Drug-loaded liposome constructed based on saikosaponin D as well as preparation method and application of drug-loaded liposome

Abstract

The invention discloses a drug-loaded liposome constructed on the basis of saikoside D, and a preparation method and application of the drug-loaded liposome. The liposome comprises soybean lecithin, saikoside D, phosphatidic acid, an active drug and poloxamer 407. According to the invention, the saikosaponin D with antitumor activity is used for completely replacing cholesterol in the traditional liposome, so that the potential health risk of the cholesterol is avoided, the integration of medicines and auxiliary medicines is realized, and the saikosaponin D and entrapped medicines can generate a synergistic antitumor effect. The modification of the poloxamer 407 endows the liposome with the long circulation characteristic. The preparation method adopts a film dispersion-ultrasonic method, and is simple and convenient to operate. The prepared lipidosome is small in particle size, narrow in distribution and good in stability, the drug loading capacity is about 1.5-7.0%, the tumor cell inhibition effect superior to that of traditional cholesterol lipidosome and free drugs is shown in vitro, and the lipidosome has wide application prospects in the aspect of preparation of anti-tumor drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical preparations, in particular to a drug-loaded liposome based on saikosaponin D and a preparation method and application thereof. BACKGROUND

[0002] Liposomes are microcapsules composed of phospholipid bilayers, which are widely used as drug delivery carriers due to their good biocompatibility and encapsulation capacity. Cholesterol is an indispensable component in traditional liposomes, which is used to stabilize the phospholipid bilayer and improve the rigidity and encapsulation efficiency of liposomes. However, when liposomes are injected intravenously, the cholesterol in them may be absorbed by the body, which may pose potential health risks to patients with high cholesterol levels or dyslipidemia. Therefore, finding a safe and effective substitute for cholesterol is an important research direction in the field of liposome technology.

[0003] Saikosaponin D is an active ingredient extracted from the traditional Chinese medicine Bupleurum. Modern research shows that it has significant anti-tumor activity. Studies have found that saikosaponin D has a similar steroidal structure to cholesterol, which can theoretically embed in the phospholipid bilayer and play a role in stabilizing the membrane structure. This provides the possibility of using it to replace cholesterol to construct a functional liposome that combines medicine and adjuvant.

[0004] In addition, conventional liposomes are easily cleared by the reticuloendothelial system in the body, have a short circulation time, and affect their target site enrichment effect. Poloxamer 407 is an amphiphilic block copolymer that can anchor its hydrophobic segment on the liposome membrane, and the hydrophilic segment forms a hydration layer on the surface, effectively prolonging the circulation time of the liposome in the body.

[0005] Based on the above background, the present application aims to develop a new drug-loaded liposome that replaces cholesterol with saikosaponin D and is modified with poloxamer 407, in order to improve safety while achieving efficient drug delivery and synergistic anti-tumor effect. SUMMARY

[0006] One of the purposes of the present application is to provide a drug-loaded liposome with higher safety, good stability and synergistic anti-tumor effect.

[0007] The second purpose of the present application is to provide a preparation method of the above-mentioned drug-loaded liposome.

[0008] The third purpose of the present application is to provide the use of the above-mentioned drug-loaded liposome in the preparation of anti-tumor drugs.

[0009] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: In a first aspect, the application provides a drug-loaded liposome based on saikosaponin D, which comprises soybean lecithin, saikosaponin D, phosphatidic acid, an active drug and poloxamer 407.

[0010] Preferably, the mass ratio between the components is: the mass ratio of soybean lecithin to saikosaponin D is 20:2~3.33, the mass ratio of soybean lecithin to phosphatidic acid is 20:1.67~2.5, and the mass ratio of soybean lecithin to poloxamer 407 is 20:3.33~10.

[0011] Preferably, the active drug is any one of anti-tumor drugs, such as curcumin, paclitaxel, vincristine, maytansine.

[0012] Preferably, the average particle size of the liposome is less than 200 nm.

[0013] Preferably, the drug loading capacity of the drug-loaded liposome is 1.5-7.0%, and further preferably 3.46-4.11%.

