Felodipine liposome, and preparation and use thereof

Abstract

A felodipine liposome, and the preparation and use thereof. The felodipine liposome comprises felodipine, a phospholipid and cholesterol, wherein the mass ratio of the felodipine to the phospholipid is 1:8 to 1:20; and the mass ratio of the phospholipid to the cholesterol is 5:1 to 20:1. Furthermore, the felodipine liposome may further contain octadecylamine, wherein the mass ratio of the cholesterol to the octadecylamine is 1:1 to 1:3. The use of the felodipine or felodipine liposome or felodipine liposome lyophilized powder in the preparation of an anti-tumor drug. The tumor is colorectal cancer, liver cancer, ovarian cancer or breast cancer, and in particular, the felodipine or felodipine liposome or felodipine liposome lyophilized powder exhibits the most potent inhibitory effect on colorectal cancer.

Description

Felodipine liposome and preparation and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to Felodipine liposome and preparation and application thereof. BACKGROUND

[0002] Felodipine is a calcium channel blocker developed by Astra Company, which is a dihydropyridine calcium channel blocker (CCB) capable of inhibiting the activity of cardiovascular muscle membrane calcium ions, continuously controlling the excitation-contraction coupling, expanding peripheral blood vessels, thereby reducing blood pressure, and continuously expanding the level of glomerular arterioles, and is mainly used for the treatment of cardiovascular and cerebrovascular diseases such as hypertension and angina pectoris. The drug has the advantages of high vascular selectivity, can significantly improve the perfusion rate of the kidney, and can effectively reduce the damage to the target organ.

[0003] At present, the marketed products of Felodipine are oral preparations, including ordinary tablets, sustained-release tablets, sustained-release capsules and controlled-release preparations. In June 1996, the company developed a sustained-release preparation of the product and marketed it under the trade name Plendil TM ), with a molecular formula of C 18 H 19 Cl2NO4, a molecular weight of 384.25400, and a structural formula as follows:

[0004] Felodipine directly and selectively acts on arteriolar vascular smooth muscle cells to inhibit the influx of extracellular Ca 2+ , reduce resistance blood vessels, and dilate arterioles, and is therefore widely used in the treatment of hypertension in the clinic. The drug has high selectivity for blood vessels, and under the conventional dose, by acting on the peripheral arteries, the whole body vascular resistance is reduced, the perfusion rate of the kidney is significantly improved, the blood pressure is reduced, and the damage to the target organ is effectively reduced. Felodipine has been widely used in the treatment of various types of hypertension and congestive heart failure. However, the side effects of Felodipine are severe hypotension and bradycardia, as well as mild to moderate edema of the foot and ankle, facial flushing, headache, dizziness, palpitation and fatigue. Therefore, for elderly patients, treatment drug monitoring means needs to be taken.

[0005] Felodipine belongs to the typical biopharmaceutics classification system class II drug, which has the characteristics of low solubility and high permeability. The low solubility is the main reason for the poor water solubility of such compounds. Felodipine is completely absorbed orally, but there is serious first-pass elimination, and the bioavailability is only 20%, thereby limiting the clinical application of Felodipine.

[0006] It has been a great challenge to solve the formulation problem of poorly soluble drugs such as felodipine. Surfactants are widely used to increase drug solubility, but they generally increase the drug's side effects. Currently, the strategy for increasing the solubility of poorly soluble drugs is mainly divided into three categories: ion pairs, amphiphilic materials can increase the solubility; nano-pulverization, nano-crystallization and other methods can change the physical state of the drug; nano-carrier delivery drug.

[0007] In the exploration of the solubilization strategy of felodipine, many studies have been devoted to changing the traditional tablets to new dosage forms that can improve their solubility and bioavailability. Researchers have applied solid dispersion, cyclodextrin inclusion, liposomes and proliposomes, self-emulsifying drug delivery system, microemulsion and other dosage forms; in addition, osmotic pump tablets, sustained-release pellets, in-situ gel can prolong the action time of the drug in the body. Reducing the particle size of the raw material is also a significant solubilization strategy, for example, when the particle size of felodipine tablets is reduced to 1 / 8, its bioavailability increases by nearly 1.5 times, and the bioavailability of nano-suspension and solid dispersion can be increased by 2-3 times. Nanoscale drugs or nanocrystals can increase the dissolution kinetics of poorly soluble drugs at a fast enough speed, so that drug molecules are continuously released during the dissolution process, thereby improving the water solubility of poorly soluble drugs.

[0008] Liposomes, as one of the most successful nano-preparations, have a structure similar to cell membranes, with the hydrophilic head of the bilayer forming the inner surface of the membrane, and the lipophilic tail being in the middle of the membrane. This structure of liposomes can encapsulate a variety of hydrophilic and lipophilic substances, which can improve the solubility of drugs in aqueous media.

[0009] Therefore, felodipine can be prepared into liposomes to improve the solubility of felodipine and thereby improve its bioavailability. SUMMARY

[0010] In order to overcome the defects of the prior art, the present application provides a felodipine liposome, which can significantly improve the bioavailability of felodipine, and the felodipine liposome can also significantly improve the anti-tumor activity of felodipine.

[0011] The present application is realized by the following technical solutions:

[0012] The present application provides a felodipine liposome, which comprises felodipine, phospholipid and cholesterol, the mass ratio of the felodipine to the phospholipid is 1:8-1:20, and the mass ratio of the phospholipid to the cholesterol is 5:1-20:1.

[0013] Further, the felodipine liposome further comprises octadecylamine, and the mass ratio of the cholesterol to the octadecylamine is 1:1-1:3.

[0014] The phospholipid is selected from the group consisting of egg yolk lecithin, hydrogenated soybean phospholipid, egg yolk phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, soybean lecithin, egg yolk sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and the like.

[0015] Further, the non-felodipine liposome contains 40-60 mg of phospholipid, 2-6 mg of cholesterol, 4-5 mg of non-felodipine, and 0-6 mg of octadecylamine per 5 mL of liposome.

[0016] Preferably, the non-felodipine liposome contains 40-60 mg of phospholipid, 2-4 mg of cholesterol, and 4-5 mg of non-felodipine per 5 mL of liposome.

[0017] The mass ratio of non-felodipine to phospholipid is 1:10-1:20, preferably 1:15-1:20.

[0018] The mass ratio of phospholipid to cholesterol is 5:1-20:1, preferably 15:1-20:1.

[0019] Preferably, the non-felodipine liposome contains 40-60 mg of phospholipid, 2-6 mg of cholesterol, 4-5 mg of non-felodipine, and 2-6 mg of octadecylamine per 5 mL of liposome.

[0020] The mass ratio of non-felodipine to phospholipid is 1:8-1:16, preferably 1:8-1:12.

[0021] The mass ratio of phospholipid to cholesterol is 20:1-20:3.

[0022] The mass ratio of cholesterol to octadecylamine is 1:1-1:3.

[0023] Further, the present application provides a preparation method of non-felodipine liposome, comprising the following steps:

[0024] (1) Weigh the prescribed amount of phospholipid, cholesterol, non-felodipine, and add or not add octadecylamine to a tomato-shaped flask, add anhydrous ethanol, vortex, and dissolve by ultrasonic for 10 min.

[0025] (2) Form a thin film of lipids in the bottle by vacuum rotary evaporation, continue vacuum rotary evaporation for 1 h to obtain a uniform thin film.

