A prodigiosin pH-sensitive liposome, its preparation method and application

Abstract

The present invention relates to the field of application of nanomaterials, and provides a violacein pH-sensitive liposome, which comprises a wrapping material liposome and violacein encapsulated in the liposome; wherein, the liposome is prepared from phosphatidylethanolamine, cholesterol hemisuccinate and cholesterol. The present invention also provides a preparation method and application of the above violacein pH-sensitive liposome. By preparing a pH-sensitive liposome to encapsulate violacein, the present invention increases the dispersibility of violacein in water and the application prospect of violacein as a medicament; meanwhile, the violacein pH-sensitive liposome provided by the present invention has a simple preparation method and is easy to implement, can effectively improve the stability of violacein, and enhances its antibacterial and antitumor effects.

Description

Technical Field

[0001] The present invention relates to the field of application of nanomaterials, and particularly to a violacein pH-sensitive liposome, a preparation method thereof and an application thereof. Background Art

[0002] Violacein is a kind of water-insoluble secondary metabolite obtained by microorganisms through oxidative condensation of tryptophan molecules as precursor substances. It can not only be used as a stain, but also has a variety of biological activities: (1) It has a strong inhibitory effect on leukemia, lymphoma cells and colon cancer cells, etc.; it can induce apoptosis of MRC-5 cells (human embryonic lung fibroblasts) and HeLa cells (a cell line of cervical cancer cells) by increasing the membrane potential of mitochondria; (2) It can inhibit the growth of bacteria and fungi, and has obvious antibacterial activity against Gram-positive bacteria such as Bacillus sp., Staploylococcous aureus, Mycobacterium and Streptococcus sp. Based on the above characteristics, violacein has broad application prospects in the fields of food, biomedicine, printing and dyeing industry, etc. However, since violacein is a water-insoluble substance, when it is subsequently used as a medicine in the field of biomedicine, this characteristic will greatly inhibit its development.

[0003] Liposomes are spherical vesicles, which can be made of cholesterol, sphingolipids, glycolipids or long-chain fatty acids or even membrane proteins, etc. When liposomes are dispersed in water, the phospholipid bilayer will spontaneously form a closed structure, and the internal aqueous environment is restricted by the phospholipid bilayer membrane. Therefore, if liposomes can be used as carriers to encapsulate violacein therein, the dispersibility of violacein in water can be further improved, and the problem that it is insoluble in water as a water-insoluble substance can be solved.

[0004] At the same time, combined with the anti-tumor effect of violacein, a pH-sensitive lipid material can be further selected to synthesize a pH-sensitive liposome. Since the pH of the tumor stroma is lower than that of normal tissue cells, a pH-sensitive liposome can be designed to better fuse with the membrane and release drugs in the tumor environment with a reduced pH, thereby increasing its targeting. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a violacein pH-sensitive liposome, a preparation method thereof and an application thereof. By preparing a novel pH-sensitive liposome to encapsulate violacein, the dispersibility of violacein in water can be increased, and at the same time, a pH-sensitive material is designed to release the internal bioactive substances in a weak acid environment, providing new ideas for the research and development of anti-tumor drugs.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A prodigiosin pH-sensitive liposome, comprising a liposome and prodigiosin encapsulated within the liposome; wherein, the liposome is prepared from phosphatidylethanolamine (PE), cholesteryl succinate (CHEMS) and cholesterol (CHOL).

[0008] As one of the preferred embodiments of the present invention, in the composition of the liposome:

[0009] Phosphatidylethanolamine is a pH-sensitive material with a pKa of 5.0 - 6.0; when the pH value is acidic, phosphatidylethanolamine exerts its pH-sensitive effect;

[0010] Cholesteryl succinate is used as an emulsifier and a liposome carrier material;

[0011] Cholesterol functions as a biomembrane material in the liposome.

[0012] As one of the preferred embodiments of the present invention, in the composition of the liposome: the mass ratio of phosphatidylethanolamine, cholesteryl succinate and cholesterol is (6 - 8):(2 - 4):(1 - 2).

[0013] As one of the preferred embodiments of the present invention, as the encapsulated bioactive ingredient, the addition amount of prodigiosin is 2% - 5% of the total mass of phosphatidylethanolamine, cholesteryl succinate and cholesterol.

