5-fluorouracil long-circulating liposome and preparation method thereof

Abstract

The invention provides a composite liposome. The composite liposome is prepared from composite phospholipid, cholesterol and DSPE-PEG2000 (distearoyl phospholipids-polyethylene glycol 2000). Through continuous tests, the inventor finds that the liposome prepared by adopting the composite phospholipid has better encapsulation efficiency and lower leakage rate, the structure is firmer and more stable, the quality stability and bioavailability are relatively improved, the drug does not leak during long-term storage, and the liposome has better application prospects; pEG modification is adopted, so that an anti-PEG-antibody can be induced to be combined with the PEGylated liposome, a complement system is activated, immune response reaction is caused, and the effects of long-acting targeting and synergistic attenuation are achieved.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a 5-fluorouracil long-circulating liposome and a preparation method and application thereof. Background Art

[0002] 5-Fluorouracil (5-Fu), also known as 2,4-dihydroxy-5-fluoropyrimidine, has a chemical formula of C4H3FN2O2. As a pyrimidine-based cell cycle-specific anti-metabolite, it acts in the S phase of cells, replaces uracil or thymine to enter DNA, and causes DNA double-strand breaks. It is clinically used to treat digestive system cancers (colorectal cancer, gastric cancer), primary liver cancer, breast cancer and other cancers.

[0003] 5-Fu has low bioavailability and plasma t 1 / 2 About 10-20 minutes. In order to maximize the killing of tumor cells, continuous administration is often required. However, continuous infusion not only brings inconvenience to the treatment process, but the sudden increase in blood drug concentration during administration will make the adverse reactions of the drug more obvious. Therefore, the clinical application of ordinary 5-fluorouracil injection has great limitations.

[0004] Therefore, a method for preparing a 5-fluorouracil injection with good stability for clinical application needs to be studied. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] Due to the limitation of 5-Fu's own properties, its oral absorption is incomplete and its bioavailability is low. After rapid injection of 5-Fu, the main mechanism of 5-Fu can cause the death of thymine-free cells, and the plasma t 1 / 2 About 10-20 minutes, so continuous infusion is more likely to maximize tumor cell killing, and continuous administration is often required. However, continuous infusion not only brings inconvenience to the treatment process, but also the sudden increase in blood drug concentration during administration makes the adverse drug reactions occur more quickly and obviously, and its clinical application has great limitations.

[0007] Liposomes, also known as liposomes, are new drug preparations with targeted drug delivery functions. At present, the preparation of 5-Fu liposomes mostly uses relatively simple phospholipid materials, such as natural lecithin. However, studies have found that lipid bilayers prepared from pure single-component phospholipids usually undergo multiple thermally induced phase transitions when heated under normal pressure conditions, resulting in poor quality stability of the prepared product, and the bioavailability has not been significantly improved compared to 5-fluorouracil injection. Composite phospholipid liposomes are a relatively new liposome preparation method at this stage. Composite phospholipids refer to bimolecular membrane structures composed of two different types of phospholipids and cholesterol, with the two phospholipids arranged alternately. Since two phospholipids with different phase transition temperatures are used as membrane materials, it is necessary to include a saturated phospholipid Tc higher than 37°C and an unsaturated phospholipid Tc lower than 4°C. The inventors added phospholipids with high phase transition temperature during the preparation process of this study, which affected the properties of the liposome bilayer membrane. The temperature was controlled at 55°C during the hydration process. Under this condition, the phospholipid membrane material simultaneously existed in the colloidal phase and the liquid crystal phase, which could appropriately adjust the deformation and rigidity of the membrane. The obtained liposomes had better encapsulation rate and lower leakage rate, and the structure was more firm and stable, which relatively improved the quality stability and bioavailability. The drug did not leak during long-term storage, and had good application prospects.

[0008] Therefore, the inventors have found through continuous experiments that the 5-Fu composite lipid long-circulation liposomes prepared by the pH gradient method combined with the reverse evaporation method and the polyethylene glycol (DSPE-PEG2000) modified composite lipid bilayer have good stability. Many studies have shown that PEGylated liposomes can induce anti-PEG-antibodies, which can bind to PEGylated liposomes, activate the complement system, and cause immune response reactions, thereby playing a long-term targeting role and producing synergistic and toxic effects. At the same time, the drug is encapsulated inside the liposome, and the unique advantages of the liposome particle size and liposome dosage form are utilized to make it less susceptible to degradation by various enzyme systems, thereby delaying the duration of action. At the same time, the liposome dosage form has unique advantages such as effectively improving drug stability, reducing drug toxicity, sustained release and targeting. In the experiment, the preparation and characterization of 5-Fu composite lipid long-circulation liposomes were preliminarily explored to prepare a 5-Fu composite lipid long-circulation liposome dosage form with more stable properties, providing a scientific basis for in-depth development and utilization.