[0026] (3) Add deionized water, vortex, and hydrate after ultrasonic, and finally obtain a non-felodipine liposome solution under the probe ultrasonic at 300-350 W.

[0027] In the step (1), the concentration of the phospholipid in the anhydrous ethanol is 8-10 mg / mL.

[0028] In the step (3), the hydration temperature is 45-55℃, preferably 45-50℃.

[0029] In the step (3), the hydration time is 30-50min.

[0030] In the step (3), the probe is ultrasonated for 10-20min, works for 2s and stops for 1s.

[0031] The non-felodipine liposome of the present application can be added with a freeze-drying protective agent to prepare a non-felodipine liposome freeze-dried powder, and the freeze-drying protective agent is selected from one or more of mannitol, dextran, sucrose and trehalose. Preferably, the freeze-drying protective agent is trehalose, and the mass ratio of trehalose to phospholipid is 2:1-3:1.

[0032] The present application also provides the use of the non-felodipine liposome or the non-felodipine liposome freeze-dried powder in improving the bioavailability of a drug.

[0033] The present application also provides the use of non-felodipine or the non-felodipine liposome or the non-felodipine liposome freeze-dried powder in preparing an anti-tumor drug.

[0034] The tumor is colorectal cancer, liver cancer, ovarian cancer or breast cancer, preferably colorectal cancer.

[0035] The present application has the following beneficial technical effects:

[0036] The present application provides a non-felodipine liposome, and the preparation method of the liposome is simple, and the prepared liposome can improve the bioavailability of non-felodipine. Further, the addition of octadecylamine to the liposome modifies the phospholipid material, and the quaternary ammonium salt cation on the liposome surface makes the liposome surface positively charged, which affects the potential and stability of the liposome. Moreover, the surface of the cationic liposome is positively charged, and can wrap the negatively charged genes or drugs through electrostatic action. The positive charge also endows the cationic liposome with the ability to penetrate the biological barrier. After malignant transformation, the membrane of cancer cells changes, and the negatively charged components such as phosphatidylserine, proteoglycan and membrane protein in the outer layer of the plasma membrane are much higher than those of normal cells. The cationic liposome has higher targeting property to the membrane of cancer cells because of the positive charge. Experimental results show that the non-felodipine liposome of the present application can significantly improve the bioavailability of non-felodipine. The non-felodipine cationic liposome prepared by adding octadecylamine has the best release effect, and the pH at the tumor site is acidic. After reaching the tumor site, the drug is released more completely, and can reach a greater drug treatment concentration. The non-felodipine cationic liposome has better tumor targeting property and significant anti-tumor activity. BRIEF DESCRIPTION OF DRAWINGS

[0037] Fig. 1 is the effect of the drug-lipid ratio on the encapsulation efficiency and particle size of the non-felodipine liposome (n=3).

[0038] Figure 2 is the effect of the amount of cholesterol on the encapsulation efficiency and particle size of felodipine liposomes (n = 3).

[0039] Figure 3 is the effect of hydration temperature on the encapsulation efficiency and particle size of felodipine liposomes (n = 3).

[0040] Figure 4 is the in vitro release curve of felodipine liposomes and felodipine raw material.

[0041] Figure 5 is the effect of felodipine liposomes on the survival rate of human kidney epithelial cells HEK-293 cells.

[0042] Figure 6 is the drug-time curve of SD rats after oral administration of felodipine liposomes and tablets (n = 6).

[0043] Figure 7 is the in vitro release of the felodipine liposomes of Example 8;

[0044] A: Release of the dissolution medium is 0.30% SDS solution;

[0045] B: Release of the dissolution medium is 1.0% Tween 80-pH 5.8 PBS buffer-20% anhydrous ethanol solution;

[0046] Data are expressed as Mean ± SD; n = 3; **P < 0.01, ***P < 0.001.

[0047] Figure 8 is the effect of each experimental group on the viability of mouse COC cells detected by CCK-8 experiment;

[0048] A, B Felodipine, FLD-Lip-Nps - and FLD-Lip-Nps + on the viability of CT26 cells;

[0049] C, D Felodipine, FLD-Lip-Nps - and FLD-Lip-Nps + on the viability of HCT116 cells;

[0050] E, F Felodipine, FLD-Lip-Nps - and FLD-Lip-Nps + on the viability of HT29 cells;

[0051] Data are expressed as Mean ± SD; n = 3; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0052] Figure 9 is the effect of each experimental group on the viability of human tumor cells detected by CCK-8 experiment;

[0053] A, B felodipine, FLD-Lip-Nps - and FLD-Lip-Nps + Effect on HUH7 cell viability;

[0054] C, D felodipine, FLD-Lip-Nps - and FLD-Lip-Nps + Effect on OVCAR3 cell viability;

[0055] E, F felodipine, FLD-Lip-Nps - and FLD-Lip-Nps + Effect on MCF7 cell viability;

[0056] Data are presented as Mean ± SD; n = 3; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0057] Figure 10 is a Blank-Lip-Nps + Safety evaluation in HEK293 and CT26 cells;

[0058] A Blank-Lip-Nps + and PEI on HEK293 cell viability; B Blank-Lip-Nps + and PEI on CT26 cell viability; Data are presented as Mean ± SD; n = 3; ****P < 0.0001.

[0059] Figure 11 is a cell morphology of CT26 cells uptake of C6-FLD-Lip-Nps - , C6-FLD-Lip-Nps + ;

[0060] Green represents C6-loaded nanofomulation, blue represents DAPI-stained nucleus;

[0061] Figure A, B is a C6-FLD-Lip-Nps - treated group, the scale is 100 pm, 10 pm, respectively;

[0062] Figure C, D is a C6-FLD-Lip-Nps + treated group, the scale is 100 pm, 10 pm, respectively.

[0063] Figure 12 is the effect of felodipine, FLD-Lip-Nps + on CT26 cell migration;

[0064] A. Cell morphology under an inverted microscope; B. Cell migration rate in different treatment groups; Data are expressed as Mean ± SD; n = 3; **P < 0.01.

[0065] Figure 13 shows felodipine and FLD-Lip-Nps. + Inhibits apoptosis in CT26 cells;

[0066] A. Apoptosis of CT26 cells in different drug administration groups; B. Statistical graph of total apoptosis rate of cells in different drug administration groups;

[0067] Data are expressed as Mean±SD; n=3; *P<0.05; **P<0.01.

[0068] Figure 14 shows physiological saline, FLD, and FLD-Lip-Nps. + Inhibition of tumor growth in mice after drug administration; data are expressed as mean ± SD; n = 8.

[0069] Figure 15 shows physiological saline, FLD, and FLD-Lip-Nps. + Survival time curves of CT26 tumor-bearing mice after drug administration; data are expressed as mean ± SD; n = 8; * P < 0.05; *** P < 0.001.

[0070] Figure 16 shows the serum biochemical analysis of mice in each treatment group;

[0071] A: AST index statistics of CT26 tumor-bearing mice; B: ALT index statistics of CT26 tumor-bearing mice;

[0072] C: Statistical chart of UREA index in CT26 tumor-bearing mice; D: Statistical chart of CREA index in CT26 tumor-bearing mice; Data are expressed as Mean±SD; n=3.