[0014] A method for preparing a prodigiosin pH-sensitive liposome according to any one of claims 1 - 4, comprising the following steps:

[0015] (1) Weigh prodigiosin and dissolve it in chloroform until completely dissolved;

[0016] (2) Respectively take phosphatidylethanolamine, cholesteryl succinate and cholesterol and dissolve them in the solution obtained in step (1);

[0017] (3) Place the mixture obtained in step (2) in a rotary evaporator under vacuum to remove the organic solvent until a transparent film is formed on the bottle wall;

[0018] (4) Place it in a vacuum drying oven for a certain time to remove the residual organic solvent;

[0019] (5) Add a buffer solution and hydrate it at room temperature until the transparent film can fall off; then, use a probe to sonicate for 15 - 30 min to obtain the target prodigiosin pH-sensitive liposome.

[0020] As one of the preferred embodiments of the present invention, in the step (1), violacein is directly purchased, and the weighed amount is 0.9 - 3.5 mg, and the volume of chloroform for dissolving violacein is 5 - 8 mL.

[0021] As one of the preferred embodiments of the present invention, in the step (2), the addition amounts of phosphatidylethanolamine, cholesterol hemisuccinate, and cholesterol are 30 - 40 mg, 10 - 20 mg, and 5 - 10 mg respectively.

[0022] As one of the preferred embodiments of the present invention, in the step (3), the temperature of the rotary evaporator under vacuum is 30 - 45 °C;

[0023] In the step (4), the temperature of the vacuum drying oven is 30 - 45 °C, and the placement time is 8 - 15 h.

[0024] As one of the preferred embodiments of the present invention, in the step (5), the buffer solution is ultrapure water, and the addition amount is 5 - 10 mL.

[0025] An application of the violacein pH-sensitive liposome according to any one of claims 1 - 4, wherein the violacein pH-sensitive liposome is used for the treatment of HeLa cervical cancer.

[0026] The advantages of the present invention compared with the prior art are as follows:

[0027] (1) Aiming at the problem that violacein, as a lipophilic substance, is insoluble in water, the present invention prepares a novel pH-sensitive liposome to encapsulate violacein, increasing the dispersibility of violacein in water and the application prospect of violacein as a medicament; among them, the pH-sensitive liposome can promote the cytosolic release of membrane-impermeable molecules, and when combined with a targeting ligand, this property can promote receptor-mediated endocytosis for targeted cytosolic delivery of bioactive substances;

[0028] (2) The present invention selects cholesterol hemisuccinate as the pH-sensitive material, which will only exert its pH-sensitive effect when the pH value is acidic, enabling the encapsulating material to specifically release the violacein inside, thereby playing the anti-tumor function of violacein and exerting the anti-tumor effect; it provides a new research idea for the research and development of anti-tumor drug liposomes;

[0029] (3) Phosphatidylethanolamine is mainly used as an emulsifier and liposome carrier material in the medicament. Since violacein is encapsulated inside, it not only solves the problem that violacein is easily decomposed by light, significantly improves the stability of the liposome, but also has extremely high bioavailability, can delay the release performance of the liposome, and further improves the application performance of the composite liposome;

[0030] (4) The violacein pH-sensitive liposomes prepared by the present invention are uniform in size, and the preparation method is simple, easy to operate, and environmentally friendly. Description of the Drawings

[0031] Figure 1 It is the scanning electron micrograph of violacein in Test Example 1;

[0032] Figure 2 It is the transmission electron micrograph of violacein pH-sensitive liposomes in Test Example 1;

[0033] Figure 3 It is the result of the ultraviolet absorption spectra of violacein and violacein pH-sensitive liposomes in Test Example 2 (in the figure, violacein represents violacein, and violacein-Lip represents violacein pH-sensitive liposomes);

[0034] Figure 4 It is the result of the infrared absorption spectra of violacein and violacein pH-sensitive liposomes in Test Example 2 (in the figure, violacein represents violacein, and violacein-Lip represents violacein pH-sensitive liposomes);

[0035] Figure 5 It is the in vitro release curve of violacein pH-sensitive liposomes in Test Example 3;

[0036] Figure 6 It is the inhibition results of different concentrations of violacein on Staphylococcus aureus S.aureus, Bacillus subtilis Bacillus subtilis, and Micrococcus tetragenus Micrococcus tetragenus in Test Example 4;