[0009] In addition, the inventors also investigated the effects of the ratio of complex phospholipids, the ratio of phospholipids to cholesterol, the drug-lipid ratio, the pH value of the external aqueous phase, the drug loading temperature and time on the encapsulation rate of 5-Fu liposomes based on single-factor experiments, and determined the optimal liposome preparation process. The use of this process not only improves the stability of the liposome preparation, but also improves the encapsulation rate of the liposome.

[0010] Therefore, in one aspect of the present invention, the present invention proposes a composite liposome. According to an embodiment of the present invention, the composite liposome comprises a composite phospholipid, cholesterol and DSPE-PEG2000. The inventors have found through continuous experiments that the use of composite phospholipids can make the prepared liposomes have better encapsulation rate and lower leakage rate, the structure is more solid and stable, the quality stability and bioavailability are relatively improved, the drug does not leak during long-term storage, and has good application prospects; the use of PEG modification can induce anti-PEG-antibodies to bind to PEGylated liposomes, activate the complement system, and cause an immune response, thereby playing a long-term targeting role and producing a synergistic and toxic effect.

[0011] It should be noted that the "complex phospholipid" described in the present application is composed of two phospholipids with different phase transition temperatures as membrane materials, and needs to contain a saturated phospholipid with a Tc higher than 37°C and an unsaturated phospholipid with a Tc lower than 4°C.

[0012] According to an embodiment of the present invention, the composite liposome may further include at least one of the following additional technical features:

[0013] According to an embodiment of the present invention, the composite phospholipid includes egg yolk lecithin E80 and hydrogenated soybean phospholipid HSPC. According to a specific embodiment of the present invention, two phospholipids with different phase transition temperatures are used as membrane materials, so that the prepared liposomes have better encapsulation efficiency and lower leakage rate, and the structure is more firm and stable, and the quality stability and bioavailability are relatively improved. The drug does not leak during long-term storage, and has good application prospects.

[0014] According to an embodiment of the present invention, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (15-1): 1. The inventors have found through continuous experiments that the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC in this range can make the prepared liposome have a higher encapsulation rate and a lower leakage rate, and the structure is relatively firm and stable.

[0015] According to an embodiment of the present invention, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (12-1):1.

[0016] According to a specific embodiment of the present invention, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is 8:1.

[0017] According to an embodiment of the present invention, the mass ratio of the composite phospholipid to cholesterol is (20-1): 1. The inventors have found through continuous experiments that the mass ratio of the composite phospholipid to cholesterol in this range can make the prepared liposome have a higher encapsulation rate and a lower leakage rate, and the structure is relatively firm and stable.

[0018] According to an embodiment of the present invention, the mass ratio of the complex phospholipid to cholesterol is (15-1):1.

[0019] According to a specific embodiment of the present invention, the mass ratio of the complex phospholipid to cholesterol is 12:1.

[0020] According to an embodiment of the present invention, based on the total mass of the composite liposome, the mass ratio of DSPE-PEG2000 is 1-15%. The inventors have found through continuous experiments that the use of DSPE-PEG2000 in this range can enable the prepared liposome to play a long-term targeting, synergistic and toxicity-reducing role in the body.

[0021] According to an embodiment of the present invention, the mass ratio of DSPE-PEG2000 is 4% to 10%.

[0022] According to an embodiment of the present invention, the mass ratio of the DSPE-PEG2000 is 6%.

[0023] In another aspect of the present invention, the present invention proposes a drug liposome. According to an embodiment of the present invention, the drug liposome includes the composite liposome described above and 5-fluorouracil. The inventors found that encapsulating the drug inside the liposome and utilizing the unique advantages of the liposome particle size and liposome dosage form can prevent the drug from being degraded by various enzyme systems, thereby delaying the duration of action. At the same time, the liposome dosage form has unique advantages such as effectively improving drug stability, reducing drug toxicity, sustained release and targeting.

[0024] According to an embodiment of the present invention, the drug liposome may further include at least one of the following additional technical features:

[0025] According to an embodiment of the present invention, the mass ratio of 5-fluorouracil to the composite liposome is 1:(1-18). The inventors have found through continuous experiments that the mass ratio of 5-fluorouracil to the composite liposome in this range can make the prepared liposome have a higher encapsulation rate and a lower leakage rate, and the structure is relatively firm and stable.

[0026] According to an embodiment of the present invention, the mass ratio of 5-fluorouracil to the composite liposome is 1:(1-14).

[0027] According to a specific embodiment of the present invention, the mass ratio of 5-fluorouracil to the composite liposome is 1:10.

[0028] In yet another aspect of the present invention, the present invention provides the use of the aforementioned complex liposomes in improving the stability of drug liposomes.

[0029] According to an embodiment of the present invention, the drug liposome is as defined above.

[0030] In another aspect of the present invention, the present invention proposes a method for preparing drug liposomes. According to an embodiment of the present invention, the method includes: performing a first mixing treatment on a composite phospholipid, cholesterol, DSPE-PEG2000 and a first solvent to obtain a first mixed solution; performing a second mixing treatment on 5-fluorouracil and a second solvent to obtain a second mixed solution; performing a third mixing treatment on the first mixed solution and the second mixed solution to obtain a third mixed solution; performing a fourth mixing treatment on the third mixed solution and a PBS buffer to obtain the drug liposomes. The drug liposomes prepared by this method can prevent the drug from being degraded by various enzyme systems, thereby delaying the duration of action. At the same time, the liposome dosage form has unique advantages such as effectively improving drug stability, reducing drug toxicity, sustained release and targeting.