[0014] In a second aspect, the application provides a preparation method of the above drug-loaded liposome, comprising the following steps: (1) dissolving soybean lecithin, saikosaponin D, phosphatidic acid and a therapeutic drug with an organic solvent to form an organic phase, wherein the organic solvent is anhydrous ethanol or methanol; (2) removing the organic solvent from the organic phase obtained in step (1) under reduced pressure at 40~55℃ to form a uniform film on the inner wall of the container; (3) dissolving poloxamer 407 in ultrapure water, and then adding it to the container in which the film is formed in step (2), and performing hydration film washing in a 40~55℃ water bath for 28~32 min to form a liposome suspension; (4) performing ice bath ultrasonic treatment on the liposome suspension obtained in step (3) for 5~20 min; (5) filtering the liposome suspension treated in step (4) through 0.45μm and 0.22μm microporous filter membranes in sequence to obtain the drug-loaded liposome based on saikosaponin D.

[0015] The preparation process flow chart is shown in Figure 1 .

[0016] In a third aspect, the application provides the use of the above drug-loaded liposome in the preparation of a drug for inhibiting the proliferation of tumor cells.

[0017] The application has the following advantages: High safety: the biologically active saikosaponin D completely replaces cholesterol, avoiding potential health risks caused by cholesterol intake, and improving the safety of intravenous drugs.

[0018] Drug combination and synergistic effect: saikosaponin D itself has anti-tumor activity, which can produce synergistic effect with the encapsulated anti-tumor drug (such as curcumin) as a membrane stabilizer, and can inhibit tumor cell proliferation together, which is better than traditional cholesterol liposomes and free drugs.

[0019] Long circulation and high stability: poloxamer 407 forms a hydrophilic protective layer on the surface of the liposome, effectively prolongs the circulation time of the liposome in the body, and improves the physical stability of the liposome through steric hindrance effect. The introduction of phosphatidic acid gives the liposome negative charge, which further enhances the stability of the system.

[0020] Excellent formulation performance: through optimization of prescription and process, the prepared liposome has small particle size (<200 nm), uniform distribution and ideal drug loading capacity, which is conducive to enrichment in tumor tissue through enhanced permeability and retention effect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Preparation process flow chart of drug-loaded liposome based on saikosaponin D; Figure 2 Transmission electron microscopy of each drug-loaded liposome based on saikosaponin D; Figure 3 Particle size distribution of each drug-loaded liposome based on saikosaponin D; Figure 4 Potential distribution of each drug-loaded liposome based on saikosaponin D; Figure 5 In vitro release properties of each drug-loaded liposome based on saikosaponin D; Figure 6 Inhibition rate of curcumin liposome based on saikosaponin D on HepG2 cell proliferation. DETAILED DESCRIPTION

[0022] The present application will be further described in detail below in conjunction with the drawings and examples, but in no way limits the present application, any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0023] The process, conditions, reagents, experimental methods, etc. of the present application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the present application has no special limitation. The experimental methods not specified in the specific conditions in each example are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0024] Unless otherwise specified, the meanings of all professional terms and scientific terms used in this specification are the same as those generally understood by the skilled person in the technical field to which the present application belongs. However, if there is a conflict, the present specification containing the definition shall prevail.

[0025] The following is the main raw material source and specification information in the examples: Soybean lecithin was purchased from Shanghai Maikelin Biotechnology Co., Ltd., HPLC≥98%; saikosaponin D was purchased from Sichuan Weikeqi Biological Technology Co., Ltd., HPLC≥98%; phosphatidic acid was purchased from Shanghai Yuanye Biological Technology Co., Ltd., HPLC≥98%; curcumin was purchased from Beijing Solabio Technology Co., Ltd., HPLC≥98%; poloxamer was purchased from Shanghai Yuanye Biological Technology Co., Ltd., HPLC≥98%; paclitaxel (Shanghai Yuanye Biological Technology Co., Ltd., HPLC≥98%; vincristine was purchased from Shanghai Yuanye Biological Technology Co., Ltd., HPLC≥98%; maytansine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., HPLC≥98%.

[0026] Example 1: Preparation of curcumin-loaded liposomes Prescription: Soybean lecithin: 20 mg; Saikosaponin D: 2.5 mg; Phosphatidic acid: 1.67 mg; Curcumin: 1.5 mg; Poloxamer 407: 10 mg; Anhydrous ethanol: 5 mL; Ultrapure water: 5 mL.

[0027] Preparation method: Precisely weigh the prescribed amount of soybean lecithin, saikosaponin D, phosphatidic acid and curcumin, and place them in a round-bottom flask. Add 5 mL of anhydrous ethanol and vortex to dissolve completely to form an organic phase.

