Drug-loadable liposome and preparation method thereof

Abstract

The invention relates to the technical field of pharmaceutical preparations. The invention provides a drug-loadable liposome and a preparation method thereof, and the preparation method comprises the following steps: (1) mixing lecithin, dipalmitoyl phosphatidylcholine, cholesterol and an organic solvent, and carrying out ultrasonic dissolution to obtain a phase A; (2) mixing polyoxyethylene sorbitan monostearate with a phosphate buffer solution to obtain a phase B; and (3) adding the phase A prepared in the step (1) into the phase B prepared in the step (2), stirring, evaporating, and filtering to obtain the product. The liposome prepared from the raw materials and the technical scheme is more stable in performance, better in drug loading stability and higher in drug entrapment capacity, and the drug release time can be prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a drug-carrying liposome and a preparation method thereof. Background Art

[0002] As a new type of drug carrier, drug-loaded liposomes have been widely used in the pharmaceutical industry in recent years. Its advantage is that it can control the release rate of drugs, reduce the side effects of drugs, and improve the therapeutic effect of drugs. Liposome technology has been applied to many fields, such as anti-tumor drugs, cardiovascular drugs, antibiotics, etc., especially in the study of anti-tumor drugs, liposome anti-tumor drugs have become a hot topic of research. Although drug-loaded liposomes have many advantages, there are still some problems in their practical application. For example, the stability of liposomes, biodegradability, and metabolic processes in the body all need further study. In addition, the production cost of liposomes is high, which also limits their wide application. Summary of the invention

[0003] The object of the present invention is to provide a drug-loaded liposome and a preparation method thereof. The liposome obtained by the raw materials and preparation method of the present invention has more stable performance, better drug loading stability, higher drug loading capacity, and can also prolong drug release time.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing drug-carrying liposomes, comprising the following steps:

[0006] (1) mixing lecithin, dipalmitoylphosphatidylcholine, and cholesterol with an organic solvent, and dissolving them by ultrasonication to obtain phase A;

[0007] (2) mixing polyoxyethylene sorbitan monostearate with phosphate buffer to obtain phase B;

[0008] (3) Add phase A obtained in step (1) to phase B obtained in step (2), stir, evaporate, and filter to obtain the product.

[0009] Preferably, the mass ratio of lecithin, dipalmitoylphosphatidylcholine and cholesterol is (35-50):(5-8):(10-15).

[0010] Preferably, the volume ratio of the total mass of the lecithin, dipalmitoylphosphatidylcholine and cholesterol to the organic solvent is (62-72) mg:(18-25) mL.

[0011] Preferably, the organic solvent in step (1) includes diethyl ether, ethanol, tert-butanol or chloroform.

[0012] Preferably, the frequency of the ultrasound in step (1) is 20-40 KHz, the time of the ultrasound is 5-10 min; and the temperature of the dissolution is 40-60°C.

[0013] Preferably, the mass volume ratio of the polyoxyethylene sorbitan monostearate to the phosphate buffer in step (2) is (5-8) mg:(15-25) mL; and the concentration of the phosphate buffer is 0.01-0.04 mol / L.

[0014] Preferably, the stirring speed in step (3) is 700-850 r / min.

[0015] Preferably, the filtering specification in step (3) is 500-800nm.

[0016] The invention also provides drug-carrying liposomes prepared by the preparation method.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects:

[0018] In the technical solution of the present invention, lecithin, dipalmitoylphosphatidylcholine, cholesterol and ethanol are selected and used in combination under scientific proportions, which not only ensures biocompatibility, but also increases the stability of liposome performance, enhances the flexibility of liposome, and reduces the easy oxidation characteristics; at the same time, it better improves the drug loading stability, reduces drug leakage, and increases the drug loading capacity. In addition, the drug-loaded liposomes of the present invention can also prolong the release time of the drug. DETAILED DESCRIPTION

[0019] The present invention provides a method for preparing drug-carrying liposomes, comprising the following steps:

[0020] (1) mixing lecithin, dipalmitoylphosphatidylcholine, and cholesterol with an organic solvent, and dissolving them by ultrasonication to obtain phase A;

[0021] (2) mixing polyoxyethylene sorbitan monostearate with phosphate buffer to obtain phase B;

[0022] (3) Add phase A obtained in step (1) to phase B obtained in step (2), stir, evaporate, and filter to obtain the product.

