°C to 15°C to obtain a mixed stirring solution. The volume ratio of the sodium percarbonate solution to the non-polar organic solvent in step (1) is 0.8 to 1.2:100;

[0010] (4) Add a calcium hydroxide solution with a concentration of 1.4 mg / ml to 1.8 mg / ml to the mixed stirring solution, stir at 10°C to 15°C for 8 min to 12 min, and ultrasonically mix for 18 min to 25 min at 10°C to 15°C to obtain a calcium hydroxide solution mixture. The volume ratio of the calcium hydroxide solution to the non-polar organic solvent in step (1) is 9 to 11:100;

[0011] (5) Let the calcium hydroxide solution mixture stand at 2°C to 4°C for 1.8 h to 2.2 h, vortex, and mix under ultrasonic waves for 28 min to 33 min to obtain a low-temperature mixture;

[0012] (6) Put the low-temperature mixture into a round-bottom flask and perform rotary evaporation to remove the non-polar organic solvent to obtain a rotary evaporation product;

[0013] (7) Add ultrapure water to the rotary evaporation product and mix under ultrasonic waves to obtain a rotary evaporation product mixed solution. The volume ratio of the ultrapure water to the non-polar organic solvent in step (1) is 0.8 to 1.2:1;

[0014] (8) Pour the rotary evaporation product mixed solution into a centrifuge tube, add ultrapure water, and mix under ultrasonic waves for 18 min to 25 min. Then centrifuge at a temperature of 10°C to 15°C and a rotation speed of 4000 r / min to 6000 r / min for 8 min to 12 min, and take the supernatant to obtain the oxygen-producing nanoliposomes.

[0015] Further, the non-polar organic solvent in step (1) is one or more of chloroform, cyclohexane, ether, benzene, trichloromethane, and n-hexane.

[0016] Further, in step (1), add soybean lecithin to the non-polar organic solvent, vortex, and mix under ultrasonic waves for 5 min to obtain a mixed solution. The addition amount of soybean lecithin per milliliter of non-polar organic solvent is 14 mg.

[0017] Further, in step (2), add a pectin solution with a concentration of 25 mg / ml to the mixed solution, stir for 40 min, and mix under ultrasonic waves for 20 min to obtain a suspension. The volume ratio of the pectin solution to the non-polar organic solvent in step (1) is 1:100.

[0018] Further, in step (3), a sodium percarbonate solution at 15 mg / ml is added to the suspension, vortexed, and ultrasonically mixed at 12°C for 20 min to obtain a mixed stirring solution. The volume ratio of the sodium percarbonate solution to the non-polar organic solvent in step (1) is 1:100.

[0019] Further, in step (4), a calcium hydroxide solution at 1.6 mg / ml is added to the mixed stirring solution, stirred at 12°C for 10 min, and ultrasonically mixed at 12°C for 20 min to obtain a calcium hydroxide solution mixture. The volume ratio of the calcium hydroxide solution to the non-polar organic solvent in step (1) is 10:100.

[0020] Further, in step (5), the calcium hydroxide solution mixture is allowed to stand at 3°C for 2 h, vortexed, and ultrasonically mixed for 30 min to obtain a low-temperature mixture.

[0021] Further, in step (7), ultrapure water is added to the rotary evaporation product and ultrasonically mixed to obtain a rotary evaporation product mixed solution. The volume ratio of the ultrapure water to the non-polar organic solvent in step (1) is 1:1.

[0022] Further, in step (8), the rotary evaporation product mixed solution is poured into a centrifuge tube, ultrapure water is added, and ultrasonically mixed for 20 min, and then centrifuged at 12°C and a rotation speed of 5000 r / min for 10 min. The upper clear liquid is taken to obtain the oxygen-producing nanoliposomes.

[0023] The present invention also provides an application of the prepared oxygen-producing nanoliposomes on solid tumor cells.

[0024] A preparation method of oxygen-producing nanoliposomes in the present invention has the principle that: in a non-polar organic solvent, soybean lecithin and a pectin solution are added, and a reaction occurs between the sodium percarbonate solution and the calcium hydroxide solution at low temperature to generate peroxide, and a cross-linking reaction can also occur between the calcium hydroxide solution and the pectin solution to coat a layer of calcium pectate gel film outside the peroxide. It is made into oxygen-producing liposomes by vortex ultrasonic mixing in lecithin-non-polar organic solvent, reverse evaporation, and then adding water and vortex ultrasonic mixing.