[0037] Figure 7 It is the inhibition results of different concentrations of violacein liposome solutions on Staphylococcus aureus S.aureus, Bacillus subtilis Bacillus subtilis, and Micrococcus tetragenus Micrococcus tetragenus in Test Example 4;

[0038] Figure 8 It is the illumination diagram of the violacein solution for 6 consecutive days in Test Example 5;

[0039] Figure 9 It is the inhibition results of different concentrations of violacein on Staphylococcus aureus S.aureus, Bacillus subtilis Bacillus subtilis, and Micrococcus tetragenus Micrococcus tetragenus after 6 days of illumination in Test Example 5;

[0040] Figure 10It is the illumination diagram of violacein liposome solution in Test Example 5 after 6 consecutive days of light exposure;

[0041] Figure 11 It is the inhibition results of violacein liposomes with different concentrations after 6 days of light exposure in Test Example 5 against Staphylococcus aureus, Bacillus subtilis and Micrococcus tetragenus;

[0042] Figure 12 It is the toxic effect of violacein and violacein liposomes with different concentrations on HeLa cervical cancer cells in Test Example 6 (in the figure, violacein represents violacein, and violacein-Lip represents pH-sensitive liposome of violacein). Detailed implementation manners

[0043] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation manners and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0044] For those not specifying specific technologies or conditions in the following embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For those reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0045] Example 1

[0046] A pH-sensitive liposome of violacein in this example includes liposome and violacein encapsulated in the liposome. Among them, the liposome is prepared from phosphatidylethanolamine (PE), cholesterol succinate monoesters (CHEMS) and cholesterol (CHOL).

[0047] In the composition components of the liposome:

[0048] Phosphatidylethanolamine is a pH-sensitive material with a pKa of 5.0 - 6.0; when the pH value is acidic, phosphatidylethanolamine exerts its pH-sensitive effect;

[0049] Cholesterol succinate monoesters are used as emulsifiers and liposome carrier materials;

[0050] Cholesterol acts as a biomembrane material in the liposome.

[0051] Preparation method:

[0052] (1) Weigh 0.9 mg of violacein and dissolve it in 5 mL of chloroform until completely dissolved.

[0053] (2) Dissolve 30 mg of phosphatidylethanolamine, 10 mg of cholesterol hemisuccinate, and 5 mg of cholesterol in the solution obtained in step (1).

[0054] (3) Pour the mixture obtained in step (2) into a round-bottom flask for rotary evaporation, and place it in a rotary evaporator under vacuum at 30 °C to remove the organic solvent until a transparent film is formed on the inner wall of the round-bottom flask.

[0055] (4) Place the round-bottom flask after rotary evaporation in a vacuum drying oven at 30 °C and dry for 8 h to remove the residual organic solvent.

[0056] (5) Add 5 mL of ultrapure water to the flask and hydrate at room temperature until the transparent film can be detached; then, use probe sonication for 15 min to obtain the target violacein pH-sensitive liposomes.

[0057] Example 2

[0058] A violacein pH-sensitive liposome in this example includes liposomes and violacein encapsulated within the liposomes. Among them, the liposomes are prepared from phosphatidylethanolamine (PE), cholesterol hemisuccinate (CHEMS), and cholesterol (CHOL).

[0059] Among the components of the liposomes:

[0060] Phosphatidylethanolamine is a pH-sensitive material with a pKa of 5.0 - 6.0; when the pH value is acidic, phosphatidylethanolamine exerts its pH-sensitive effect;

[0061] Cholesterol hemisuccinate is used as an emulsifier and liposome carrier material;

[0062] Cholesterol functions as a biomembrane material in the liposomes.

[0063] Preparation method:

[0064] (1) Weigh 3.5 mg of violacein and dissolve it in 8 mL of chloroform until completely dissolved.

[0065] (2) Dissolve 40 mg of phosphatidylethanolamine, 20 mg of cholesterol hemisuccinate, and 10 mg of cholesterol in the solution obtained in step (1).

[0066] (3) Pour the mixture obtained in step (2) into a round-bottom flask for rotary evaporation, and place it in a rotary evaporator under vacuum at 45 °C to remove the organic solvent until a transparent film is formed on the inner wall of the round-bottom flask.