[0031] According to an embodiment of the present invention, the method for preparing drug liposomes may further include at least one of the following additional technical features:

[0032] According to an embodiment of the present invention, the method further comprises: subjecting the drug liposomes to ultrasonic treatment, granulation, pH adjustment and water bath incubation treatment.

[0033] According to an embodiment of the present invention, the composite phospholipid includes egg yolk lecithin E80 and hydrogenated soybean phospholipid HSPC. According to a specific embodiment of the present invention, two phospholipids with different phase transition temperatures are used as membrane materials, so that the prepared liposomes have better encapsulation efficiency and lower leakage rate, and the structure is more firm and stable, and the quality stability and bioavailability are relatively improved. The drug does not leak during long-term storage, and has good application prospects.

[0034] According to an embodiment of the present invention, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (15-1): 1. The inventors have found through continuous experiments that the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC in this range can make the prepared liposome have a higher encapsulation rate and a lower leakage rate, and the structure is relatively firm and stable.

[0035] According to an embodiment of the present invention, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (12-1):1.

[0036] According to a specific embodiment of the present invention, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is 8:1.

[0037] According to an embodiment of the present invention, the mass ratio of the composite phospholipid to cholesterol is (20-1): 1. The inventors have found through continuous experiments that the mass ratio of the composite phospholipid to cholesterol in this range can make the prepared liposome have a higher encapsulation rate and a lower leakage rate, and the structure is relatively firm and stable.

[0038] According to an embodiment of the present invention, the mass ratio of the complex phospholipid to cholesterol is (15-1):1.

[0039] According to a specific embodiment of the present invention, the mass ratio of the complex phospholipid to cholesterol is 12:1.

[0040] According to an embodiment of the present invention, in the second mixing process, the mass ratio of the total mass of the composite phospholipids, cholesterol, and DSPE-PEG2000 in the first mixed solution to the mass ratio of 5-fluorouracil in the second mixed solution is 1:(1-18). The inventors have found through continuous experiments that the mass ratio of the total mass of the composite phospholipids, cholesterol, and DSPE-PEG2000 in the first mixed solution to the mass ratio of 5-fluorouracil in the second mixed solution within the range can make the prepared liposomes have a higher encapsulation rate and a lower leakage rate, and the structure is also relatively firm and stable.

[0041] According to an embodiment of the present invention, the mass ratio is 1:(1-14).

[0042] According to a specific embodiment of the present invention, the mass ratio is 1:10.

[0043] According to an embodiment of the present invention, based on the total mass of the composite phospholipid, cholesterol and DSPE-PEG2000, the mass ratio of DSPE-PEG2000 is 1-15%. The inventors have found through continuous experiments that the use of DSPE-PEG2000 in this range can enable the prepared liposomes to play a long-term targeting, synergistic and toxicity-reducing role in the body.

[0044] According to an embodiment of the present invention, the mass ratio of DSPE-PEG2000 is 4-10%.

[0045] According to an embodiment of the present invention, the mass ratio of DSPE-PEG2000 is 6%.

[0046] According to an embodiment of the present invention, the first solvent includes chloroform and ether, which is helpful for fully dissolving the complex phospholipids, cholesterol and DSPE-PEG2000.

[0047] According to an embodiment of the present invention, the volume ratio of chloroform to ether in the first solvent is 2:1, which is helpful for fully dissolving the complex phospholipids, cholesterol and DSPE-PEG2000.

[0048] According to an embodiment of the present invention, the second solvent is a citric acid buffer solution with a pH of 4.0, which can help to keep the pH value of the solution stable.

[0049] According to an embodiment of the present invention, the concentration of 5-fluorouracil in the second mixed solution is 8 mg / ml.

[0050] According to an embodiment of the present invention, the pH of the PBS buffer is 7.8.

[0051] According to an embodiment of the present invention, the pH is adjusted by 0.2 mol NaOH.

[0052] It should be noted that the “pH adjustment” refers to the pH of the external aqueous phase.

[0053] According to an embodiment of the present invention, the pH after adjusting the pH is 7.0 to 8.0. The inventors have found through a large number of experiments that as the transmembrane pH gradient gradually increases (the pH of the inner aqueous phase remains unchanged, and the pH of the outer aqueous phase gradually increases), the transmembrane driving force is increased, the incubation effect is significantly improved, and the encapsulation rate is high. In addition, due to the presence of ester bonds in phospholipids that are sensitive to alkaline conditions, the stability of the liposome will be poor if the pH value of the outer aqueous phase is too high, so it is not advisable to adjust the pH of the outer aqueous phase to be too high alkaline.

[0054] According to a specific embodiment of the present invention, the pH after the pH adjustment is 7.4-7.8.