[0073] Figure 17 shows the particle size and potential of liposomes containing octadecylamine;

[0074] Figure 18 shows different concentrations of octadecylamine-containing cationic liposomes (Lip-Nps). + Adsorption of FAP plasmids; Lip-Nps + FAP-DNA (μl:μg)

[0075] 1-10:4, 2-8:4, 3-6:4, 4-4:4, 5-0:4

[0076] Figure 19 shows the transfection of DC2.4 cells with different concentrations of liposome-FAP plasmid.

[0077] Data are expressed as Mean ± SD; n = 3, *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0078] Example 1 Formulation screening of felodipine liposome

[0079] A certain amount of egg yolk lecithin, 4 mg of cholesterol, and 4 mg of felodipine were weighed into a 100 mL brown pear-shaped flask, and anhydrous ethanol was added to dissolve the egg yolk lecithin, so that the concentration of the egg yolk lecithin in ethanol was 8 mg / mL. After vortexing for 10 min, the solution was ultrasonically treated in a water bath for 10 min to completely dissolve the solution. The lipids were then formed into a thin film in the flask by rotary evaporation under reduced pressure. The lipids were then dried by continuing rotary evaporation for 1 h, and then placed in a vacuum desiccator overnight. After adding 5 mL of distilled water and ultrasonically treating for 10 min, the solution was vortexed for 10 min. The solution was then ultrasonically treated for 10 min at 300 W, with 1 s of work and 1 s of rest.

[0080] The entrapment efficiency of felodipine liposomes was determined by low-speed centrifugation. After centrifuging 1 mL of felodipine liposomes at 8000 rpm for 30 min, 200 μL of supernatant was diluted to 3 mL, and methanol was added to break the emulsion for 30 min. The entrapment efficiency was calculated according to the following formula. 包 总 × 100%

[0081] wherein, W 包 and W 总 represent the drug content in the liposomes and the total drug content, respectively.

[0082] (1) The effect of the ratio of drug to phospholipid on the particle size and entrapment efficiency of felodipine liposomes:

[0083] The amount of drug was kept constant, and the amount of phospholipid was changed to adjust the ratio of drug to phospholipid to 1:5, 1:10, 1:15, and 1:20. The liposomes were prepared under the same conditions, and the changes in particle size and entrapment efficiency were observed. The results are shown in Figure 1. The results showed that as the amount of phospholipid in the felodipine liposomes increased, the particle size of the liposomes decreased, and the entrapment efficiency increased. When the drug-phospholipid ratio was 1:10-1:20, the entrapment efficiency was high, and the particle size was small. When the drug-phospholipid ratio was 1:15-1:20, the entrapment efficiency was high, and the particle size was the smallest. When the drug-phospholipid ratio was 1:15, the entrapment efficiency was the highest, and the particle size was the smallest. Therefore, the ratio of drug to phospholipid in the formulation was determined to be 1:15.

[0084] (2) The effect of the amount of cholesterol on the particle size and entrapment efficiency of felodipine liposomes:

[0085] ​The drug amount was not changed, the ratio of drug and phospholipid was 1:15, the mass ratio of phospholipid and cholesterol was adjusted to 20:1, 15:1, 10:1, 5:1, and other conditions were not changed, liposomes were prepared, and the changes of particle size and encapsulation efficiency were observed. The results are shown in Figure 2. The results showed that when the mass ratio of phospholipid and cholesterol was 20:1-5:1, the particle size was less than 150 nm, and the encapsulation efficiency was greater than 95%. When the mass ratio of phospholipid and cholesterol was 15:1, the particle size was the smallest, and the difference in encapsulation efficiency was not obvious. Under the premise of ensuring good encapsulation efficiency and the smallest particle size of liposomes, the optimal mass ratio of added phospholipid and cholesterol was 15:1.

[0086] (3) Effect of hydration temperature on the particle size and encapsulation efficiency of felodipine liposomes:

[0087] The drug amount was not changed, the ratio of drug and phospholipid was 1:15, the mass ratio of phospholipid and cholesterol was 15:1, and other conditions were not changed. The hydration temperature was adjusted to 45℃, 50℃, 55℃, 60℃, liposomes were prepared, and the changes of particle size and encapsulation efficiency were observed. The results are shown in Figure 3. The results showed that the hydration temperature had little effect on the encapsulation efficiency and particle size. Considering that high temperature may damage liposomes, the hydration temperature was selected to be 50℃.

[0088] (4) Effect of hydration medium type on the particle size and encapsulation efficiency of felodipine liposomes:

[0089] The drug amount was not changed, the ratio of drug and phospholipid was 1:15, the mass ratio of phospholipid and cholesterol was 15:1, and other conditions were not changed. The hydration temperature was 50℃, and the effect of hydration medium, distilled water, pH=7.4 PBS buffer, and pH=6.5 PBS buffer, on the particle size and encapsulation efficiency of liposomes was investigated. The results showed that the liposomes prepared with pH=7.4 PBS buffer and pH=6.5 PBS buffer as hydration medium were precipitated. Therefore, distilled water was selected as the hydration medium.

[0090] Example 2 Preparation of felodipine liposomes

[0091] 60 mg of egg yolk phospholipid, 4 mg of cholesterol, and 4 mg of felodipine were precisely weighed into a 100 mL brown pear-shaped flask, and then dissolved in anhydrous ethanol to fix the concentration of egg yolk phospholipid in ethanol at 8 mg / mL. After vortexing for 10 min, water bath ultrasonic was performed for 10 min to completely dissolve the mixture. Then, the lipids were formed into a thin film in the flask by rotary evaporation under reduced pressure. Subsequently, the lipids were dried by continuing rotary evaporation for 1 h. The dried lipids were placed in a vacuum dryer overnight. 5 mL of distilled water was added, and vortexing was performed for 10 min after ultrasonic for 10 min. Finally, hydration was performed, and probe ultrasonic was continued for 10 min under the condition of 300 W, 1 s of work, and 1 s of off.

[0092] Example 3 Particle size and potential of the felodipine liposome of Example 2

[0093] The particle size, Zeta potential and PDI of the felodipine liposome prepared in Example 2 were determined by dynamic light scattering technology. Each sample was determined in triplicate, and the average particle size, Zeta potential and PDI of the sample were recorded. The average particle size was 119.3 nm, the PDI value was 0.286, and the potential value was -28.57 mV.

[0094] Example 4 In vitro release of the felodipine liposome of Example 2

[0095] According to the Chinese Pharmacopoeia 2020 edition, the release of the felodipine liposome of Example 2 was determined by paddle method,

[0096] and the commercially available felodipine raw material was used as a reference. The prepared felodipine liposome (about 2.5 mg of felodipine) and felodipine (about 2.5 mg) were loaded into a pre-treated dialysis bag (molecular weight cut-off of 3500 Da), and the dialysis bag was clamped at both ends with a clamp, and then placed in 100 mL of dissolution medium at a temperature of 37 ± 0.5°C, with a rotation speed of 100 rpm. 5 mL of sample was taken at 0.5, 1, 2, 3, 4, 5, 6, 8, 10 and 24 h (while supplementing the same temperature and the same amount of release medium), and then filtered with a 0.22 μm microporous filter and injected into HPLC for determination. Each group of experiments was performed in triplicate, and the cumulative release at each time point was calculated. The release time was taken as the abscissa, and the cumulative release percentage was taken as the ordinate, and the in vitro release curve was drawn. The results are shown in Figure 4.