[0025] The beneficial effects of a preparation method of oxygen-producing nanoliposomes in the present invention are as follows: 1) The preparation process is simple, with high repeatability, small preparation difficulty, and easy to promote and apply; 2) The oxygen-producing nanoliposomes prepared have a high oxygen production amount, can be stored for a long time under freezing conditions, and the effective oxygen release time at room temperature is up to 55 h to 60 h, with high performance stability; 3) The raw materials used have simple and non-toxic components and high safety performance. Specific embodiments

[0026] In the preparation method of the oxygen-producing nano-liposome of the present invention, the non-polar organic solvent can be one or more of chloroform, cyclohexane, ether, benzene, trichloromethane, n-hexane, etc.

[0027] The following examples only take several of the non-polar organic solvents as examples, which can help those skilled in the art to understand the present invention more comprehensively, but cannot limit the present invention in any way.

[0028] Example 1

[0029] A preparation method of an oxygen-producing nano-liposome includes the following steps:

[0030] (1) Add soy lecithin to cyclohexane, vortex, and mix under ultrasonic waves for 4 min to obtain a mixed solution, and the addition amount of soy lecithin in cyclohexane per milliliter is 10 mg;

[0031] (2) Add a pectin solution of 20 mg / ml to the mixed solution, stir for 35 min, and mix under ultrasonic waves for 18 min to obtain a suspension, and the volume ratio of the pectin solution to the cyclohexane in step (1) is 0.8:100;

[0032] (3) Add a sodium percarbonate solution of 10 mg / ml to the suspension, vortex, and mix under ultrasonic waves at 10°C for 18 min to obtain a mixed stirring solution, and the volume ratio of the sodium percarbonate solution to the cyclohexane in step (1) is 0.8:100;

[0033] (4) Add a calcium hydroxide solution of 1.4 mg / ml to the mixed stirring solution, stir at 10°C for 8 min, and mix under ultrasonic waves at 10°C for 18 min to obtain a calcium hydroxide solution mixed solution, and the volume ratio of the calcium hydroxide solution to the cyclohexane in step (1) is 9:100;

[0034] (5) Let the calcium hydroxide solution mixed solution stand at 2°C for 1.8 h, vortex, and mix under ultrasonic waves for 28 min to obtain a low-temperature mixture;

[0035] (6) Put the low-temperature mixture into a round-bottom flask and rotate and evaporate to remove cyclohexane to obtain a rotary evaporation product;

[0036] (7) Add ultrapure water to the rotary evaporation product and mix under ultrasonic waves to obtain a rotary evaporation product mixed solution, and the volume ratio of the ultrapure water to the cyclohexane in step (1) is 0.8:1;

[0037] (8) Pour the rotary evaporation product mixture into a centrifuge tube, add ultrapure water, mix under ultrasonic waves for 18 min, then centrifuge at a temperature of 10 °C and a rotation speed of 4000 r / min for 8 min, and take the supernatant to obtain the oxygen-producing nanoliposomes.

[0038] Example 2

[0039] A method for preparing oxygen-producing nanoliposomes, comprising the following steps:

[0040] (1) Add soy lecithin to chloroform, vortex, and mix under ultrasonic waves for 6 min to obtain a mixture, and the addition amount of soy lecithin per milliliter of chloroform is 15 mg;

[0041] (2) Add a 30 mg / ml pectin solution to the mixture, stir for 45 min, and mix under ultrasonic waves for 25 min to obtain a suspension, and the volume ratio of the pectin solution to the chloroform in step (1) is 1.2:100;

[0042] (3) Add a 20 mg / ml sodium percarbonate solution to the suspension, vortex, and mix under ultrasonic waves at 15 °C for 25 min to obtain a mixed stirring solution, and the volume ratio of the sodium percarbonate solution to the chloroform in step (1) is 1.2:100;

[0043] (4) Add a 1.8 mg / ml calcium hydroxide solution to the mixed stirring solution, stir at 15 °C for 12 min, and mix under ultrasonic waves at 15 °C for 25 min to obtain a calcium hydroxide solution mixture, and the volume ratio of the calcium hydroxide solution to the chloroform in step (1) is 11:100;