[0055] According to a specific embodiment of the present invention, the pH after adjusting the pH is 7.6.

[0056] It should be noted that the "inner aqueous phase" described in the present application refers to a citric acid buffer solution at pH 4.0, and the "external aqueous phase" may refer to a PBS buffer solution or a 0.2 mol NaOH solution.

[0057] According to an embodiment of the present invention, the water bath incubation temperature is 40-70° C. The inventors found that the drug loading process requires a specific temperature range to improve the fluidity and permeability of the lipid membrane and reduce the energy barrier to ensure that the drug is maximally encapsulated into the liposome, and it was found that within this temperature range, the encapsulation rate increases with increasing temperature.

[0058] According to a specific embodiment of the present invention, the temperature of the water bath incubation is 60°C.

[0059] According to an embodiment of the present invention, the water bath incubation time is 15 to 95 minutes. The inventors also found that the length of the drug loading time may also cause the drug to fail to fully enter the liposome or affect the stability of the lipid membrane, thereby affecting the encapsulation rate. It was found that the encapsulation rate was better within this time range.

[0060] According to a specific embodiment of the present invention, the water bath incubation time is 60 minutes.

[0061] In another aspect of the present invention, the present invention provides a drug liposome. According to an embodiment of the present invention, the drug liposome is prepared by the method described above. The features and advantages described above for the method for preparing the drug liposome are also applicable to the drug liposome, and will not be repeated here.

[0062] In another aspect of the present invention, the present invention proposes the use of the aforementioned complex liposome or the aforementioned drug liposome in the preparation of a drug for preventing or treating digestive system cancer, primary liver cancer or breast cancer.

[0063] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. DETAILED DESCRIPTION

[0064] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0065] Example 1: Thin film dispersion method

[0066] Table 1

[0067]

[0068] Accurately weigh the organic phase solutes egg yolk lecithin E80, hydrogenated soybean lecithin (HSPC), cholesterol, and 4% DSPE-PEG2000 in the prescription ratio in Table 1 and dissolve them in a chloroform-ether (2:1) mixed solvent as membrane material, wherein the mass ratio of egg yolk lecithin E80 to hydrogenated soybean lecithin (HSPC) is 6:1, and the mass ratio of the total mass of egg yolk lecithin E80 and hydrogenated soybean lecithin (HSPC) to cholesterol is 8:1. Place them in a small beaker and dissolve them in a water bath until clear. Slowly add pH 4.0 citric acid buffer (8 mg / ml) containing 5-Fu to the organic phase solution, where the mass ratio of 5-Fu to the total mass of the organic phase solute is 1:8, mix with the inner aqueous phase solution by shearing and ultrasound to form a stable W / O emulsion, and remove the solvent by vacuum rotary evaporation to form a lipid film; add the outer aqueous phase phosphate buffer (PBS buffer) pH 7.8 to a round-bottom flask and hydrate under the same conditions; use an ultrasonic cell crusher to disperse the liposome particles, use 0.2 mol NaOH to adjust the sample pH to 7.4, incubate in a 55°C water bath for 30 minutes, and the preparation is completed.

[0069] Example 2: Reverse evaporation combined with pH gradient method

[0070] 5-Fu long-circulating liposomes were prepared by the reverse evaporation method combined with the pH gradient method: the organic phase solutes egg yolk lecithin E80, hydrogenated soybean lecithin (HSPC), cholesterol, and 6% DSPE-PEG2000 as membrane materials in the prescription ratio in Table 1 were accurately weighed and dissolved in chloroform-ether (2:1) mixed solvent, wherein the mass ratio of egg yolk lecithin E80 to hydrogenated soybean lecithin (HSPC) was 8:1, and the mass ratio of the total mass of egg yolk lecithin E80 and hydrogenated soybean lecithin (HSPC) to cholesterol was 12:1. The mixture was placed in a small beaker and dissolved in a water bath until clear. Slowly add pH4.0 citric acid buffer (8mg / ml) containing 5-Fu to the organic phase solution, where the mass ratio of 5-Fu to the total mass of organic phase solute is 1:10, shear ultrasound to form a stable W / O type emulsion, evaporate under reduced pressure to remove the organic solvent, until a gel film is formed on the bottle wall, add pH7.8 PBS buffer as the external aqueous phase for hydration, and obtain the initial liposome product. After ultrasound, pass through a microporous filter membrane in turn for granulation, and obtain a liposome solution. Use 0.2molNaOH to adjust the sample pH to 7.6, put it in a 60℃ water bath for incubation for 60min, and then put it in an ice bath below 10℃ for 30min to obtain the final sample.