[0023] In the present invention, lecithin, dipalmitoylphosphatidylcholine, cholesterol and an organic solvent are uniformly mixed. The organic solvent described in the present invention preferably includes ether, ethanol, tert-butyl alcohol or chloroform, and is further preferably ether, ethanol or chloroform, and is further preferably chloroform or ethanol. The mass ratio of lecithin, dipalmitoylphosphatidylcholine and cholesterol described in the present invention is preferably (35-50):(5-8):(10-15), and is further preferably (38-48):(5.5-7.5):(11-14), and is further preferably 47:7:13. The volume ratio of the total mass of lecithin, dipalmitoylphosphatidylcholine and cholesterol described in the present invention to the organic solvent is preferably (62-72) mg:(18-25) mL, and is further preferably (64-70) mg:(18.5-23) mL, and is further preferably 67 mg:21 mL.

[0024] In the present invention, the mixed solution is ultrasonically dissolved to obtain phase A. The frequency of the ultrasound in the present invention is preferably 20-40KHz, more preferably 22-38KHz, and more preferably 32KHz; the time of the ultrasound is preferably 5-10min, more preferably 6-9min, and more preferably 8min. The temperature of the dissolution in the present invention is preferably 40-60°C, more preferably 43-56°C, and more preferably 48°C.

[0025] In the present invention, polyoxyethylene sorbitan monostearate is mixed with phosphate buffer to obtain phase B. The concentration of the phosphate buffer in the present invention is preferably 0.01-0.04 mol / L, more preferably 0.015-0.03 mol / L, and more preferably 0.025 mol / L. The mass volume ratio of the polyoxyethylene sorbitan monostearate to the phosphate buffer in the present invention is preferably (5-8) mg:(15-25) mL, more preferably (5.5-7.5) mg:(16-24) mL, and more preferably 6.5 mg:22 mL.

[0026] In the present invention, the prepared phase A is added to the phase B and stirred evenly. The stirring speed is preferably 700-850r / min, further preferably 720-820r / min, and further preferably 760r / min. After stirring evenly in the present invention, evaporation is performed to obtain a liposome suspension, and then filtration is performed. The evaporation is preferably performed by water bath rotary evaporation. The temperature of the water bath is preferably 22-28°C, further preferably 23-27°C, and further preferably 24-26°C; the speed of the rotary evaporation is preferably 80-110rpm, further preferably 85-105rpm, and further preferably 95rpm. The use of water bath rotary evaporation under the conditions of the present invention can better ensure the uniformity of the film formed after evaporation and remove the organic solvent. The filtration of the present invention is preferably performed using a polycarbonate membrane, and the specification of the filtration is preferably 500-800nm, further preferably 600-760nm, and further preferably 700nm.

[0027] The invention also provides drug-carrying liposomes prepared by the preparation method.

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1

[0030] A method for preparing drug-carrying liposomes, comprising the following steps:

[0031] (1) mixing lecithin, dipalmitoylphosphatidylcholine and cholesterol in a volume ratio of 67 mg:21 mL of ethanol, wherein the mass ratio of lecithin, dipalmitoylphosphatidylcholine and cholesterol is 46:6.5:13, and after mixing evenly, dissolving by ultrasound at 48° C. and 32 kHz for 8 min to obtain phase A;

[0032] (2) mixing polyoxyethylene sorbitan monostearate with 0.025 mol / L phosphate buffer at a ratio of 6.5 mg:22 mL to obtain phase B;

[0033] (3) Add phase A to phase B, stir evenly at 760 r / min, and then perform rotary evaporation in a 24° C. water bath at 95 rpm until all ethanol is removed, and then squeeze through a 700 nm polycarbonate membrane for filtration to obtain the product.

[0034] Example 2

[0035] A method for preparing drug-carrying liposomes, comprising the following steps:

[0036] (1) mixing lecithin, dipalmitoylphosphatidylcholine and cholesterol in a volume ratio of 62 mg:18 mL of ethanol, wherein the mass ratio of lecithin, dipalmitoylphosphatidylcholine and cholesterol is 40:5:10, and after mixing evenly, dissolving by ultrasound at 40° C. and 20 kHz for 10 min to obtain phase A;

[0037] (2) mixing polyoxyethylene sorbitan monostearate with 0.02 mol / L phosphate buffer at a ratio of 8 mg:25 mL to obtain phase B;

[0038] (3) Add phase A to phase B, stir at 700 r / min, and then perform rotary evaporation in a 25°C water bath at 80 rpm until all ethanol is removed. Then, squeeze through an 800 nm polycarbonate membrane for filtration to obtain the product.

