CN101780043B - Preparation method of sanguinarine liposome with acid sensitivity - Google Patents

Abstract

The invention relates to the field of pharmaceutical preparation, in particular to a preparation method of sanguinarine liposome with acid sensitivity, which is characterized by selecting citric acid ammonium salt for preparing the sanguinarine liposome when an ammonium salt gradient method is adopted for preparation. The sanguinarine liposome prepared by the method of the invention has acid sensitivity and is favorable to the antitumor effect.

Description

A kind of preparation method of acid-sensitive sanguinarine liposome

technical field

The invention relates to the field of pharmaceutical preparations, in particular to a method for preparing sanguinarine liposomes, and the sanguinarine liposomes prepared by the method of the invention have acid sensitivity.

Background technique

Sanguinarine was discovered in the early 19th century, its molecular formula is C ₂₀ H ₁₄ NO ₄ , and its relative molecular mass is 332. As a benylisoquinoline alkaloid, it has a wide range of antibacterial, antifungal and antiinfective properties. Recent studies have shown that sanguinarine has good inhibitory activity on a variety of human cancer cells. It can reduce the proliferation of blood vessels around cancer cells by inhibiting endothelial growth factor, and can inhibit the growth of cancer cells without affecting normal cells at micromolar concentrations. It can effectively block the MDR (multidrug resistance effect) of most tumor cells, so it is expected to play a role in clinical tumor treatment. See (Mackraj, ThirumalaGovender, Prem Gathiram. Sanguinarine [J]. Cardiovascular Therapeutics, 2008, 26: 75-83) (Haseeb Ahsan, Shannon Reagan-Shaw, Jorien Breur, Nihal Ahmad, Sanguinarine induces apoptosis of human pancreatic carcinoma BxPC-1 and -3cells via modulations in Bcl-2family proteins[J]. Cancer Letters, 2007, 249(2): 198-208.)

The drug is toxic like most tumor drugs, and its adverse reactions include nausea and vomiting, limb swelling and erythema, heart failure, etc. Therefore, it is necessary to choose a suitable drug delivery system to reduce its toxicity and improve its curative effect in vivo. Liposome, as a biodegradable and biocompatible drug delivery system, is the preferred drug delivery system for anticancer drugs.

Although liposomes have a series of advantages such as reducing the systemic toxic and side effects of anticancer drugs and improving the curative effect, they also have many problems such as being easily cleared by the RES system, insufficient targeting, and immunogenicity. One of them is that most common liposomes enter cells by endocytosis, first exist in the endosome (endosome), and then are transported to the lysosome (lysosome) under the mediation of the microtubule system. In the enzyme body, liposomes and the active ingredients contained in them are degraded, which directly leads to the loss of anticancer drugs.

The pH value of human blood is 7.4, the pH value of tumor tissue is about 6.5, and the pH value of endosome is about 5.5. If the liposome can be triggered by the lower pH value of endosome, the active ingredient contained in it will Released and escaped into the cytoplasm, it can prevent the drug from being destroyed by lysosomes, thereby improving the curative effect.

Acid-sensitive liposome is a functional liposome that uses the above-mentioned pH difference to trigger the release of active ingredients in the liposome into the cytoplasm and avoid being degraded by lysosomes. At present, there are two main principles of acid-sensitive liposomes, one is the application of pH-sensitive lipids, such as DOPE, oleic acid, cholesterol hemisuccinate, etc.; the other is the application of pH-sensitive polyelectrolytes combined with lipids. Plastid surface, such as poly(2-ethylacrylic acid), etc. The conformation of these substances changes with pH, triggering liposome membrane instability, aggregation, fusion, and release of contents.

Contents of the invention

The invention discloses a preparation method of acid-sensitive sanguinarine liposome.