[0071] Example 3: Reverse Phase Evaporation Method

[0072] Accurately weigh the organic phase solutes egg yolk lecithin E80, hydrogenated soybean lecithin (HSPC), cholesterol, and 8% DSPE-PEG2000 in the prescription ratio in Table 1 and dissolve them in a chloroform-ether (2:1) mixed solvent as membrane material, wherein the mass ratio of egg yolk lecithin E80 to hydrogenated soybean lecithin (HSPC) is 10:1, and the mass ratio of the total mass of egg yolk lecithin E80 and hydrogenated soybean lecithin (HSPC) to cholesterol is 10:1. Place them in a small beaker and dissolve them in a water bath until clear. Slowly add pH 4.0 citric acid buffer (8 mg / ml) containing 5-Fu to the organic phase solution, where the mass ratio of 5-Fu to the total mass of the organic phase solute is 1:12, mix with the inner aqueous phase solution by shearing and ultrasound to form a stable W / O emulsion, and remove the solvent by vacuum rotary evaporation to form a lipid film; add the outer aqueous phase buffer to a round-bottom flask and hydrate under the same conditions; use an ultrasonic cell crusher to disperse the liposome particles, use 0.2 mol NaOH to adjust the sample pH to 7.6, incubate in a 55°C water bath for 30 minutes, and the preparation is completed.

[0073] Example 4: Ethanol injection method

[0074] The organic phase solutes egg yolk lecithin E80, hydrogenated soybean lecithin (HSPC), cholesterol, and 10% DSPE-PEG2000 in the prescription ratio in Table 1 were accurately weighed as membrane materials, and the ethanol mixed solvent was heated and ultrasonically dissolved, wherein the mass ratio of egg yolk lecithin E80 to hydrogenated soybean lecithin (HSPC) was 12:1, and the mass ratio of the total mass of egg yolk lecithin E80 and hydrogenated soybean lecithin (HSPC) to cholesterol was 15:1, placed in a small beaker, and dissolved in a water bath until clear. The pH 4.0 citric acid buffer (8 mg / ml) containing 5-Fu was slowly injected into the organic phase solution below the liquid surface with a syringe, wherein the mass ratio of 5-Fu to the total mass of the organic phase solute was 1:15, and the organic solvent ethanol was removed by continuous stirring for 2 hours, and the external aqueous phase buffer was added and stirred for 35 minutes for incubation; the liposome particles were dispersed by an ultrasonic cell crusher, and the sample pH was adjusted to 7.8 with 0.2 mol NaOH, and the preparation was completed.

[0075] Comparative Example 1:

[0076] Table 2

[0077]

[0078] The organic phase solutes egg yolk lecithin E80, cholesterol, and 6% DSPE-PEG2000 in the prescription ratio in Table 2 were accurately weighed and dissolved in a chloroform-ether (2:1) mixed solvent, where the mass ratio of egg yolk lecithin E80 to cholesterol was 8:1, placed in a small beaker, and dissolved in a water bath until clear. Slowly add pH4.0 citric acid buffer (8 mg / ml) containing 5-Fu to the organic phase solution, where the mass ratio of 5-Fu to the total mass of the organic phase solute was 1:10, mixed with the inner aqueous phase solution by shearing and ultrasound to form a stable W / O type emulsion, and the solvent was removed by vacuum rotary evaporation to form a lipid membrane; the outer aqueous phase PBS buffer was added to a round-bottom flask and hydrated under the same conditions; the liposome particles were dispersed using an ultrasonic cell crusher, and the sample pH was adjusted to 7.6 using 0.2 mol NaOH, and incubated in a 55°C water bath for 30 minutes to complete the preparation.

[0079] Comparative Example 2:

[0080] Weigh the organic phase solutes hydrogenated soybean lecithin HSPC, cholesterol, and 8% DSPE-PEG2000 in the prescription ratio in Table 2 respectively as membrane materials and dissolve them in a chloroform-ether (2:1) mixed solvent, where the mass ratio of hydrogenated soybean lecithin HSPC to cholesterol is 10:1, place them in a small beaker, and dissolve them in a water bath until they are clear. Slowly add pH4.0 citric acid buffer (8 mg / ml) containing 5-Fu to the organic phase solution, where the mass ratio of 5-Fu to the total mass of organic phase solutes is 1:12, shear ultrasound to form a stable W / O type emulsion, and evaporate the organic solvent under reduced pressure to form a gel film on the bottle wall, then add pH7.8 PBS buffer as the external aqueous phase for hydration to obtain the initial liposomes. After ultrasound, pass through a microporous filter membrane for granulation in turn to obtain a liposome solution. Use 0.2 mol NaOH to adjust the sample pH to 7.6, incubate in a 60°C water bath for 60 minutes, and then put it in an ice bath below 10°C for 30 minutes to obtain the final sample.

[0081] Comparative Example 3:

[0082] The organic phase solutes egg yolk lecithin and cholesterol in the prescription ratio in Table 2 were accurately weighed as membrane materials and dissolved in a chloroform-ether (2:1) mixed solvent, wherein the mass ratio of egg yolk lecithin E80 to cholesterol was 8:1, placed in a small beaker, and dissolved in a water bath until clear. Slowly add pH4.0 citric acid buffer (8 mg / ml) containing 5-Fu to the organic phase solution, wherein the mass ratio of 5-Fu to the total mass of the organic phase solute was 1:12, shear ultrasound to form a stable W / O type emulsion, and evaporate the organic solvent under reduced pressure until a gel film is formed on the bottle wall, and then add pH7.8 PBS buffer as the external aqueous phase for hydration to obtain the initial liposome product, and after ultrasound, pass through a microporous filter membrane for granulation in turn to obtain a liposome solution. Use 0.2 mol NaOH to adjust the sample pH to 7.6, put it in a 60°C water bath for incubation for 60 minutes, and then put it in an ice bath below 10°C for 30 minutes to obtain the final sample.