[0028] Place the round-bottom flask in a rotary evaporator and evaporate under reduced pressure in a 40°C water bath to remove the organic solvent and form a uniform film on the bottle wall.

[0029] Precisely weigh the poloxamer 407 and dissolve it in 5 mL of ultrapure water. Add it to the above-mentioned round-bottom flask with a thin film.

[0030] Place the round-bottom flask in a 40°C water bath and wash the film for 30 min to obtain a liposome suspension.

[0031] Place the above-mentioned liposome suspension in an ultrasonic cleaner and ice-bath ultrasonic for 10 min.

[0032] Filter the liposome suspension after ultrasonic through 0.45 μm and 0.22 μm microporous filter membranes for 3 times respectively, to obtain curcumin liposomes (Cur-SSD-Lps).

[0033] Example 2 Preparation method of paclitaxel-loaded liposomes Prescription: Soybean lecithin: 20 mg; Saikosaponin D: 2 mg; Phosphatidic acid: 2.5 mg; Paclitaxel: 1.5 mg; Poloxamer 407: 3.33 mg; Anhydrous ethanol: 5 mL; Ultrapure water: 5 mL.

[0034] Preparation method: Precisely weigh the prescription amount of soybean lecithin, saikosaponin D, phosphatidic acid and paclitaxel, and place them in a round-bottom flask. Add 5 mL of anhydrous ethanol and vortex to dissolve completely to form an organic phase.

[0035] Place the round-bottom flask in a rotary evaporator and evaporate under reduced pressure in a 40°C water bath to remove the organic solvent and form a uniform film on the bottle wall.

[0036] Precisely weigh the poloxamer 407 and dissolve it in 5 mL of ultrapure water. Add it to the above-mentioned round-bottom flask with the film.

[0037] Place the round-bottom flask in a 40°C water bath for 28 minutes to obtain a crude liposome suspension.

[0038] Place the above-mentioned liposome suspension in an ultrasonic cleaner and ice-bath ultrasonic for 20 min.

[0039] Filter the ultrasonicated liposome suspension through 0.45 μm and 0.22 μm microporous filters for 3 times each to obtain paclitaxel liposomes.

[0040] Preparation method of vincristine liposomes Prescription: Soybean lecithin: 20 mg; Saikosaponin D: 3 mg; Phosphatidic acid: 2 mg; Vincristine: 1.5 mg; Poloxamer 407: 5 mg; Anhydrous ethanol: 5 mL; Ultrapure water: 5 mL.

[0041] Preparation method: Precisely weigh the prescription amount of soybean lecithin, saikosaponin D, phosphatidic acid and vincristine, and place them in a round-bottom flask. Add 5 mL of anhydrous ethanol and vortex to dissolve completely to form an organic phase.

[0042] Put the round bottom flask on the rotary evaporator and remove the organic solvent under reduced pressure in a 55°C water bath to form a uniform film on the wall of the flask.

[0043] Precisely weigh the poloxamer 407, dissolve it in 5 mL of ultrapure water, and add it to the above-mentioned round bottom flask with the film.

[0044] Put the round bottom flask in a 55°C water bath to hydrate the film for 32 minutes to obtain a crude liposome suspension.

[0045] Put the above-mentioned liposome suspension in an ultrasonic cleaner and ice-bath ultrasonic for 5 min.

[0046] Filter the liposome suspension after ultrasonic through 0.45 μm and 0.22 μm microporous filter membranes for 3 times, respectively, to obtain vincristine liposomes.

[0047] Example 4 Preparation method of maytansine liposomes Prescription: Soybean lecithin: 20 mg; Saikosaponin D: 3 mg; Phosphatidic acid: 2.2 mg; Maytansine: 1.5 mg; Poloxamer 407: 8 mg; Anhydrous ethanol: 5 mL; Ultrapure water: 5 mL.

[0048] Preparation method: Precisely weigh the prescribed amount of soybean lecithin, saikosaponin D, phosphatidic acid, and maytansine, and place them in a round bottom flask. Add 5 mL of anhydrous ethanol and vortex to dissolve completely to form an organic phase.

[0049] Put the round bottom flask on the rotary evaporator and remove the organic solvent under reduced pressure in a 40°C water bath to form a uniform film on the wall of the flask.