We found in the process of adopting the ammonium salt gradient method to prepare sanguinarine liposomes: adopt conventional ammonium sulfate, the obtained sanguinarine liposomes have no acid sensitivity; and adopt ammonium citrate, the obtained The sanguinarine liposome has acid sensitivity, which is obviously beneficial to improve the antitumor effect of sanguinarine.

The preparation method of the invention comprises: dissolving phospholipids and cholesterol in an organic solvent, evaporating under reduced pressure to remove the organic solvent, adding ammonium citrate aqueous solution to hydrate to form a suspension, homogenizing, dialysis, and incubating with sanguinarine aqueous solution to obtain the product. Wherein the organic solvent is preferably one or more of chloroform, dichloromethane, methanol, ethanol, ether, acetone. Chloroform, methanol, dichloromethane or ethanol are more preferred.

The preparation method of the present invention can also be: dissolving phospholipids and cholesterol in an organic solvent, dripping an ammonium citrate aqueous solution under stirring conditions, evaporating under reduced pressure to remove the organic solvent, homogenizing, dialysis, and incubating with a sanguinarine aqueous solution to obtain the . The organic solvent is preferably ethanol or ether.

The phospholipids described in the present invention are preferably soybean phospholipids.

The ratios involved in the present invention are all molar ratios.

In the present invention, the ratio of sanguinarine to phospholipids and cholesterol directly affects the quality of liposomes, and the concentration of ammonium citrate used in the preparation process also affects its quality.

When other parameters in the fixed prescription and preparation process are constant, only examine the ratio of medicine and phospholipid, the average particle size and encapsulation efficiency of sanguinarine liposome are as shown in Table 1.

Table 1. Effect of the ratio of sanguinarine/phospholipid on liposome particle size and encapsulation efficiency (n=3)

As can be seen from Table 1, the molar ratio of sanguinarine and phospholipids is within the scope of 1: 1～5, and the encapsulation efficiency is lower than 80%; when the molar ratio of the two is within the scope of 1: 6～20, the encapsulation efficiency It is greater than 80%, which meets the requirements; but when the ratio of the two exceeds 1:15, due to the high concentration of phospholipids in the liposome, the liquid is viscous and easily separated. Therefore, the ratio of sanguinarine/phospholipid in the present invention is preferably 1:6-15.

When other factors in the fixed prescription were kept constant and only the amount of cholesterol was changed, the average particle size and encapsulation efficiency of sanguinarine liposomes were also different. The results are shown in Table 2.

Table 2. Effect of the ratio of sanguinarine/cholesterol on liposome particle size and encapsulation efficiency (n=3)

As can be seen from Table 2, when the molar ratio of drug and cholesterol is less than 1:0.1, the encapsulation efficiency is lower than 80%; when the ratio of the two is in the range of 1:0.5～2.5, the encapsulation efficiency is above 80%; When the molar ratio of the two is in the range of 1:5-15, the encapsulation efficiency decreases with the increase of cholesterol, and the particle size gradually increases, and the prepared liposome is unstable and easy to separate. Therefore, the molar ratio of drug/cholesterol in the present invention is preferably 1:0.5-2.5.

When other factors in the fixed prescription were constant and only the concentration of ammonium citrate used in the preparation process was changed, the average particle size and encapsulation efficiency of sanguinarine liposomes were also different. The results are shown in Table 3.

Table 3. Effect of ammonium citrate concentration on sanguinarine liposome particle size and encapsulation efficiency (n=3)

As can be seen from Table 3, different concentrations of ammonium citrate aqueous solutions have little effect on liposome particle size, but have a significant effect on encapsulation efficiency. As the concentration of ammonium citrate increases, the encapsulation efficiency increases gradually. When the ammonium citrate concentration is 0-200mmol/L, the encapsulation efficiency is lower than 80%; when the ammonium citrate concentration is 250-500mmol/L, the encapsulation efficiency is higher than 80%, and the concentration is 400mmol/L When the encapsulation efficiency is close to 100%. But when the ammonium citrate concentration is above 500mmol/L, the prepared liposomes are unstable and easily delaminated. Therefore, the preferred concentration range of ammonium citrate in the present invention is 250～500mmol/L.