[0083] Comparative Example 4:

[0084] Accurately weigh the organic solutes egg yolk lecithin, hydrogenated soybean lecithin HSPC, and cholesterol in the prescription ratio in Table 2 as membrane materials and dissolve them in a chloroform-ether (2:1) mixed solvent, wherein the mass ratio of egg yolk lecithin E80 to hydrogenated soybean lecithin (HSPC) is 10:1, and the mass ratio of the total mass of egg yolk lecithin E80 and hydrogenated soybean lecithin (HSPC) to cholesterol is 8:1. Place them in a small beaker and dissolve them in a water bath until clear. Slowly add pH 4.0 citric acid buffer (8 mg / ml) containing 5-Fu to the organic phase solution, where the mass ratio of 5-Fu to the total mass of the organic phase solute is 1:10, mix with the inner aqueous phase solution by shearing and ultrasound to form a stable W / O emulsion, and remove the solvent by vacuum rotary evaporation to form a lipid film; add the outer aqueous phase PBS buffer to a round-bottom flask and hydrate under the same conditions; use an ultrasonic cell crusher to disperse the liposome particles, use 0.2 mol NaOH to adjust the sample pH to 7.6, incubate in a 55°C water bath for 30 minutes, and the preparation is completed.

[0085] Example 5: Stability Study

[0086] 1. Morphological observation, particle size and Zeta potential determination

[0087] The liposome morphology was observed using a transmission electron microscope (TEM) using the negative staining method.

[0088] The particle size and distribution of liposomes will directly affect the stability of liposome preparations, as well as their distribution and disposal in the body. Zeta potential is a measure of the strength of mutual repulsion or attraction between particles in the system, and is also an important parameter for evaluating the stability of liposomes. A mature preparation process should stabilize the particle size and potential, and present a normal distribution with a narrow distribution range. Use a pipette to measure 1 ml of the test sample, add it to the square sample cell of the Malvern potentiometric particle size analyzer, and use dynamic light scattering to detect the particle size and particle size distribution.

[0089] 2. Determination of encapsulation efficiency and drug loading

[0090] Since 5-Fu is a hydrophilic dibasic weak acid drug with pKa1=8 and pKa2=13, the 2020 edition of the "Chinese Pharmacopoeia" (Part II) uses ultraviolet spectrophotometry (General Rule 0401) for the content determination of 5-Fu APIs, injections and creams. This experiment also uses ultrafiltration-ultraviolet spectrophotometry to determine the liposome drug loading and encapsulation efficiency. Measure 1.0 ml of liposome solution and put it into an ultrafiltration tube. Ultrafiltration is used to separate the drug and free drug encapsulated in the liposomes, and then the amount of 5-Fu loaded on the liposomes is determined. The maximum absorption wavelength of 5-Fu scanned by the ultraviolet spectrophotometer is 265 nm, so the measurement wavelength is selected at 265 nm. The absorbance of the 5-Fu aqueous solution is determined by ultraviolet method, and the blank liposomes are detected by the same method. The results can confirm that excipients such as phospholipids have no ultraviolet absorption at the same wavelength to eliminate the interference of experimental excipients. Linear regression was performed on 5-Fu absorbance (A) and mass concentration (c) to obtain a standard curve and calculate the encapsulation efficiency and drug loading.

[0091] Accurately weigh a certain amount of drug-loaded 5-Fu long-circulating liposomes, dilute and measure its ultraviolet absorbance at a wavelength of 265nm, and calculate the encapsulation efficiency (Eencapsulation Efficiency E%) and drug loading (Loading Content, L%) according to the following formula. Where: W1 is the mass of the drug detected in the liposome (mg), W2 is the mass of the added drug (mg), and W3 is the mass of the lipid carrier (mg).

[0092]

[0093]

[0094] 3. Determination of leakage rate

[0095] The prepared liposomes were placed in a refrigerator at 4°C and the release rate (R%) of the sample drug was measured. The measurement was repeated 3 times and the release rate was calculated using the following formula:

[0096]

[0097] The liposome solutions prepared in Examples 1-4 and Comparative Examples 1-4 were sterilized by a 0.22 μm filter membrane, placed in a cillin bottle, sealed and capped, and placed at 4°C for 60 days. The appearance, particle size, potential, encapsulation rate, drug loading and leakage rate changes were taken at 0 days, 3 days and 30 days respectively. The measurement results are shown in Table 3 below. It can be seen from the table that the liposomes prepared in Examples 1 to 4 have higher encapsulation rates and drug loading than the liposomes prepared in Comparative Examples 1 to 4, and the leakage rate is also lower after being placed for 30 days. By comparing the liposomes obtained by the methods of Examples 1 to 4, it is found that the liposomes prepared in Example 2 have higher drug loading and encapsulation rates, and the leakage rate is also lower after being placed for 30 days. Therefore, it is shown that the liposomes prepared under the conditions of E80: HSPC (w / w) of 8:1, drug-lipid ratio (w / w) of 1:10, phospholipid: cholesterol of 12:1, DSPE-PEG2000 of 6%, and external aqueous phase pH of 7.6 have higher encapsulation efficiency and drug loading capacity, and have lower leakage rate during storage.