[0044] (5) Let the calcium hydroxide solution mixture stand at 4 °C for 2.2 h, vortex, and mix under ultrasonic waves for 33 min to obtain a low-temperature mixture;

[0045] (6) Put the low-temperature mixture into a round-bottom flask for rotary evaporation to remove chloroform and obtain a rotary evaporation product;

[0046] (7) Add ultrapure water to the rotary evaporation product and mix under ultrasonic waves to obtain a rotary evaporation product mixture, and the volume ratio of the ultrapure water to the chloroform in step (1) is 1.2:1;

[0047] (8) Pour the rotary evaporation product mixture into a centrifuge tube, add ultrapure water, and mix under ultrasonic waves for 25 min, then centrifuge at a temperature of 15 °C and a rotation speed of 6000 r / min for 12 min, and take the supernatant to obtain the oxygen-producing nanoliposomes.

[0048] Example 3

[0049] A method for preparing oxygen-producing nanoliposomes, comprising the following steps:

[0050] (1) Add soy lecithin to a mixed solvent of chloroform and cyclohexane, vortex, and mix under ultrasonic waves for 5 min to obtain a mixed solution. The addition amount of soy lecithin in each milliliter of the mixed solvent of chloroform and cyclohexane is 25 mg, and the volume ratio of chloroform to cyclohexane in the mixed solvent of chloroform and cyclohexane is chloroform: cyclohexane = 1:1;

[0051] (2) Add a 25 mg / ml pectin solution to the mixed solution, stir for 40 min, and mix under ultrasonic waves for 20 min to obtain a suspension. The volume ratio of the pectin solution to the mixed solvent of chloroform and cyclohexane in step (1) is 1:100;

[0052] (3) Add a 15 mg / ml sodium percarbonate solution to the suspension, vortex, and mix under ultrasonic waves at 12 °C for 20 min to obtain a mixed stirring solution. The volume ratio of the sodium percarbonate solution to the mixed solvent of chloroform and cyclohexane in step (1) is 1:100;

[0053] (4) Add a 1.6 mg / ml calcium hydroxide solution to the mixed stirring solution, stir at 12 °C for 10 min, and mix under ultrasonic waves at 12 °C for 20 min to obtain a calcium hydroxide solution mixed solution. The volume ratio of the calcium hydroxide solution to the mixed solvent of chloroform and cyclohexane in step (1) is 10:100;

[0054] (5) Let the calcium hydroxide solution mixed solution stand at 3 °C for 2 h, vortex, and mix under ultrasonic waves for 30 min to obtain a low-temperature mixture;

[0055] (6) Put the low-temperature mixture into a round-bottom flask and perform rotary evaporation to remove the mixed solvent of chloroform and cyclohexane to obtain a rotary evaporation product;

[0056] (7) Add ultrapure water to the rotary evaporation product and mix under ultrasonic waves to obtain a rotary evaporation product mixed solution. The volume ratio of the ultrapure water to the mixed solvent of chloroform and cyclohexane in step (1) is 1:1;

[0057] (8) Pour the rotary evaporation product mixed solution into a centrifuge tube, add ultrapure water, and mix under ultrasonic waves for 20 min. Then centrifuge at a temperature of 12 °C and a rotation speed of 5000 r / min for 10 min, and take the upper clear liquid to obtain the oxygen-producing nanoliposomes.

[0058] Example 4

[0059] A method for preparing oxygen-producing nanoliposomes, comprising the following steps:

[0060] (1) Add soy lecithin to diethyl ether, vortex, and mix under ultrasonic waves for 5.5 min to obtain a mixed solution. The addition amount of soy lecithin in each milliliter of diethyl ether is 40 mg;

[0061] (2) Add a 25 mg / ml pectin solution to the mixed solution, stir for 38 min, and mix under ultrasonic waves for 19 min to obtain a suspension. The volume ratio of the pectin solution to the diethyl ether in step (1) is 1.05:100;

[0062] (3) Add a 15 mg / ml sodium percarbonate solution to the suspension, vortex, and mix under ultrasonic waves at 13 °C for 18 min to obtain a mixed stirring solution. The volume ratio of the sodium percarbonate solution to the diethyl ether in step (1) is 1.06:100;