[0097] From the release curve, it can be seen that the felodipine liposome group is in the rapid release period from 3 to 6 h, and in the slow release period from 6 to 24 h. The cumulative release amount rises rapidly in 3-6 h due to the rapid release of felodipine in the liposome, and the release curve is flat in 6-24 h due to the slow diffusion of felodipine into the release medium. The cumulative release of the felodipine liposome group can reach about 45% at 8 h, while the cumulative release of the felodipine raw material is less than 5%. Moreover, the felodipine liposome can continuously accumulate release from 8 h to 48 h. Therefore, the felodipine liposome has a certain delay or sustained release effect, and the cumulative release amount of the felodipine liposome is significantly higher than that of the felodipine raw material. Therefore, the felodipine liposome improves the bioavailability of felodipine.

[0098] Example 5 Effect of felodipine liposome on the survival rate of human renal epithelial cells HEK-293

[0099] CCK-8 method was used to detect the effect of felodipine raw material, blank liposome and the liposome of Example 2 on the survival rate of human renal epithelial cells HEK-293. Human embryonic kidney cells HEK-293 in logarithmic growth phase were digested with trypsin containing EDTA, and after termination of digestion, the cells were collected by centrifugation, diluted with 1 ml of DMEM medium containing 10% FBS and 1% double antibody, and the number of cells was calculated. The counted cell suspension was added to a 96-well plate at 5x10 3 After the cells grew well, the raw material of felodipine, blank liposome and felodipine liposome with a concentration range of 20, 40, 80, 120, 160 and 200 μM were used for treatment, 4 replicates for each concentration group, and 4 PBS groups were set as blank controls, and 4 zero drug groups were set as negative controls, and then incubated at 37°C in a 5% CO2 incubator for 24 h. After 24 h of action, the drug-containing culture solution was removed by changing the solution, 10 μL of CCK-8 and 90 μL of incomplete culture solution were added to each well, and incubated for 1 h. The OD value at 450 nm was detected by a microplate reader, and the cell survival rate was calculated according to the formula. The experiment was repeated three times, and the whole process of cell culture was kept sterile, and all the related reagents and instruments used were treated by high temperature sterilization or alcohol disinfection.

[0100] Cell viability (%) = (OD 实验组 - OD 空白组 ) / (OD 对照组 - OD 空白组 ) x 100%

[0101] The experimental results are shown in Figure 5, which shows that the toxicity of the felodipine liposome to HEK-293 cells is lower than that of the raw material of felodipine, indicating that the safety of the drug is increased after the preparation of the liposome.

[0102] Example 6 In vivo pharmacokinetic study of rats

[0103] 1. Sample processing method

[0104] Experimental group: felodipine liposome of Example 2

[0105] Control group: felodipine commercial tablet, Beijing Union Pharmaceutical Factory. The felodipine commercial tablet was prepared into a suspension.

[0106] Felodipine dose for rats: 5 mg / kg.

[0107] 2. Dosing regimen

[0108] Twelve SPF level cultured SD rats (about 300 g) were randomly divided into two groups, A and B, with 6 rats in each group, half male and half female, and fasted for 12 h before administration. The next day at 8:00, the A group was given non-felodipine liposomes and the B group was given non-felodipine commercial tablets by gavage. Water was available 2 h after administration, and food was given uniformly 4 h later.

[0109] 3. Collection of plasma samples

[0110] Gavage time: 8:00 am, blood collection time: 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48 h, blood collection method: about 0.5 mL of blood was taken from the canthus at each time point, and then the blood sample was immediately placed in a dry centrifuge tube treated with sodium heparin. Centrifuge at 4000 rpm for 10 min to separate the plasma, take the supernatant, and store at -20℃ for testing.

[0111] 4. Processing of experimental data

[0112] Data analysis was performed using Winnonlin 8.1 to calculate the time to peak (T max ), peak concentration (C max ), elimination half-life (T 1 / 2 ), area under the plasma concentration-time curve (AUC 0-∞ ), and mean residence time (MRT).

[0113] After gavage with non-felodipine liposomes and non-felodipine tablets, the concentration of non-felodipine in the plasma of normal rats was detected, the main pharmacokinetic parameters were calculated using Winnonlin 8.1, and the drug concentration-time curve of FDP in the plasma of normal rats was plotted using GraphPad Prism 8, as shown in Table 1 and Figure 6. The maximum concentration (C max ) and area under the curve (AUC 0-∞ ) of the non-felodipine liposome group were significantly greater than those of the non-felodipine tablet group, 3.27 times and 1.97 times, respectively, indicating that non-felodipine liposomes increased the total amount of drug absorption in vivo and increased the bioavailability. Although the T max and mean residence time (MRT) of the liposome group were smaller than those of the commercial tablet group, the difference was not large, and they could still function for a relatively long time.

[0114] Table 1 Main pharmacokinetic parameters of non-felodipine liposomes Table 1 Main pharmacokinetic parameters of non-felodipine liposomes

[0115] a h, b ng / mL, c h·ng / mL, d mL / kg, e mL / h / kg

[0116] Example 7 Formulation screening of felodipine cationic liposome

[0117] The prescribed amount of phospholipid, cholesterol, felodipine and octadecylamine were weighed into a 100 mL round flask, 5 mL of anhydrous ethanol was added, vortexed, and dissolved by ultrasonic for 10 min. The solution was evaporated under vacuum at 50 rpm and 45°C until completely evaporated, and then the temperature was raised to 50°C and the evaporation was continued under vacuum for 1 h to obtain a uniform film. 5 mL of deionized water was added, vortexed, and dissolved by ultrasonic for 5 min. Subsequently, the solution was hydrated at 50°C for 30 min, and then sonicated at 330 W for 10 min with a probe sonicator, with a working time of 2 s and a pause of 1 s. The amount of each component in the formulation was changed to investigate the effect of the amount of each component on the particle size and encapsulation efficiency of the liposome. The results are shown in Tables 2-4.

[0118] Table 2 Screening of phospholipid ratio (mg) (n = 3)

[0119] Table 3 Screening of cholesterol ratio (mg) (n = 3)

[0120] Table 4 Screening of octadecylamine ratio (mg) (n = 3)

[0121] The results show that when the mass ratio of felodipine to phospholipid is 1:8-1:16, the particle size of the liposome is less than 250 nm and the encapsulation efficiency is greater than 90%; when the mass ratio of felodipine to phospholipid is 1:8-1:12, the particle size is less than 200 nm and the encapsulation efficiency is greater than 95%; when the mass ratio of phospholipid to cholesterol is 20:1-20:3, the particle size is less than 200 nm and the encapsulation efficiency is greater than 90%; and when the mass ratio of cholesterol to octadecylamine is 1:1-1:3, the particle size is less than 200 nm and the encapsulation efficiency is greater than 95%.

[0122] Based on the above results, the most preferred mass ratio of felodipine to phospholipid is 1:8-1:12, the mass ratio of phospholipid to cholesterol is 20:1-20:3, and the mass ratio of cholesterol to octadecylamine is 1:1-1:3, in which case the particle size of the felodipine liposome is less than 200 nm and the encapsulation efficiency can be greater than 95%.