[0098] Table 3

[0099]

[0100]

[0101] Example 6: Pharmacodynamics Experiment

[0102] In vitro pharmacodynamics testing:

[0103] Three types of tumor cells in logarithmic growth phase (1×10 5 / mL) (Hela, MFC and 4T1) were spread on a 96-well plate, with 100 μL per well. Four concentration groups were set up, including a blank control group, a negative control group, a positive control group, and a test sample. Each concentration group had three parallel wells. After 24 hours of culture, the drugs were added in groups. After 48 hours of continuous culture, MTS was added. After a certain period of continuous culture, the absorbance (OD value) at a wavelength of 490 nm was detected by an enzyme marker. The cell proliferation inhibition rate (IR) and cell half inhibitory concentration (IC 50 ), and compare the inhibitory effects of the test sample and the positive control on the proliferation of different cells in vitro. The reference substance is fluorouracil injection (2022S012) produced by Shenyang Yaoda Leiyunshang Pharmaceutical Co., Ltd.

[0104] As can be seen from Table 4, Examples 1-4 showed an inhibitory effect superior to that of fluorouracil injection in the three tumor cells. 50 The values ​​are lower than those of fluorouracil injection. It can be seen that in the in vitro efficacy experiment, Examples 1-4 are superior to the existing fluorouracil injection and are even better than Comparative Examples 1-4.

[0105] In vivo pharmacodynamics testing:

[0106] Sources of cells and experimental animals:

[0107] Mouse breast cancer cell 4T1 was derived from Zhongqiao Xinzhou, and the culture conditions were: RPMI1640+10% FBS.

[0108] Species & Strain: BALB / c mice.

[0109] Body weight at the time of tumor inoculation: about 18-22 g, slightly smaller than the control group.

[0110] Gender and number: Male, 15 mice.

[0111] Model Building

[0112] After the cells are revived, they are cultured in RPMI1640 complete medium containing 10% FBS. The cells are subcultured every 2 to 3 days. The cells in the logarithmic growth phase are collected and centrifuged, and then counted after staining with 0.4% trypan blue. The viable cell concentration is adjusted to about 10 in serum-free medium. 7 / mL, 0.1mL of cell suspension was taken and inoculated into the right axilla of 52 balb / c mice with a final volume of 0.2mL / mouse. When the tumor volume began to increase, the mice were randomly divided into 3 groups according to the tumor volume, with 5 mice in each group. The mice were given the drug once every 6 days with an interval of 5 days, for a total of 3 times. The dosage was 0.1mg·kg -1 .

[0113] Tumor diameter measurement

[0114] Detection time: grouping (i.e. the day of first administration is D1), every day after the first administration, the long and short diameters of the tumor are measured and recorded with a vernier caliper, the tumor volume is calculated, and the tumor growth curve is drawn based on the tumor volume.

[0115] The tumor volume was calculated according to the following formula:

[0116] V = 1 / 2 × major diameter × minor diameter × minor diameter

[0117] Efficacy evaluation based on tumor volume

[0118] The relative tumor volume (RTV) and relative tumor proliferation rate T / C (%) were calculated according to the following formula:

[0119] RTV=V t / V0

[0120] V t :The tumor volume was measured every day

[0121] V0: initial tumor volume (before drug administration)

[0122] T / C (%) = average RTV value of the drug administration group / average RTV value of the model group × 100%

[0123] If T / C≤40%, the difference between the RTV in the drug-treated group and the RTV in the model group was P<0.05 after statistical analysis, indicating that there was an inhibitory effect on tumor growth; conversely, if T / C≥40%, there was no inhibitory effect on tumor growth.

[0124] Efficacy evaluation based on tumor weight

[0125] After the experiment, the tumor nodules were peeled off and weighed, and the differences in tumor weight between the groups were compared to further calculate the tumor inhibition rate IRTW. IRTW>60% was used as the reference index for effectiveness. The calculation formula is as follows:

[0126] IRTW (%) = (W blank control group - W drug administration group) / W blank control group × 100%

[0127] In the 4T1 tumor model experiment, Examples 1-4 showed an inhibitory effect superior to that of fluorouracil injection. However, the inhibitory effects of Comparative Examples 1-4 were all lower than those of fluorouracil injection. It can be seen that in the 4T1 in vivo efficacy experiment, Examples 1-4 showed an effect superior to that of fluorouracil injection, and even superior to that of Comparative Examples 1-4.