When adopting the ammonium salt gradient method to prepare sanguinarine liposomes, ammonium sulfate is used more as the ammonium salt in this field, but this test found that: when adopting the ammonium salt gradient method to prepare sanguinarine liposomes, use citron The sanguinarine liposomes prepared by ammonium citrate and ammonium sulfate have certain differences, and the sanguinarine liposomes prepared by ammonium citrate have obvious acid sensitivity, which can be proved by in vitro release test.

1. In vitro release experiment:

Configure HEPES buffer (0.14mol/LNaCl, 0.01mol/L HEPES) as release medium, adjust pH to 7.4, 6.5, 5.5 with NaOH solution. These three pHs respectively simulate the physiological fluid (pH7.4), tumor tissue (pH6.5), and the environment after endosome ingestion (pH5.5).

Precisely pipette 1.5 mL of sanguinarine liposomes, place them in a dialysis bag, tie both ends tightly, place them in 40 mL of HEPES buffer, stir magnetically at a constant temperature of 37°C, sample 0.5 mL at regular intervals, and replenish fresh release medium in time. After the sample is centrifuged at high speed, the supernatant is drawn, the drug content is determined by HPLC, the cumulative release rate is calculated, and the time release curve is drawn.

The two tested liposomes were sanguinarine liposomes prepared by ammonium sulfate gradient method and sanguinarine liposomes prepared by ammonium citrate gradient method. See Figures 1 and 2. Among them, the ammonium sulfate group hardly showed acid sensitivity, and there was no significant difference in the release under the three pH environments; but the liposomes in the ammonium citrate group showed obvious acid sensitivity, and with the decrease of solution pH , the release of the drug in liposomes was significantly accelerated.

Two, using human cervical cancer Hela cells to compare the anti-tumor experiments of two liposomes in vitro

The human cervical cancer Hela cells in the logarithmic growth phase were digested with 0.02% EDTA, and the live cells were counted with trypan blue to make a cell suspension, and the H22 cells were directly collected by centrifugation. Add 0.5×10 ⁴ /mL cells into a 96-well culture plate, 100 μL per well, set up triplicate wells, place in a 5% CO ₂ incubator at 37°C for overnight culture, add 3, 5, 7, 9 μg·mL ^- The sanguinarine solution of ¹ , the sanguinarine liposome prepared by ammonium sulfate, and the sanguinarine liposome of the present invention have no other positive controls, and the negative control group adds equal-volume blank liposomes. The cell suspension was used as a blank control, 100 μL/well, incubated at 37°C in a 5% CO ₂ incubator for 44 h, adding 20 μL of 5 mg·mL ^-1 MTT solution to each well, and continuing to culture for 4 h. Discard all supernatants of tumor cell adherent cells directly. H22 suspended cells were centrifuged at 1000rpm in a plate centrifuge for 10min, inverted on absorbent paper, and the supernatant was sucked off. Add 100 μL/well of DMSO, vibrate on a micro-oscillator for 5 minutes to completely dissolve the crystals, and measure the absorbance value (A) at a wavelength of 570 nm by an enzyme-linked analyzer. The higher the A value, the more the number of viable cells. According to A, the growth inhibition rate of the drug on the cells can be calculated, and the results are shown in Table 4.

Table 4 The effect of sanguinarine solution and liposomes on human cervical cancer Hela cells for 24 hours

*P<0.05, **P<0.01 Compared with the liposome group prepared by the liposome of the present invention and the ammonium sulfate gradient method

After the two groups of liposomes acted on human cervical cancer Hela cells for 24 hours, they all had obvious tumor-inhibiting effects, and there was a concentration-dependent effect. Wherein the liposome of the present invention has significantly higher antitumor effect than the sanguinarine liposome prepared by ammonium sulfate at the same administration concentration, and there is a significant difference.