[0123] Example 8 Preparation of felodipine liposome (FLD-Lip-Nps + )

[0124] Weigh 40 mg of phospholipid, 2 mg of cholesterol, 5 mg of felodipine, and 2 mg of octadecylamine into a 100 mL round-bottom flask. Add 5 mL of anhydrous ethanol, vortex, and sonicate for 10 min to dissolve. Vacuum rotary evaporate at 50 rpm and 45 °C until the solution is completely evaporated. Raise the temperature to 50 °C and continue vacuum rotary evaporation for 1 h to obtain a uniform film. Add 5 mL of deionized water, vortex, and sonicate for 5 min to dissolve. Subsequently, hydrate at 50 °C for 30 min, and sonicate at 330 W with a probe for 10 min, working for 2 seconds and pausing for 1 second.

[0125] Example 9: Preparation of Felodipine Liposome Lyophilized Powder

[0126] Using dextran and trehalose as freeze-drying protectants, 1 mL of the felodipine liposome solution prepared according to the method in Example 8 was taken, and dextran and trehalose were added respectively at glycolipid ratios of 1:1, 2:1, and 3:1, and vortexed to dissolve. The solution was transferred to a brown vial, frozen overnight at -80°C, and then freeze-dried for 48 hours to obtain a dried sample. The sample was reconstituted with 1 mL of deionized water, diluted 10-fold, and the encapsulation efficiency, particle size, and potential were measured.

[0127] The results showed that when trehalose was added and the sugar-lipid ratio was 2:1-3:1, the particle size, potential and encapsulation efficiency of the freeze-dried nano-formulation did not change much before and after the addition, which could protect the stability of the nano-formulation.

[0128] Example 10: In vitro release of felodipine liposomes

[0129] Accurately weigh 3.0 mg of felodipine and dissolve it in 3 mL of 0.3% SDS solution and 1.0% Tween 80-pH 5.8 PBS buffer-20% anhydrous ethanol medium, respectively; separately take 3 mL of FLD-Lip-Nps from Example 8. + Solution: Dissolve commercially available tablets containing 5 mg felodipine in 5 mL of either of the above two media solutions, then place them into an 8–10 KD dialysis bag and clamp both ends. Place the bag into 100 mL of 0.30% SDS solution and 1.0% Tween 80-pH 5.8 PBS buffer-20% anhydrous ethanol dissolution medium, respectively. Shake at 37°C and 100 rpm on a constant temperature shaker. Take 1 mL samples at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, and 72 h, and add 1 mL of media solution. The results are shown in Figure 7.

[0130] As shown in Figure 7, as illustrated in Figure A, felodipine and FLD-Lip-Nps... +The cumulative release amounts of felodipine tablets in this dissolution medium for 72h were 15.13%, 16.59%, and 14.26%, respectively, and the release amounts were not much different and the drug could not be normally released. Therefore, as shown in Figure B, using 1.0% Tween 80-pH 5.8 PBS buffer-20% anhydrous ethanol as the dissolution medium, the release amounts of felodipine, FLD-Lip-Nps + The cumulative release amounts of felodipine tablets in this dissolution medium for 72h were 59.81%, 96.44%, and 64.44%, respectively, and the cumulative release amount of the drug for 72h was statistically analyzed. The release amount of FLD-Lip-Nps + was obviously stronger than that of felodipine and felodipine tablets ** (P<0.01, *** P<0.001), and it can be seen that the liposome can obviously enhance the release of felodipine and has a certain sustained-release effect.

[0131] Example 11 Anti-tumor activity of felodipine liposome

[0132] 1. Cell strains: CT26, HEK293, HT29, HCT116, HUH7, OVCAR3, and MCF7 cell strains were purchased from the China Academy of Sciences Cell Bank.

[0133] 2. Experimental animals: 7-week-old Balb / c mice, female, weighing 20±2g, SPF level, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0134] 3. Experimental method:

[0135] (1) Solution preparation:

[0136] Felodipine stock solution: 3.84mg of felodipine was weighed (precisely weighed), and 200μL of DMSO solution was added for dissolution.

[0137] FLD-Lip-Nps + : Example 8.

[0138] FLD-Lip-Nps - : The scheme of Example 8 without octadecylamine and with the same other conditions.

[0139] CCK-8 solution: CCK-8 stock solution and incomplete culture solution were prepared according to 1:10.

[0140] (2) CCK-8 method for measuring the effects of FLD, FLD-Lip-Nps - and FLD-Lip-Nps + on the activity of mouse CT26 cells, human HCT116 and HT29 cells

[0141] Logarithmic growth phase cells were inoculated in 96-well plates at 5x10 3 cells / well with 100 μL complete medium and incubated at 37 °C, 5% CO2 for 24 h. The above solution was diluted to the desired concentration gradient with complete medium, the solution in the 96-well plate was discarded, 100 μL drug solution was added to each well, shaken, and incubated at 37 °C, 5% CO2 for 24 h. The solution in the 96-well plate was discarded, 100 μL CCK-8 solution was added to each well, shaken, and incubated at 37 °C for 30 min. The OD value was measured at 450 nm using a microplate reader, the cell survival rate was calculated, the inhibition curve was drawn, and the IC 50 value was calculated.

[0142] Feldipine, FLD-Lip-Nps - and FLD-Lip-Nps + were used to treat CT26, HCT116 and HT29 cells at a certain gradient concentration for 24 h.

[0143] The results showed that the cell activity of the three cells decreased with the increase of drug concentration, and the effect of FLD-Lip-Nps + on the activity of the three COC cells was significantly stronger than that of feldipine and FLD-Lip-Nps - (Fig. 8A, C, E), and the IC 50 values were 34.78, 27.77 and 34.15 μM, respectively (Fig. 8B, D, F). In addition, HCT116 and HT29 had certain drug resistance to FLD-Lip-Nps - The above results may be due to the better solubility of the drug in FLD-Lip-Nps + and the easier combination of FLD-Lip-Nps + with the anion and phosphatidylserine (PS) on the cell membrane surface, thereby improving the drug efficacy.

[0144] (3) CCK-8 method for detecting the effect of feldipine, FLD-Lip-Nps - and FLD-Lip-Nps + on the activity of human tumor cells

[0145] The effect of feldipine, FLD-Lip-Nps - and FLD-Lip-Nps + on the activity of human hepatoma HUH7, ovarian cancer OVCAR3 and breast cancer MCF7 cells was detected according to the method of (2).

[0146] The results showed that with the increase of drug concentration, FLD-Lip-Nps +The viability of the three kinds of cells decreased significantly (Fig. 9A, C, E), IC 50 The values were 32.65, 34.15, and 49.16 μM, respectively (Fig. 9B, D, F); felodipine had a greater effect on HUH7 and MCF7, but OVCAR3 showed a certain degree of drug resistance; FLD-Lip-Nps - The effects on the three cells were not ideal. The above results may be due to the fact that FLD-Lip-Nps + The drug solubility was better, and FLD-Lip-Nps + was more easily combined with anions and PS on the surface of the cell membrane, thereby improving the drug efficacy.

[0147] Example 12 Safety evaluation of felodipine liposomes

[0148] Polyetherimide (PEI) was used as a positive control, and the concentration gradient was set to 10, 20, 30, 40, 50, and 60 μg / mL. The safety of Blank-Lip-Nps + on CT26 and HEK293 cells was studied.

[0149] Cationic liposomes are commonly used as transfection reagents in cell studies and have a certain toxic effect on cells. In this experiment, the commonly used transfection reagent PEI was used as a positive control to investigate the safety of Blank-Lip-Nps + on cells. As shown in Fig. 10, Blank-Lip-Nps + The survival rate of HEK293 cells decreased with increasing concentration, and the cell viability was significantly stronger than that of the PEI treatment group at around 80%. Blank-Lip-Nps + had a lower effect on CT26 cells, and the cell survival rate was around 110%, which was also significantly stronger than that of the PEI treatment group. The results showed that Blank-Lip-Nps + had a certain safety on HEK293 cells and CT26 cells.