[0128] In this study, DSPE-PEG2000 was used to modify liposomes as drug carriers, which can avoid phagocytosis by the reticuloendothelial system and increase the blood circulation time of liposomes, thereby prolonging the circulation time of liposomes in the blood, achieving the purpose of continuous and slow release of drugs, reducing the number of dosing times, improving efficacy, and reducing adverse reactions.

[0129] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0130] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A composite liposome, characterized in that: Including complex phospholipids, cholesterol and DSPE-PEG2000.

2. The complex liposome according to claim 1, characterized in that The compound phospholipids include egg yolk lecithin E80 and hydrogenated soybean lecithin HSPC; Optionally, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (15-1):1; Optionally, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (12-1):1; Preferably, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is 8:

1.

3. The complex liposome according to claim 1, characterized in that The mass ratio of the composite phospholipid to cholesterol is (20-1):1; Optionally, the mass ratio of the complex phospholipid to cholesterol is (15-1):1; Preferably, the mass ratio of the complex phospholipid to cholesterol is 12:

1.

4. The complex liposome according to claim 1, characterized in that Based on the total mass of the composite liposome, the mass ratio of the DSPE-PEG2000 is 1% to 15%; Optionally, the mass ratio of DSPE-PEG2000 is 4% to 10%; Preferably, the mass ratio of the DSPE-PEG2000 is 6%.

5. A drug liposome, characterized in that: The invention comprises the composite liposome according to claim 1 and 5-fluorouracil.

6. The pharmaceutical liposome according to claim 5, characterized in that: The mass ratio of 5-fluorouracil to the composite liposome is 1:(1-18); Optionally, the mass ratio of 5-fluorouracil to the composite liposome is 1:(1-14); Preferably, the mass ratio of 5-fluorouracil to the composite liposome is 1:

10.

7. Use of the composite liposome according to claim 1 in improving the stability of drug liposomes; Optionally, the drug liposome is as defined in any one of claims 5 to 6.

8. A method for preparing drug liposomes, characterized in that: include: Performing a first mixing treatment on the composite phospholipid, cholesterol, DSPE-PEG2000 and the first solvent to obtain a first mixed solution; Performing a second mixing process on 5-fluorouracil and a second solvent to obtain a second mixed solution; Performing a third mixing process on the first mixed solution and the second mixed solution to obtain a third mixed solution; The third mixed solution is subjected to a fourth mixing treatment with a PBS buffer to obtain the drug liposome.

9. The method according to claim 8, characterized in that Further including: The drug liposomes are subjected to ultrasonic treatment, granulation, pH adjustment and water bath incubation.

10. The method according to claim 8, characterized in that The compound phospholipids include egg yolk lecithin E80 and hydrogenated soybean lecithin HSPC; Optionally, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (15-1):1; Optionally, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is (12-1):1; Preferably, the mass ratio of the egg yolk lecithin E80 to the hydrogenated soybean lecithin HSPC is 8:

1.

11. The method according to claim 8, characterized in that The mass ratio of the composite phospholipid to cholesterol is (20-1):1; Optionally, the mass ratio of the complex phospholipid to cholesterol is (15-1):1; Preferably, the mass ratio of the complex phospholipid to cholesterol is 12:

1.

12. The method according to claim 8, characterized in that In the second mixing process, the mass ratio of the total mass of the composite phospholipid, cholesterol, and DSPE-PEG2000 in the first mixed solution to the mass ratio of 5-fluorouracil in the second mixed solution is 1: (1～18)； Optionally, the mass ratio is 1:(1-14); Preferably, the mass ratio is 1:

10.

13. The method according to claim 8, characterized in that Based on the total mass of the composite phospholipid, cholesterol and DSPE-PEG2000, the mass ratio of the DSPE-PEG2000 is 1 to 15%; Optionally, the mass ratio of DSPE-PEG2000 is 4 to 10%; Preferably, the mass ratio of DSPE-PEG2000 is 6%.

14. The method according to claim 8, characterized in that The first solvent includes chloroform and ether; Optionally, the volume ratio of chloroform to diethyl ether in the first solvent is 2:1; Optionally, the second solvent is a citric acid buffer at pH 4.0; Optionally, the concentration of 5-fluorouracil in the second mixed solution is 8 mg / ml; Optionally, the pH of the PBS buffer is 7.

8.

15. The method according to claim 9, characterized in that The pH is adjusted by 0.2 mol NaOH; Optionally, the pH after adjusting the pH is 7.0 to 8.0; Optionally, the pH after adjusting the pH is 7.4 to 7.8; Preferably, the pH after adjusting the pH is 7.6; The water bath incubation temperature is 40-70°C; Preferably, the water bath incubation temperature is 60°C; Optionally, the water bath incubation time is 15 to 95 min; Preferably, the water bath incubation time is 60 min.

16. A drug liposome, characterized in that: It is prepared by the method described in any one of 8 to 15.

17. Use of the composite liposome according to any one of claims 1 to 4 or the drug liposome according to any one of claims 5 to 6 and 16 in the preparation of a drug for preventing or treating digestive system cancer, primary liver cancer or breast cancer.

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