Analyzing the reason, after liposome endocytosis enters the cell, because the liposome prepared by the ammonium citrate gradient method has acid sensitivity, under the trigger of the low pH of the endosome, the liposome membrane and the endosome Membrane fusion releases sanguinarine and enters the cytoplasm, thereby exerting an antitumor effect; while the sanguinarine liposomes prepared by the ammonium sulfate gradient method are not acid-sensitive, and more are transferred from endosomes to lysosomes, Drugs are degraded by lysosomes, so the antitumor effect is poor.

3. The mice bearing Heps tumor were used to evaluate the anti-tumor effect of the two liposomes in vivo.

Take 50 ICR mice, 18-22g, half male and half male. According to the transplanted tumor research method, inoculate Heps solid tumor (take the tumor block under aseptic operation, weigh it, grind it with a glass tissue homogenizer, put it into a sterile container after grinding, add normal saline to dilute to 1: 3, the container was placed on ice, and aspirated with an empty needle, the cells were mixed evenly before each aspiration, and 0.2 mL was inoculated subcutaneously in the axilla of the right forelimb of each mouse), and the mice were weighed 24 hours after inoculation, and Divide into 5 groups at random, blank control group, cyclophosphamide solution group (20mg/kg) are negative and positive control group respectively, sanguinarine solution group, adopt the sanguinarine liposome prepared by ammonium sulfate, the serum of the present invention Rhizine liposome (dosage is 14mg/kg), after inoculation 24 hours tail vein injection administration, once a day, administration altogether 6 times, put to death tumor-bearing mice on the 2nd day after drug withdrawal, weigh, The tumor mass was separated and weighed, and the obtained data were processed statistically (t test). The results are shown in Table 5.

)(n＝10)The inhibitory effect of table 5 sanguinarine solution and liposome iv on mouse transplanted tumor Heps (

)(n=10)

*P<0.05, **P<0.01 compared with blank group

Compared with the solution group, both liposomes significantly inhibited the growth of Heps tumor (P<0.01). However, the in vivo tumor inhibition rate of the liposome of the present invention is significantly better than that of the non-acid-sensitive sanguinarine liposome prepared by ammonium sulfate.

This further illustrates that the sanguinarine liposome prepared by the citric acid gradient method significantly improves its curative effect in vivo due to its acid sensitivity.

The sanguinarine liposome prepared by the invention has an encapsulation rate greater than 80% and a particle diameter within the range of 100-300nm within the optimized prescription range.

The invention has the advantages that: the sanguinarine liposome is prepared by the citric acid gradient method, the preparation process is simple, not only the liposome has a high encapsulation efficiency, but also the liposome has the characteristics of acid sensitivity, Thereby significantly improving its tumor inhibition rate in vivo and in vitro.

Description of drawings

Figure 1 is the release situation of sanguinarine liposomes prepared by ammonium sulfate under three pH conditions

Fig. 2 is the release situation of the sanguinarine liposome prepared by the method of the present invention under three pH conditions

Fig. 3 is the particle size distribution figure of sanguinarine liposome of the present invention

Fig. 4 is the transmission electron micrograph of sanguinarine liposome of the present invention

Detailed ways

Example 1

Take by weighing 1.6g soybean lecithin (purity is greater than 99% phosphatidylcholine), 160mg cholesterol is dissolved in 50mL ethanol, ultrasonic, dissolve; /L). The mixed solution was rotary evaporated at 50° C. for 2 h, and the ethanol was completely evaporated to dryness, and homogenized under high pressure to reduce the particle size (5000 psi, 3 times), to obtain blank liposomes before drug loading. Then the blank liposomes were placed in a dialysis bag, the two ends were tied tightly, put into 500 mL of normal saline for 2 hours, and the normal saline was changed every hour. Finally, dissolve 90 mg of sanguinarine in 90 mL of distilled water, mix it with the above-mentioned liposomes after dialysis, incubate in a water bath at 30°C for 5 min, and after sterile filtration (the pore size of the membrane filter is 0.2 μm), divide the subsequent filtrate into vials That's it. The molar ratio of sanguinarine: soybean lecithin: cholesterol is 1:7.5:1.5.