[0150] Example 13 Uptake of FLD-Lip-Nps - and FLD-Lip-Nps + by CT26 cells

[0151] Precise weighing of phospholipid, cholesterol, felodipine (mass ratio 40:2:5) in 100 mL of a tomato-shaped flask, cationic liposome additionally added 2 mg of octadecylamine, dissolved in 5 mL of anhydrous ethanol solution, vortexed and ultrasonicated for 10 min, added 30 μL of C6 solution (0.5 mg / mL) per portion. Evaporated under vacuum at 50 rpm and 45°C, after the solution was completely evaporated, the temperature was raised to 50°C and vacuum evaporation was continued for 1 h to obtain a uniform film. Added 5 mL of deionized water, vortexed and ultrasonicated for 5 min, continued to hydrate at 50°C for 30 min, ultrasonicated at 330 W for 10 min, probe 2 s, stop 1 s, finally obtained C6-FLD-Lip-Nps - and C6-FLD-Lip-Nps + , the whole process needs to avoid light operation.

[0152] Cells in the logarithmic phase of growth were inoculated in a confocal dish, 1.6×10 5 cells / well, cultured with 1 mL of complete medium at 37°C, 5% CO2 for 24 h, the culture solution was discarded, replaced with 200 μL of incomplete medium, added 4 μL of C6-FLD-Lip-Nps - solution, 4 μL of C6-FLD-Lip-Nps + solution, continued to culture at 37°C, 5% CO2 for 2 h; then washed with 1 mL of PBS solution for 3 times to remove the residual culture solution, added 200 μL of 4% paraformaldehyde solution, fixed for 20 min, then continued to wash with 1 mL of PBS solution for 3 times; added 100 μL of DAPI dye, placed for 5 min, then washed with 1 mL of PBS solution for 3 times, finally added 200 μL of PBS solution, and photographed under a confocal microscope.

[0153] The cell morphology chart photographed under a confocal microscope after 2 h of drug treatment of CT26 cells is shown in Figure 11.

[0154] The results show that compared with anionic liposomes, the green fluorescence of cells is stronger after cationic liposome administration, indicating that cationic liposomes are more easily taken up by cells, thereby exerting a drug effect.

[0155] In the cell uptake experiment, C6 has green fluorescence, which enters the cytoplasm after being taken up by cells, and the cells also have green fluorescence, FLD-Lip-Nps + Because of the positive charge, it is easier to fuse with the negatively charged cell membrane surface, improving the efficiency of entering cells and the effect of drugs. In the study of the anti-tumor activity of near-infrared two-zone imaging drug-loaded liposomes, the analysis of liposome cell uptake behavior and the tumor cell inhibition experiment proved that octadecylamine can promote the uptake of liposomes by cells and synergistically enhance the anti-tumor effect with drugs.

[0156] Example 14 Felodipine and FLD-Lip-Nps + Effect on CT26 cell migration

[0157] Draw horizontal lines evenly on the back of the 6-well plate with a ruler, at least 5 lines per well. Cells in the logarithmic growth phase were inoculated in a 6-well plate, 6x10 5 cells / well, 1 mL of complete medium was used to culture at 37°C, 5% CO2 for 24 h, until the cell amount was 90%-100%. A 200 μL gun head was used to draw a line perpendicular to the horizontal line on the back of the plate, and the line was drawn evenly. 1 mL of PBS solution was used to wash 3 times to remove the cells that fell off, 1 mL of incomplete medium was added to the blank group, and 1 mL of 5 μM / L felodipine and FLD-Lip-Nps solution was added to the experimental group + , and the cells were placed in the incubator for a few minutes before taking pictures. After 24 h of continuous culture and observation, the cells were photographed again.

[0158] Effect of felodipine and FLD-Lip-Nps on cell migration was investigated by cell scratch test + , and the cells were photographed again. + + The results showed that FLD-Lip-Nps could significantly inhibit the migration of CT26 cells. + +

[0159] Example 15 Flow cytometry detection of felodipine and FLD-Lip-Nps + Effect on CT26 cell apoptosis

[0160] Cells in the logarithmic growth phase were inoculated in a 6-well plate, 5x10 5 cells / well, 2 mL of complete medium was used to culture at 37°C, 5% CO2 until about 80%, and blank cells, PI staining, FITC staining, control group, felodipine drug group (20, 40, 60 μM), FLD-Lip-Nps + ​​​Drug groups (20, 40, 60 μM), 1 mL per well, continue to culture at 37℃, 5% CO2 for 48 h. First, transfer the supernatant solution of each well into 5 mL centrifuge tube respectively, wash with 1 mL PBS solution for 2 times per well, add 200 μL trypsin without EDTA for digestion, terminate the digestion with 1 mL culture solution, transfer all the solution of each well into the above-mentioned 5 mL centrifuge tube respectively, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with 1 mL PBS solution, centrifuge at 1000 rpm for 5 min, discard the supernatant, repeat the washing for 2 times. Add 5 μL PI to the PI single staining tube, add 10 μL FITC to the FITC single staining tube, add 5 μL PI and 10 μL FITC to the control group and drug groups, mix well and stain for 15 min, add 400 μL Binding buffer, finally detect by flow cytometry.

[0161] Non-felodipine and FLD-Lip-Nps were detected by flow cytometry + The effect on cell apoptosis at 48 h, as shown in FIG. 13A, the early apoptosis percentage of CT26 cells induced by felodipine group at low, medium and high concentrations was 12.8%, 14.1% and 15.9% respectively; the late apoptosis percentage was 11.6%, 20.5% and 12.1% respectively; the total apoptosis percentage was 24.4%, 34.6% and 28.0% respectively. The early apoptosis percentage of CT26 cells induced by FLD-Lip-Nps + group at low, medium and high concentrations was 17.2%, 17.3% and 19.7% respectively; the late apoptosis percentage was 19.9%, 27.5% and 26.3% respectively; the total apoptosis percentage was 37.1%, 44.8% and 46.0% respectively. With the increase of concentration, the early apoptosis percentage and the total apoptosis percentage of felodipine group and FLD-Lip-Nps + group were gradually improved, and the apoptosis percentage of FLD-Lip-Nps + group was significantly higher than that of felodipine group. The difference analysis of total apoptosis rate of the two groups of cells was shown in FIG. 13B, and there was significant difference between low concentration group and high concentration group (*P<0.05, **P<0.01). The results showed that FLD-Lip-Nps + had significant apoptosis induction effect on CT26, and the effect was improved with the increase of concentration.

[0162] In the in vitro anti-tumor activity study of felodipine and FLD-Lip-Nps + , both of them induced apoptosis of CT26 cells and inhibited the migration of CT26 cells, but the solubilization effect of FLD-Lip-Nps + , the enhanced uptake of the negative charge of cationic liposome targeting the cell membrane surface and the combination with PS all made FLD-Lip-Nps +showed stronger anti-tumor activity in vitro.

[0163] Example 16 FLD and FLD-Lip-Nps + Evaluation of in vivo anti-tumor activity of CT26 tumor-bearing mice

[0164] 7-week-old Balb / c mice, female, weighing 20±2g, SPF level, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0165] FLD solution: accurately weigh FLD-Lip-Nps + Medium amount of FLD, dissolved with 10% DMSO, and then add physiological saline containing 2% Tween 80 (90%).