[0067] (4) Place the round-bottom flask after rotary evaporation in a vacuum drying oven at 45 °C and dry for 15 h to remove the residual organic solvent.

[0068] (5) Add 10 mL of ultrapure water into the bottle and hydrate at room temperature until the transparent film can be detached; then, use probe sonication for 30 min to obtain the prodigiosin pH-sensitive liposomes required for the target.

[0069] Example 3

[0070] A kind of prodigiosin pH-sensitive liposome in this example includes liposomes and prodigiosin encapsulated in the liposomes. Among them, the liposomes are prepared from phosphatidylethanolamine (PE), cholesteryl succinate (CHEMS), and cholesterol (CHOL).

[0071] Among the components of the liposomes:

[0072] Phosphatidylethanolamine is a pH-sensitive material with a pKa of 5.0 - 6.0; when the pH value is acidic, phosphatidylethanolamine exerts its pH-sensitive effect;

[0073] Cholesteryl succinate is used as an emulsifier and liposome carrier material;

[0074] Cholesterol acts as a biomembrane material in the liposomes.

[0075] Preparation method:

[0076] (1) Weigh 2 mg of prodigiosin and dissolve it in 7 mL of chloroform until completely dissolved.

[0077] (2) Respectively take 33 mg of phosphatidylethanolamine, 13 mg of cholesteryl succinate, and 6 mg of cholesterol and dissolve them in the solution obtained in step (1).

[0078] (3) Pour the mixture obtained in step (2) into a round-bottom flask for rotary evaporation and place it in a 40 °C vacuum rotary evaporator to remove the organic solvent until a transparent film is formed on the inner wall of the round-bottom flask.

[0079] (4) Place the round-bottom flask after rotary evaporation treatment in a 40 °C vacuum drying oven and dry for 12 h to remove the residual organic solvent.

[0080] (5) Add 8 mL of ultrapure water into the bottle and hydrate in the dark at room temperature until the transparent film can be detached; then, use probe sonication for 18 min to obtain the prodigiosin pH-sensitive liposomes required for the target.

[0081] Test Example 1

[0082] This test example is used to illustrate the morphological characteristics of prodigiosin and prodigiosin pH-sensitive liposomes, and at the same time verify from the morphology whether the present invention can encapsulate prodigiosin.

[0083] 1. Drop the violacein solution on a mica flake. After drying at room temperature, place it on a double-sided conductive adhesive for fixation and perform vacuum gold plating treatment. Put the treated sample into a scanning electron microscope to observe its morphological characteristics. The results are as Figure 1 shown.

[0084] 2. Take 100 μL of violacein pH-sensitive liposomes (taking the violacein pH-sensitive liposomes prepared in Example 3 as an example), add 900 μL of ultrapure water to dilute it, drop the sample on a dry copper grid, dry it at a certain distance under an infrared light source. After the copper grid is completely dry, drop a drop of 1% phosphotungstic acid solution on the copper grid for counterstaining, and blot the excess phosphotungstic acid on the copper grid with filter paper. After complete drying, observe and photograph it under a transmission electron microscope. The results are as Figure 2 shown.

[0085] From Figure 2 the results, it can be seen that the violacein pH-sensitive liposomes prepared by the present invention are uniform in size, with a diameter of 150 - 200 nm. And at the same time, combining Figure 1 and Figure 2 it can be known that the present invention can successfully encapsulate violacein.

[0086] Test Example 2

[0087] This test example is used to illustrate the changes in the ultraviolet and infrared absorption spectra of violacein and violacein pH-sensitive liposomes, and verify whether the present invention successfully encapsulates violacein from the spectra.

[0088] 1. Accurately weigh 5 mg of violacein, dissolve it with absolute ethanol and dilute it to an appropriate concentration. Using absolute ethanol as the blank solution, scan in the wavelength range of 300 - 700 nm, record the ultraviolet absorption spectrum after scanning, and determine the maximum absorption wavelength of violacein; then take 5 mg of violacein pH-sensitive liposomes (taking the violacein pH-sensitive liposomes prepared in Example 3 as an example), dissolve it with pure water to an appropriate concentration, use pure water as the blank solution, scan in the wavelength range of 300 - 700 nm, and record the ultraviolet absorption spectrum after scanning to determine the maximum absorption wavelength of violacein liposomes. The results are as Figure 3 shown.