The encapsulation efficiency of the prepared liposome is 94.45%, the average particle diameter is 110.5nm, and the polydispersity coefficient is 0.18.

Finished product traits: This product is orange-red suspension liquid.

Particle size measurement: This product uses physiological saline as a dilution solvent, and after dilution several times, the particle size is measured by PCS method (MalvernZetasizer3000HS). The particle size distribution diagram is shown in Figure 3.

Morphological observation: This product uses isotonic saline as a dilution solvent, and is observed under a transmission electron microscope after being diluted dozens of times. As shown in Figure 4, the liposomes are regular spheres with good roundness and uniform lipid membrane.

Example 2

It is basically the same as Example 1, but there are the following changes: when preparing blank liposomes in the early stage, the operation process is: take 1g of soybean phospholipid (purity greater than 99% phosphatidylcholine), dissolve 160mg of cholesterol in 50mL of ether, ultrasonically, Dissolving; under the condition of 300rpm magnetic stirring, slowly inject the above solution into 30mL of ammonium citrate solution (300mmol/L), and the mixed solution was rotatated at 50°C for 2h, and the ether was completely evaporated to dryness, and the water bath was sonicated to reduce the particle size to obtain Blank liposomes before drug loading. Then the blank liposomes were placed in a dialysis bag, the two ends were tied tightly, put into 500 mL of normal saline for 2 hours, and the normal saline was changed every hour. Afterwards, the sanguinarine aqueous solution prepared in Example 1 was mixed with the above solution, incubated in a water bath at 30°C for 5 minutes, and after sterile filtration (the pore size of the membrane filter was 0.2 μm), the subsequent filtrate was dispensed into vials. The molar ratio of sanguinarine: soybean lecithin: cholesterol is 1:7.5:1.5.

The encapsulation efficiency of the prepared sanguinarine liposomes was 92.71%, the average particle size was 104.3nm, and the polydispersity coefficient was 0.15.

Example 3

Take by weighing 1.6g soybean lecithin (purity is greater than 99%), 160mg cholesterol is dissolved in the dichloromethane, this solution is placed in the eggplant shape bottle of grind mouth, removes organic solvent under reduced pressure on 30 ℃ constant temperature water baths, makes phospholipid, Cholesterol forms a uniform film on the bottom of the bottle, and then put it in a vacuum desiccator to vacuum overnight, and set aside. In addition, 30 mL of 300 mmol/L ammonium citrate solution was added to the above-mentioned eggplant-shaped bottle, and the membrane was washed by rotating at 30° C. until milky white liposome suspension was formed, homogenized under high pressure to reduce the particle size (5000 psi, 3 times), to obtain blank liposomes before drug loading. Then the blank liposomes were placed in a dialysis bag, the two ends were tied tightly, put into 500 mL of normal saline for 2 hours, and the normal saline was changed every hour. Mix the sanguinarine aqueous solution in Example 1 with the above-mentioned blank liposome suspension, incubate in a water bath at 30° C. for 5 minutes, and perform sterile filtration (the pore size of the membrane filter is 0.2 μm). The molar ratio of sanguinarine: soybean lecithin: cholesterol is 1:7.5:1.5.

The encapsulation efficiency of the prepared sanguinarine liposome is 93.78%, the particle diameter is 113.2nm, and the polydispersity coefficient is 0.174.