[0050] Precisely weigh the poloxamer 407, dissolve it in 5 mL of ultrapure water, and add it to the above-mentioned round bottom flask with the film.

[0051] Put the round bottom flask in a 40°C water bath to hydrate the film for 30 minutes to obtain a crude liposome suspension.

[0052] Put the above-mentioned liposome suspension in an ultrasonic cleaner and ice-bath ultrasonic for 10 min.

[0053] Filter the liposome suspension after ultrasonic through 0.45 μm and 0.22 μm microporous filter membranes for 3 times, respectively, to obtain maytansine liposomes.

[0054] Comparative Example 1 The saikosaponin D in the prescription was replaced by cholesterols in equal amount, and other components, usage and preparation process were the same as in Example 1 to prepare curcumin-loaded liposomes (Cur-Chol-Lps) with cholesterols as the membrane material Effect test example 1 Characterization of liposomes (1) Microscopic morphology observation: The drug-loaded liposomes based on saikosaponin D prepared in Examples 1-4 were observed by transmission electron microscopy (TEM), and the results are shown in Figure 2 It can be seen that the drug-loaded liposomes based on saikosaponin D have a diameter of about 100-200 nm, are round or oval, have no obvious aggregation, and are uniform in size.

[0055] (2) Particle size, dispersibility and Zeta potential investigation: The particle size, polydispersity coefficient (PDI) and Zeta potential of the drug-loaded liposomes based on saikosaponin D prepared in Examples 1-4 and the liposomes prepared in Comparative Example 1 were determined by laser particle size analyzer, and the results are shown in Figure 3 and Figure 4 The liposome particle size distribution is concentrated, the dispersibility is good, and the absolute value of Zeta potential is high.

[0056] The average particle size of the curcumin-loaded liposomes prepared in Example 1 was 186.90±5.62 nm, the polydispersity coefficient was 0.156±0.051, and the Zeta potential was -47.82±1.95 mV.

[0057] The average particle size of the paclitaxel-loaded liposomes prepared in Example 2 was 178.43±6.57 nm, the polydispersity coefficient was 0.186±0.042, and the Zeta potential was -42.51±2.37 mV.

[0058] The average particle size of the vincristine-loaded liposomes prepared in Example 3 was 145.13±5.95 nm, the polydispersity coefficient was 0.230±0.039, and the Zeta potential was -43.82±3.27 mV.

[0059] The average particle size of the maytansine-loaded liposomes prepared in Example 4 was 162±7.04 nm, the polydispersity coefficient was 0.245±0.031, and the Zeta potential was -46.30±4.15 mV.

[0060] The average particle size of the liposomes prepared in Comparative Example 1 was 195.34±6.82 nm, the polydispersity coefficient was 0.205±0.030, and the Zeta potential was -49.64±2.17 mV.

[0061] (3) Determination of liposome encapsulation efficiency and drug loading Accurately pipette 1 mL of curcumin liposome prepared in Example 1-4 based on saikosaponin D and the liposome solution prepared in Comparative Example 1, extract with 2 mL of ethyl acetate, and accurately pipette 500 μL of the lower liposome solution into a 10 mL volumetric flask. Dilute to the calibration mark with anhydrous ethanol, sonicate to break the emulsion, determine the absorbance, and calculate the content of the curcumin encapsulated in the liposome (M1) by substituting into the linear regression equation. Take another 500 μL of the drug-loaded liposome solution, do not extract and elute, and place in a 10 mL volumetric flask. Dilute to the calibration mark with anhydrous ethanol, sonicate to break the emulsion, determine the absorbance, and calculate the total content of curcumin in the liposome. The results are shown in Table 1.

[0062] Table 1 Determination results of liposome encapsulation rate and drug loading

[0063] (3) In vitro release property investigation Take an appropriate amount of free curcumin (Cur) anhydrous ethanol solution and Cur-SSD-Lps solution prepared in Example 1, and place in dialysis bags (molecular weight cut-off 3500). Tie the two ends of the dialysis bag tightly. Incubate at 37°C and 150 r / min, take samples at 1, 2, 4, 8, 12, 24, 36, 48, and 72 h, and supplement with an equal volume of release medium at the same temperature. Determine the absorbance at 430 nm at each time point, calculate the cumulative release degree, and plot the in vitro release curve. The results are shown in Figure 2. Figure 5 As can be seen from Figure 2, free curcumin (Cur) in the release medium shows a rapid release state, and the cumulative release degree at 36 h is 88.84%. However, the cumulative release degree of Cur-SSD-Lps at 72 h is only 47.5%, indicating that Cur-SSD-Lps has obvious sustained-release characteristics compared with free curcumin, can improve the in vivo bioavailability and stability of curcumin, and prolong the circulation time of curcumin in the body.