[0166] FLD-Lip-Nps + Aqueous solution: the solution of Example 8, after freeze-drying, reconstituted with deionized water, centrifuged to remove unencapsulated free drugs, and obtained.

[0167] 8-week-old Balb / c female mice, each injected with 1 million (100 μL) cell suspension on the right back, and the tumor growth of mice was observed and recorded. When the tumor volume reached about 100 mm 3 Intratumoral administration (V=length x width x width / 2) was started. The tumor volume of all mice was recorded, and divided into 3 groups, physiological saline, FLD, and FLD-Lip-Nps + Each group of 8 mice, on days 1, 3, 5, 7, and 9, administered 2 mg per mouse (FLD content), 60 μL per mouse. Before each day of administration, each mouse was weighed, and the change in tumor volume was recorded. On day 15, the mouse heart blood was taken, and the tumor, heart, liver, spleen, lung, and kidney were dissected and weighed. The anti-tumor effect of FLD different preparations was evaluated with body weight and tumor volume as the evaluation index.

[0168] CT26 tumor-bearing mice were administered physiological saline, FLD, and FLD-Lip-Nps + Intratumoral injection administration treatment, according to the change in tumor volume and the survival time of mice, to evaluate the in vivo anti-tumor effect of FLD and FLD-Lip-Nps + .

[0169] The tumor volume of mice was recorded daily and the tumor inhibition curve was plotted, the results showed that the tumor volume of the control group of mice grew rapidly, and on day 15, the average tumor volume reached 1288.28 mm 3 , greater than 1092.23 mm 3 of the FLD group and 809.01 mm + of the FLD-Lip-Nps 3(FIG. 14A); compared with the saline control group, FLD and FLD-Lip-Nps + The relative tumor volume (RTV) decreased after the end of the group treatment, FLD-Lip-Nps + showed effective anti-tumor activity, and the tumor growth inhibition value (TGI) was 32.20%, stronger than the TGI of the FLD group: 9.26%, FLD-Lip-Nps + showed better anti-tumor activity than the FLD group * P<0.05). The tumors of the mice in each group at the end of the experiment are shown in FIG. 14B. Although there were individual tumors with larger volumes in the preparation groups, the tumor volumes in the FLD-Lip-Nps + group were relatively smaller compared with the saline and FLD groups, indicating that FLD had certain anti-COC activity, and the anti-cancer activity of the preparation group was stronger than that of the raw material drug group.

[0170] Analysis and comparison of the body weight of the mice found that after 3 doses, the body weight of the mice in the FLD-Lip-Nps + group began to decrease, and after the end of the 5th dose, the body weight of the mice began to gradually increase, while the body weight of the mice in the control and FLD groups fluctuated less, and the body weight change rates of the mice in the three groups were 7.76%, 7.50%, and 6.03%, respectively, and the body weight of the mice showed an upward trend. Overall, intratumoral injection of FLD and FLD-Lip-Nps + had little effect on the body weight of the mice.

[0171] Example 17 FLD and FLD-Lip-Nps + Effect on the survival time of CT26 tumor-bearing mice

[0172] 8-week-old Balb / c female mice were injected with 100 million (100 μL) of cell suspension on the right back, and the tumor growth of the mice was observed and recorded. When the tumor volume reached about 100 mm 3 , intratumoral administration was started (V = length x width x width / 2). The tumor volumes of all the mice were recorded, and they were divided into 3 groups: saline, FLD, and FLD-Lip-Nps + , each with 8 mice, and the mice were administered on days 1, 3, 5, 7, and 9, 2 mg / mouse (FLD content), 60 μL / mouse. The body weight and tumor volume of the mice were recorded every other day, and when the tumor volume reached 2000 mm 3 , the experiment was terminated, the tumors were dissected and weighed, and they were stored in paraformaldehyde and RNA lysis-preventing solution, the survival time of each mouse was recorded, and the survival curve was plotted.

[0173] The body weight and tumor volume of the mice were recorded the following day. The tumor volume was recorded when it reached 2000 mm². 3 The experiment was terminated around the 12th and 15th day, and the survival time of the mice was recorded to plot the survival rate curve. As shown in Figure 15, during the survival experiment, mice in the saline and FLD groups began to die on day 12 and day 15, respectively, while mice in the FLD-Lip-Nps group died on day 15. + The first mouse death occurred on day 17; all mice in the saline and FLD groups died on days 24 and 32, respectively, while all mice in the FLD-Lip-Nps group died. + Four mice from the group survived on day 41, and FLD-Lip-Nps... + All mice in the group died, and the survival time of the mice was extended by 17 days, which was significantly different from the control group and the FLD group. *** P < 0.001, * P < 0.05. The above results indicate that the saline group, lacking effective drug treatment, experienced rapid tumor growth, resulting in the shortest survival time for mice. Drug treatment in the FLD group prolonged the survival time of mice. (FLD-Lip-Nps) + The mice in this group survived longer than those in the previous two groups. FLD-Lip-Nps + Treatment with this drug significantly prolonged the survival time of mice, demonstrating strong anticancer activity.

[0174] In the in vivo antitumor effect on tumor-bearing mice, FLD-Lip-Nps + It inhibited tumor growth and prolonged the survival time of mice.

[0175] In summary, FLD-Lip-Nps + It has stronger anti-cancer activity, providing a research basis and evidence for the clinical treatment of COC.

[0176] Example 18: Determination of serum biochemical parameters in CT26 tumor-bearing mice

[0177] Mouse blood samples were collected and incubated at room temperature for 1 hour until the blood separated into layers. The samples were then centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. The supernatant was then centrifuged again at 12000 rpm for 3 minutes to obtain mouse serum. This serum was then analyzed using FLD-Lip-Nps. +The mouse serum biochemical indicators were measured to evaluate the effect of the drug on the liver and kidney function of the mice. The indicators included AST, ALT, BUN, and CREA. The results showed that the AST was between 134 and 215 U / L, the ALT was between 28 and 55 U / L, the BUN was between 14 and 26 mmol / L, and the CREA was between 23 and 34 mmol / L (FIG. 16A-D). The normal range of the mouse related indicators was as follows: AST: 36.31-235.48 U / L; ALT: 10.06-96.47 U / L; UREA: 10.81-34.74 mmol / L; and CREA: 10.91-85.09 mmol / L. All the indicators were within the normal reference range of the mouse blood biochemical indicators, indicating that the nano-treatment had no damage to the liver and kidney function.

[0178] Example 19 Flow cytometry detection of transfection of mouse dendritic cells by octadecylamine-containing liposomes (Lip-Nps + ) on mouse dendritic cells

[0179] 1. Cell culture

[0180] The mouse dendritic cells DC2.4 were cultured in a 37°C, 5% CO2 incubator using RMPI-1640 medium containing 10% fetal bovine serum (FBS) and 1% double-antibiotic. When the cells grew to 70%-80% of the culture dish, they were digested and passaged with a trypsin solution containing 0.02% EDTA. The whole process of cell culture was kept sterile, and all the related reagents and equipment used were subjected to high-temperature high-pressure sterilization or alcohol disinfection.