[0063] (4) Add a 1.6 mg / ml calcium hydroxide solution to the mixed stirring solution, stir at 12 °C for 9.5 min, and mix under ultrasonic waves at 12 °C for 19.5 min to obtain a calcium hydroxide solution mixed solution. The volume ratio of the calcium hydroxide solution to the diethyl ether in step (1) is 10.2:100;

[0064] (5) Let the calcium hydroxide solution mixed solution stand at 3 °C for 2.2 h, vortex, and mix under ultrasonic waves for 30 min to obtain a low-temperature mixture;

[0065] (6) Put the low-temperature mixture into a round-bottom flask and perform rotary evaporation to remove diethyl ether to obtain a rotary evaporation product;

[0066] (7) Add ultrapure water to the rotary evaporation product and mix under ultrasonic waves to obtain a rotary evaporation product mixed solution. The volume ratio of the ultrapure water to the diethyl ether in step (1) is 1.03:1;

[0067] (8) Pour the rotary evaporation product mixed solution into a centrifuge tube, add ultrapure water, and mix under ultrasonic waves for 20 min. Then centrifuge at a temperature of 12.5 °C and a rotation speed of 5400 r / min for 9 min, and take the supernatant to obtain the oxygen-producing nanoliposomes.

[0068] Example 5

[0069] A method for preparing oxygen-producing nanoliposomes, comprising the following steps:

[0070] (1) Add soy lecithin to chloroform, vortex, and mix under ultrasonic waves for 4.6 min to obtain a mixed solution. The addition amount of soy lecithin in each milliliter of the chloroform mixed solvent is 30 mg;

[0071] (2) Add a pectin solution with a concentration of 25.5 mg / ml to the mixture, stir for 42 min, and mix under ultrasonic waves for 21 min to obtain a suspension. The volume ratio of the pectin solution to the chloroform in step (1) is 1.04:100;

[0072] (3) Add a sodium percarbonate solution with a concentration of 15 mg / ml to the suspension, vortex, and mix under ultrasonic waves at 12 °C for 20 min to obtain a mixed stirring solution. The volume ratio of the sodium percarbonate solution to the chloroform in step (1) is 1.09:100;

[0073] (4) Add a calcium hydroxide solution with a concentration of 1.6 mg / ml to the mixed stirring solution, stir at 12 °C for 10 min, and mix under ultrasonic waves at 12 °C for 20 min to obtain a calcium hydroxide solution mixture. The volume ratio of the calcium hydroxide solution to the chloroform in step (1) is 10.5:100;

[0074] (5) Let the calcium hydroxide solution mixture stand at 3 °C for 2.6 h, vortex, and mix under ultrasonic waves for 29 min to obtain a low-temperature mixture;

[0075] (6) Place the low-temperature mixture in a round-bottom flask and perform rotary evaporation to remove chloroform to obtain a rotary evaporation product;

[0076] (7) Add ultrapure water to the rotary evaporation product and mix under ultrasonic waves to obtain a rotary evaporation product mixture. The volume ratio of the ultrapure water to the chloroform in step (1) is 1.07:1;

[0077] (8) Pour the rotary evaporation product mixture into a centrifuge tube, add ultrapure water, and mix under ultrasonic waves for 22 min. Then centrifuge at a temperature of 12 °C and a rotation speed of 5600 r / min for 8 min, and take the upper clear liquid to obtain the oxygen-producing nanoliposomes.

[0078] The particle size and potential test results of the oxygen-producing nanoliposomes prepared in Examples 1 - 5 are shown in Table 1 below:

[0079]

[0080] The test results of the dissolved oxygen amount of the oxygen-producing nanoliposomes prepared in Examples 1 - 5 changing with time at room temperature are shown in Table 2 below:

[0081] Table 2

[0082]

[0083] As can be seen from the above performance test results, for the preparation method of an oxygen-producing nanoliposome of the present invention, the prepared oxygen-producing nanoliposome has a high oxygen production amount, and the effective oxygen release time at room temperature is as high as 55h to 60h. It can be stored for a long time under freezing conditions, and has high performance stability.