[0064] Example 2 CCK-8 method for detecting the effect of liposomes on the proliferation of HepG2 liver cancer cells HepG2 cells in the logarithmic growth phase were diluted to 2×10 4The cells were seeded in 96-well plates at a density of cells / well and cultured for 24 h. The original culture medium was discarded, and different concentrations of SSD-Lps (liposomes without curcumin, liposomes prepared according to the prescription of Example 1 without active drug curcumin), Cur-Chol-Lps (liposomes prepared according to Comparative Example 1) and Cur-SSD-Lps (liposomes prepared according to Example 1) were added, respectively (the concentration of SSD or Chol was 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 μg / mL), 5 replicate wells were set for each concentration, and the culture was continued for 24 h. 10 μl of CCK-8 solution was added to each well, and incubation was carried out for 2 h. The absorbance was measured at 450 nm by using an enzyme-labeled instrument.

[0065] The experimental results are shown in Table 1. Figure 6 As shown in Table 1, SSD-Lps and Cur-SSD-Lps had obvious proliferation inhibition effect on HepG2 cells, and the effect was concentration-dependent, while Cur-Chol-Lps had less proliferation inhibition effect on HepG2 cells, and the cell inhibition rate remained above 80% with the change of curcumin concentration. When the mass concentration of SSD was 2-6 μg / mL, Cur-SSD-Lps had more significant tumor cell killing ability than Cur-Chol-Lps and SSD-Lps (P<0.05) in the treatment of HepG2 cells. It was calculated that the IC50 of SSD-Lps and Cur-SSD-Lps on HepG2 cells was 4.709 μg / mL and 4.281 μg / mL, respectively.

[0066] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A drug-loaded liposome constructed based on saikosaponin D, characterized in that, It includes soybean lecithin, saikosaponin D, phosphatidic acid, active pharmaceutical ingredients, and poloxamer 407.

2. The drug-loaded liposome according to claim 1, characterized in that, The mass ratio of soybean lecithin to saikosaponin D is 20:2~3.33, the mass ratio of soybean lecithin to phosphatidic acid is 20:1.67~2.5, and the mass ratio of soybean lecithin to poloxamer 407 is 20:3.33~10.

3. The drug-loaded liposome according to claim 1, characterized in that, The active drug is an anti-tumor drug.

4. The drug-loaded liposome according to claim 3, characterized in that, The antitumor drug is any one of curcumin, paclitaxel, vincristine, and maytansine.

5. The drug-loaded liposome according to claim 1, characterized in that, The average particle size of the liposomes is less than 200 nm.

6. The drug-loaded liposome according to any one of claims 1-5, characterized in that, The drug loading capacity of the drug-loaded liposomes is 1.5-7.0%.

7. A method for preparing drug-loaded liposomes according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Soybean lecithin, saikosaponin D, phosphatidic acid and therapeutic drugs are dissolved in an organic solvent to form an organic phase, wherein the organic solvent is anhydrous ethanol or methanol; (2) Remove the organic solvent from the organic phase obtained in step (1) by rotary evaporation under reduced pressure at 40~55℃ to form a uniform film on the inner wall of the container; (3) Dissolve poloxamer 407 in ultrapure water and then add it to the container for forming the film as described in step (2). Hydrate and wash the film in a water bath at 40~55℃ to form a liposome suspension. (4) The liposome suspension obtained in step (3) is subjected to ice bath sonication. (5) The liposome suspension after step (4) is filtered through a microporous membrane to obtain the drug-loaded liposomes based on saikosaponin D.

8. The method according to claim 7, characterized in that, The hydration and washing time in step (3) is 28~32 min.

9. The method according to claim 7, characterized in that, The time for ice bath ultrasound in step (4) is 5~20 min; the filtration in step (5) is to use microporous membranes of 0.45 μm and 0.22 μm in sequence.

10. The use of the drug-loaded liposomes according to any one of claims 1-5 or the drug-loaded liposomes prepared by the preparation method according to any one of claims 7-9 in the preparation of antitumor drugs.

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