[0181] 2. Preparation of octadecylamine-containing blank liposomes (Lip-Nps + )

[0182] Phospholipid 40 mg, cholesterol 2 mg, and octadecylamine 2 mg were dissolved in 5 ml of ethanol, vortexed for 10 min until completely dissolved, and rotary evaporated (vacuum degree slowly increased, speed: 50 rpm, temperature: 45-50°C). The sample was placed in a vacuum dryer overnight, 5 ml of water was added, vortexed, and rotary evaporated at 50°C (without vacuum). The sample was hydrated for 30 min, then transferred to a 10 ml EP tube, and subjected to ultrasonic treatment with a cell disrupter for 10 min (300 w, 1 s interval). The sample was filtered through a 0.22 μm filter membrane, and the particle size and potential were measured, as shown in FIG. 17. The average particle size was 110.6 nm, and the positive potential was 41 mV.

[0183] 3. Agarose gel electrophoresis experiment

[0184] The electrophoresis buffer containing 1% agarose was heated to completely melt, then poured into the electrophoresis tank, and the electrophoresis buffer was added after the agarose was solidified. The liposome-FAP plasmid was prepared as follows: 0-4, 6-4, 8-4, 10-4, 12-4 (μl-μg), mixed and allowed to stand for 15 min to make the liposome fully combine with the FAP plasmid. After adding the sample buffer, 10-20 μl was added to each well, and the sample loading order and amount were recorded. The voltage was adjusted to 80 V, and the gel was taken out after electrophoresis for 50 min, and photographed.

[0185] 4. Liposome containing octadecylamine combined with fibroblast activation protein (FAP) plasmid transfection of mouse dendritic cells (DC2.4)

[0186] The logarithmic growth phase DC2.4 cells were taken, and a cell suspension of 200,000 / ml was prepared using RPMI-1640 medium containing 10% FBS and 1% double antibody. The DC2.4 cell suspension was inoculated in a 6-well plate with a cell density of 500,000 cells / well, and incubated in a 37°C, 5% CO2 incubator for 24 h. The cells were attached and basically recovered to the growth state. The medium was aspirated and replaced with FAP plasmid-containing cationic liposome diluted in blank medium, 700 μl per well, and the drug content was liposome-FAP plasmid: 6-4, 8-4, 10-4 (μl-μg), respectively. The control group was added with blank medium. After continuing to culture for 6 h, the drug-containing medium was aspirated, and complete medium was added for continued culture for 24 h. The cells were digested with trypsin without EDTA, collected, mixed with 100 μl of 0.5% FBS PBS, added with FAP-1 / FITC antibody, and incubated in the dark for 30 min. The liquid was removed by centrifugation, mixed with PBS, and centrifuged again. The supernatant was discarded, and the above steps were repeated twice. After mixing with 500 μl of 0.5% FBS PBS, the cell transfection degree was detected using a flow cytometer. The P2 region in the flow cytometer analysis results represented the percentage of FAP positive cells in the total number of cells.

[0187] Experimental results:

[0188] 1. Agarose gel electrophoresis experiment

[0189] The agarose gel electrophoresis method was used to detect the adsorption capacity of different concentrations of cationic liposome containing octadecylamine (Lip-Nps + ) to FAP plasmid. After the combination of liposome and plasmid, different concentrations of liposome could completely adsorb FAP plasmid (Figure 18). FAP plasmid without liposome migrated under the action of electrophoresis, and FAP plasmid with liposome did not migrate, indicating that FAP plasmid was adsorbed on the liposome at different concentrations.

[0190] 2. Detection of cationic liposome containing octadecylamine (Lip-Nps +) combined with fibroblast activation protein (FAP) plasmid (Lip-Nps) + / FAP) transfected mouse dendritic cells (DC2.4)

[0191] Lip-Nps analysis using FAP-1 / FITC single staining method + / FAP transfection performance on DC2.4 cells. The drug concentrations used were Lip-Nps, respectively. + DC2.4 cells were treated with FAP concentrations of 6-4, 8-4, and 10-4 (μl-μg) for 6 h, then cultured again in complete medium for 24 h. After transfection with different concentrations of liposomes, the FAP positivity rates in the various DC2.4 cell groups were 35.53±0.55%, 48.43±3.23%, and 64.10±2.54% (Figure 19). The results show that with the increase of Lip-Nps... + As liposome concentration increased, FAP transfection efficiency gradually improved, with the highest concentration group showing significantly higher efficiency than the control group, indicating that Lip-Nps... + Cationic liposomes can transfect FAP plasmids into DC2.4 cells (Figure 19), demonstrating their successful use as delivery vectors for nucleic acid drugs.

[0192] Therefore, nucleic acid drugs, especially fibroblast-activating protein particles, can be prepared into liposomes containing nucleic acid drugs using the octadecylamine-containing blank liposomes of this invention via conventional methods. This enhances the function of the nucleic acid drugs.

Claims

1. Liposomes of felodipine, characterized in that, The non-lipid includes non-lipid, phospholipid, and cholesterol, the mass ratio of non-lipid to phospholipid is 1:8-1:20, and the mass ratio of phospholipid to cholesterol is 5:1-20:

1.

2. The non-felodipine liposome according to claim 1, characterized by, The non-lipid also contains octadecylamine, and the mass ratio of cholesterol to octadecylamine is 1:1-1:

3.

3. The non-felodipine liposome of claim 1, characterized in that, Each 5 mL of the liposome contains 40-60 mg of phospholipid, 2-4 mg of cholesterol, and 4-5 mg of non-lipid.

4. The non-felodipine liposome of claim 2, characterized in that, Each 5 mL of the liposome contains 40-60 mg of phospholipid, 2-6 mg of cholesterol, 4-5 mg of non-lipid, and 2-6 mg of octadecylamine.

5. The felodipine liposome as set forth in claim 3, characterized by The mass ratio of non-lipid to phospholipid is 1:15-1:20, and the mass ratio of phospholipid to cholesterol is 10:1-20:

1.

6. The non-felodipine liposome of claim 4, characterized in that, The mass ratio of non-lipid to phospholipid is 1:8-1:16, preferably 1:8-1:12, the mass ratio of phospholipid to cholesterol is 20:1-20:3, and the mass ratio of cholesterol to octadecylamine is 1:1-1:

3.

7. The non-felodipine liposome according to claim 2, 4 or 6, wherein, The non-lipid can be replaced by nucleic acid drugs, preferably fibroblast activation protein plasmid.

8. A preparation method of the non-lipid liposome according to any one of claims 1-6, comprising the following steps: (1) weighing the prescribed amount of phospholipid, cholesterol, non-lipid, and octadecylamine or not in a tomato-shaped flask, adding anhydrous ethanol, vortexing, and ultrasonic dissolving; (2) forming a thin film of the lipids in the bottle by vacuum rotary evaporation, and continuing vacuum rotary evaporation to obtain a uniform thin film; (3) adding deionized water, vortexing, and ultrasonic after hydration, and finally obtaining a non-lipid liposome solution under the probe ultrasonic at 300-350 W.

9. A liposomal lyophilized powder of felodipine, characterized in that, The non-lipid liposome according to any one of claims 1-6 is added to a freeze-drying protective agent to obtain a freeze-dried preparation.

10. Use of non-lipid or the non-lipid liposome according to any one of claims 1-6 or the non-lipid liposome freeze-dried powder according to claim 8 in the preparation of an anti-tumor drug, and the tumor is preferably colorectal cancer, liver cancer, ovarian cancer, or breast cancer.

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