Example 4

Take by weighing 1.5g soybean lecithin (purity is greater than 99%), 200mg cholesterol is dissolved in the dichloromethane, this solution is placed in the eggplant shape bottle of grinding mouth, removes organic solvent by decompression evaporation on 30 ℃ constant temperature water bath, makes phospholipid, Cholesterol forms a uniform film on the bottom of the bottle, and then put it in a vacuum desiccator to vacuum overnight, and set aside. In addition, 30 mL of 300 mmol/L ammonium citrate solution was added to the above-mentioned eggplant-shaped bottle, and the membrane was washed by rotating at 30° C. until milky white liposome suspension was formed, homogenized under high pressure to reduce the particle size (5000 psi, 3 times), to obtain blank liposomes before drug loading. Then the blank liposomes were placed in a dialysis bag, the two ends were tied tightly, put into 500 mL of normal saline for 2 hours, and the normal saline was changed every hour. Mix the sanguinarine aqueous solution in Example 1 with the above-mentioned blank liposome suspension, incubate in a water bath at 30° C. for 5 minutes, and perform sterile filtration (the pore size of the membrane filter is 0.2 μm). The molar ratio of sanguinarine: soybean lecithin: cholesterol is 1:7.0:1.9.

The encapsulation efficiency of the prepared sanguinarine liposome is 91.37%, the polydispersity coefficient is 0.174, and the particle diameter is 105.1 nm.

Example 5

Take by weighing 2g soybean lecithin (purity greater than 99%), 150mg cholesterol is dissolved in the dichloromethane, this solution is placed in the ground eggplant-shaped bottle, removes organic solvent by decompression evaporation on 30 ℃ constant temperature water bath, makes phospholipid, cholesterol A uniform film was formed on the bottom of the bottle, and then placed in a vacuum desiccator to evacuate overnight and set aside. In addition, 30 mL of 300 mmol/L ammonium citrate solution was added to the above-mentioned eggplant-shaped bottle, and the membrane was washed by rotating at 30° C. until milky white liposome suspension was formed, homogenized under high pressure to reduce the particle size (5000 psi, 3 times) to obtain blank liposomes before drug loading. Then the blank liposomes were placed in a dialysis bag, the two ends were tied tightly, put into 500 mL of normal saline for 2 hours, and the normal saline was changed every hour. Mix the sanguinarine aqueous solution in Example 1 with the above-mentioned blank liposome suspension, incubate in a water bath at 30° C. for 5 minutes, and perform sterile filtration (the pore size of the membrane filter is 0.2 μm). The molar ratio of sanguinarine: soybean lecithin: cholesterol is 1:9.4:1.4.

The encapsulation efficiency of the prepared sanguinarine liposome is 97.47%, the polydispersity coefficient is 0.174, and the particle diameter is 117.2nm.

Claims (4)

1. A preparation method for sanguinarine liposomes, which is prepared by an ammonium salt gradient method, is characterized in that it comprises: dissolving phospholipids and cholesterol in an organic solvent, evaporating under reduced pressure to remove the organic solvent, adding an aqueous solution of ammonium citrate to hydrate and form Suspension, homogenization, dialysis, and incubation with sanguinarine aqueous solution; wherein the molar ratio of sanguinarine, phospholipids, and cholesterol is 1:6～15:0.5～2.5; the concentration of ammonium citrate aqueous solution is 250 ~500mmol/L. 2. the preparation method of claim 1, wherein said organic solvent is selected from one or more in chloroform, dichloromethane, methanol, ethanol, ether, acetone. 3. A preparation method for sanguinarine liposomes, which is prepared by an ammonium salt gradient method, is characterized in that it comprises: dissolving phospholipids and cholesterol in an organic solvent, dripping an aqueous solution of ammonium citrate under stirring conditions, and evaporating under reduced pressure to remove Organic solvent, homogenization, dialysis, and incubation with sanguinarine aqueous solution; wherein the molar ratio of sanguinarine, phospholipid, and cholesterol is 1:6～15:0.5～2.5; the concentration of ammonium citrate aqueous solution is 250～ 500mmol/L. 4. The preparation method according to claim 3, wherein the organic solvent is ethanol or ether.

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