[0039] Example 3

[0040] A method for preparing drug-carrying liposomes, comprising the following steps:

[0041] (1) mixing lecithin, dipalmitoylphosphatidylcholine and cholesterol in a volume ratio of 72 mg:25 mL of ethanol, wherein the mass ratio of lecithin, dipalmitoylphosphatidylcholine and cholesterol is 48:8:14, mixing evenly, and dissolving by ultrasound at 40 kHz for 5 min at 56° C. to obtain phase A;

[0042] (2) mixing polyoxyethylene sorbitan monostearate with 0.03 mol / L phosphate buffer at a ratio of 5 mg:15 mL to obtain phase B;

[0043] (3) Add phase A to phase B, stir evenly at 850 r / min, and then perform rotary evaporation in a 26° C. water bath at 105 rpm until all ethanol is removed, and then squeeze through a 600 nm polycarbonate membrane for filtration to obtain the product.

[0044] Example 4

[0045] A method for preparing drug-carrying liposomes, comprising the following steps:

[0046] (1) mixing lecithin, dipalmitoylphosphatidylcholine and cholesterol in a volume ratio of 67 mg:21 mL of chloroform, wherein the mass ratio of lecithin, dipalmitoylphosphatidylcholine and cholesterol is 46:6.5:13, and after mixing evenly, dissolving by ultrasound at 48° C. and 32 kHz for 8 min to obtain phase A;

[0047] (2) mixing polyoxyethylene sorbitan monostearate with 0.025 mol / L phosphate buffer at a ratio of 6.5 mg:22 mL to obtain phase B;

[0048] (3) Add phase A to phase B, stir at 760 r / min, and then perform rotary evaporation in a 24°C water bath at 95 rpm until the chloroform is completely removed. Then, squeeze through a 700 nm polycarbonate membrane and filter to obtain the product.

[0049] Test Example 1

[0050] (I) Liposome preparation

[0051] 1. Drug-loaded liposomes (blank liposomes)

[0052] 47 mg of lecithin, 7 mg of dipalmitoylphosphatidylcholine and 13 mg of cholesterol were added to 21 mL of ethanol solution, mixed evenly, and dissolved by ultrasound at 32 KHz for 8 min at 48 °C to obtain phase A. 6.5 mg of polyoxyethylene sorbitan monostearate was mixed evenly with 22 mL of 0.025 mol / L phosphate buffer to obtain phase B. Phase A was added to phase B, stirred evenly at 760 r / min, and then rotary evaporated in a 24 °C water bath, and rotary evaporated at 95 rpm until ethanol was completely removed, and then squeezed through a 700 nm polycarbonate membrane for filtration to obtain the product.

[0053] 2. Doxorubicin hydrochloride loaded liposomes

[0054] The same as the preparation of blank liposomes, phase A and phase B were obtained. Then phase A and 3 mg of doxorubicin hydrochloride were added to phase B, stirred evenly at 760 r / min, and then rotary evaporated in a 24°C water bath, and rotary evaporated at 95 rpm until ethanol was completely removed, and then squeezed through a 700 nm polycarbonate membrane for filtration to obtain.

[0055] (II) Encapsulation rate

[0056] The doxorubicin hydrochloride-loaded liposomes prepared as above were used as the test group, and a control group was set up based on the process of preparing the doxorubicin hydrochloride-loaded liposomes in the test group to study the effects of different condition settings on the liposome encapsulation efficiency.

[0057] Control group 1: Same as the experimental group, except that “47 mg lecithin, 7 mg dipalmitoylphosphatidylcholine and 13 mg cholesterol were added to 21 mL ethanol solution” was replaced by “54 mg lecithin and 13 mg cholesterol were added to 21 mL ethanol solution”.

[0058] Control group 2: Same as the experimental group, except that “47 mg of lecithin, 7 mg of dipalmitoylphosphatidylcholine and 13 mg of cholesterol were added to 21 mL of ethanol solution” was replaced by “54 mg of dipalmitoylphosphatidylcholine and 13 mg of cholesterol were added to 21 mL of ethanol solution”.

[0059] Control group 3: Same as the experimental group, except that “47 mg lecithin, 7 mg dipalmitoylphosphatidylcholine and 13 mg cholesterol were added to 21 mL ethanol solution” was replaced by “54 mg dipalmitoylphosphatidylcholine and 3 mg cholesterol were added to 21 mL ethanol solution”.