[0084] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for preparing oxygen-producing nanoliposomes, characterized in that: The following steps are involved: (1) adding soybean lecithin to a non-polar organic solvent, vortexing, and mixing under ultrasonication for 4 to 6 minutes to obtain a mixed solution, wherein the amount of soybean lecithin added to each milliliter of the non-polar organic solvent is 10 mg to 40 mg; the non-polar organic solvent is one or more of chloroform, cyclohexane, ether, benzene, chloroform, and n-hexane; (2) adding 20 mg / ml to 30 mg / ml pectin solution to the mixed solution, stirring for 35 min to 45 min, and mixing under ultrasonication for 18 min to 25 min to obtain a suspension, wherein the volume ratio of the pectin solution to the non-polar organic solvent in step (1) is 0.8 to 1.2:100; (3) adding 10 mg / ml to 20 mg / ml of sodium percarbonate solution to the suspension, vortexing, and ultrasonically mixing at 10°C to 15°C for 18 min to 25 min to obtain a mixed stirring liquid, wherein the volume ratio of the sodium percarbonate solution to the non-polar organic solvent in step (1) is 0.8 to 1.2:100; (4) adding 1.4 mg / ml to 1.8 mg / ml of calcium hydroxide solution to the mixed stirred liquid, stirring at 10° C. to 15° C. for 8 min to 12 min, and ultrasonically mixing at 10° C. to 15° C. for 18 min to 25 min to obtain a calcium hydroxide solution mixture, wherein the volume ratio of the calcium hydroxide solution to the non-polar organic solvent in step (1) is 9 to 11:100; (5) the calcium hydroxide solution mixture was allowed to stand at 2°C to 4°C for 1.8 hours to 2.2 hours, vortexed, and mixed under ultrasonic waves for 28 minutes to 33 minutes to obtain a low-temperature mixture; (6) placing the low-temperature mixture into a round-bottom flask and performing rotary evaporation to remove the non-polar organic solvent to obtain a rotary evaporation product; (7) adding ultrapure water to the rotary evaporation product and mixing under ultrasonication to obtain a rotary evaporation product mixture, wherein the volume ratio of the ultrapure water to the non-polar organic solvent in step (1) is 1:1; (8) Pour the rotary evaporation product mixture into a centrifuge tube, add ultrapure water, and mix under ultrasonication for 18 minutes to 25 minutes. Then, centrifuge at a temperature of 10°C to 15°C and a speed of 4000 rpm to 6000 rpm for 8 minutes to 12 minutes, and collect the supernatant to obtain the oxygen-producing nanoliposomes.

2. The method for preparing oxygen-producing nanoliposomes according to claim 1, wherein: In the step (1), soybean lecithin is added to the non-polar organic solvent, vortexed, and mixed under ultrasound for 5 minutes to obtain a mixed solution. The amount of soybean lecithin added to each milliliter of the non-polar organic solvent is 14 mg.

3. The method for preparing oxygen-producing nanoliposomes according to claim 1, wherein: In the step (2), 25 mg / ml pectin solution is added to the mixed liquid, stirred for 40 minutes, and mixed under ultrasound for 20 minutes to obtain a suspension. The volume ratio of the pectin solution to the non-polar organic solvent in step (1) is 1:

100.

4. The method for preparing oxygen-producing nanoliposomes according to claim 1, wherein: In the step (3), 15 mg / ml sodium percarbonate solution is added to the suspension, vortexed, and ultrasonically mixed at 12° C. for 20 minutes to obtain a mixed stirring liquid, wherein the volume ratio of the sodium percarbonate solution to the non-polar organic solvent in the step (1) is 1:

100.

5. The method for preparing oxygen-producing nanoliposomes according to claim 1, wherein: In the step (4), a 1.6 mg / ml calcium hydroxide solution is added to the mixed stirring liquid, stirred at 12° C. for 10 min, and ultrasonically mixed at 12° C. for 20 min to obtain a calcium hydroxide solution mixture, wherein the volume ratio of the calcium hydroxide solution to the non-polar organic solvent in step (1) is 10:

100.

6. The method for preparing oxygen-producing nanoliposomes according to claim 1, wherein: In the step (5), the calcium hydroxide solution mixture is allowed to stand at 3° C. for 2 h, vortexed, and mixed under ultrasonic waves for 30 min to obtain a low-temperature mixture.

7. The method for preparing oxygen-producing nanoliposomes according to claim 1, wherein: In step (8), the rotary evaporation product mixture is poured into a centrifuge tube, ultrapure water is added, and mixed under ultrasonic conditions for 20 minutes, and then centrifuged at a temperature of 12° C. and a speed of 5000 r / min for 10 minutes, and the supernatant is collected to obtain the oxygen-producing nanoliposomes.

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