[0060] Control group 4: Same as the experimental group, except that “ultrasonic dissolution at 32KHz at 48℃ for 8min” was replaced by “ultrasonic dissolution at 12KHz at 48℃ for 2min”, and “after stirring evenly at 760r / min, rotary evaporation was performed in a 24℃ water bath, and rotary evaporation was performed at 95rpm until the ethanol was completely removed” was replaced by “after stirring evenly at 1000r / min, rotary evaporation was performed in a 24℃ water bath, and rotary evaporation was performed at 50rpm until the ethanol was completely removed”.

[0061] Take 1 mL of the doxorubicin hydrochloride liposome suspension prepared in the test group and the control group 1-4, respectively, and apply it to the Sephadex G-50 column, and elute with PBS at pH 7.4 (flow rate 1 mL / min). Collect the liposome fraction, add 2 mL of 10% (volume ratio) Triton-X-100, heat and dissolve at 60°C, cool to room temperature and then make up to volume. Use PBS as blank to measure the absorbance value A at 485 nm. 485 Under the same conditions, the absorbance of the liposome samples of equal volume but not loaded on the column was measured, and the encapsulation efficiency of the doxorubicin hydrochloride-loaded liposomes in different groups was calculated (see Table 1).

[0062] In addition, the doxorubicin hydrochloride-loaded liposomes prepared in the test group and the control group 1-4 were placed at 25°C for 0.5h, 1h, 1.5h and 2h, respectively, and then the encapsulation efficiency of the doxorubicin hydrochloride-loaded liposomes at different placement times was determined (see Table 2).

[0063] Encapsulation efficiency (%) = absorbance of liposomes loaded on the column in each treatment group / absorbance of liposomes not loaded on the column × 100%

[0064] Table 1 Encapsulation efficiency of doxorubicin hydrochloride-loaded liposomes in different treatment groups

[0065]

[0066]

[0067] Table 2 Encapsulation efficiency of doxorubicin hydrochloride-loaded liposomes in different storage time and treatment groups

[0068]

[0069] According to Tables 1 and 2, it can be seen that the encapsulation rate of the doxorubicin hydrochloride-loaded liposomes prepared in the test example group is relatively the highest; and after being placed for the same time, the encapsulation rate of the doxorubicin hydrochloride-loaded liposomes in the test example group is still higher than that in the control group, which also shows that the drug loading has good stability and can prolong the release time of the drug.

[0070] In summary, it can be seen that the liposomes obtained by the raw materials and preparation method of the present invention have more stable performance, better drug loading stability, higher drug loading capacity, and can also prolong drug release time.

[0071] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing drug-carrying liposomes, characterized in that: The steps include: (1) mixing lecithin, dipalmitoylphosphatidylcholine, and cholesterol with an organic solvent, and dissolving them by ultrasonication to obtain phase A; (2) mixing polyoxyethylene sorbitan monostearate with phosphate buffer to obtain phase B; (3) Add phase A obtained in step (1) to phase B obtained in step (2), stir, evaporate, and filter to obtain the product.

2. The preparation method according to claim 1, characterized in that: The mass ratio of lecithin, dipalmitoylphosphatidylcholine and cholesterol is (35-50):(5-8):(10-15); The volume ratio of the total mass of the lecithin, dipalmitoylphosphatidylcholine and cholesterol to the organic solvent is (62-72) mg:(18-25) mL.

3. The preparation method according to claim 1, characterized in that: The organic solvent in step (1) includes ether, ethanol, tert-butanol or chloroform.

4. The preparation method according to claim 1, characterized in that: The frequency of the ultrasound in step (1) is 20-40 KHz, and the duration of the ultrasound is 5-10 min; The dissolving temperature is 40-60°C.

5. The preparation method according to claim 1, characterized in that: The mass volume ratio of the polyoxyethylene sorbitan monostearate to the phosphate buffer in step (2) is (5-8) mg: (15-25) mL; The concentration of the phosphate buffer is 0.01-0.04 mol / L.

6. The preparation method according to claim 1, characterized in that: The stirring speed in step (3) is 700-850r / min.

7. The preparation method according to claim 1, characterized in that: The filtering specification in step (3) is 500-800nm.

8. The drug-loaded liposome prepared by the preparation method according to any one of claims 1 to 7.

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