[0089] From Figure 3 the results, it can be seen that after the pH-sensitive liposomes encapsulate violacein, the characteristic peak undergoes a red shift, and the ultraviolet results indicate successful synthesis.

[0090] 2. For further characterization, perform infrared spectroscopy to verify the synthesis results. The results are as Figure 4 shown.

[0091] From Figure 4As a result, it can be seen that after the violacein was wrapped with the pH-sensitive material, infrared spectroscopy was combined to verify the synthesis result, and the characteristic peak underwent a red shift, indicating that the violacein was successfully encapsulated within the liposome.

[0092] Test Example 3

[0093] This test example was used to verify the pH sensitivity of the violacein pH-sensitive liposome of the present invention.

[0094] A certain amount of violacein pH-sensitive liposome (taking the violacein pH-sensitive liposome prepared in Example 3 as an example) was measured and placed in 10 mL of PBS solutions with pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, and pH 8.0 of 0.5% Tween-80. At 35 °C and 100 r / min, the solution was taken out after 72 h by the constant temperature oscillation method, and the absorbance was measured by an ultraviolet-visible spectrophotometer to draw a curve.

[0095] The in vitro release curve of the violacein liposome is as Figure 5 shown. When the pH of the solution was neutral, it could be seen from the release curve that the violacein hardly released from the liposome. When the pH value gradually decreased, the release amount of the violacein gradually increased. When the pH was lower than 5.0, it was found that the violacein was basically completely released, and when the pH decreased further, the release curve remained unchanged. The release curves of the violacein were the same when the pH of the solution was 4.5 and 3.5, indicating that the critical pH value of the violacein pH-sensitive liposome should be around 5.0, and the pKa of CHEMS (pH-sensitive material) was 5.0. That is to say, its pH-sensitive effect would only be exerted when the pH value was less than 5.0, and the experimental results were consistent with the theory.

[0096] Test Example 4

[0097] This test example was used to verify whether the activity of the encapsulated violacein was reduced.

[0098] In order to further verify that the activity of the encapsulated violacein was not reduced, bacterial viability experiments were carried out respectively for verification:

[0099] Staphylococcus aureus, Bacillus subtilis, and Micrococcus tetragenus in the logarithmic growth phase were resuspended in fresh LB liquid medium at a ratio of 1:40. Different concentrations of the drug (pH-sensitive liposomes of violacein) were added to the drug-treated group to a final concentration of 15, 30, 60 μg / mL, and they were cultured together with the blank group (using violacein instead of pH-sensitive liposomes of violacein) for 12 h under the conditions of 37 °C and 200 rpm. The next day, the cells were centrifuged at 3000 rpm for 2 min and rinsed twice with PBS buffer (to remove the culture medium). Using the LIVE / DEAD bacterial kit, the collected bacteria were stained in the dark for 30 min. Here, they were rinsed twice with PBS buffer (to remove the excess dye), and finally the bacteria were resuspended in a certain volume of pure water. 5 μL of the cell suspension was taken and placed on a glass slide. After drying, an appropriate amount of cedar oil was dropped on the bacteria, and fluorescence microscopy was used for observation and photographic recording. The results are as Figure 6 、 7 shown.

[0100] From Figure 6 、 7 the results, it can be seen that the activity of the encapsulated violacein has not decreased, and the pH-sensitive liposomes of violacein prepared in the present invention have a better antibacterial effect.

[0101] Test Example 5

[0102] This test example is used to verify whether light will affect the biological activity of the violacein of the present invention.

[0103] To further verify whether light will affect the biological activity of violacein, the viability assay of bacteria was carried out after the violacein solution and the pH-sensitive liposomes of violacein solution were placed in the sun for continuous irradiation:

[0104] A certain volume of the violacein solution and the pH-sensitive liposomes of violacein solution were placed in a sunny place and irradiated continuously for 6 days, and the color changes were recorded to study the effect of light on the antibacterial effect of violacein. The results are as Figure 8 、 9 、10, 11 shown.

[0105] From Figures 8 - 11 the results, it can be seen that when the violacein solution is continuously irradiated, it will lose its biological activity and thus has no inhibitory effect on bacteria. However, the pH-sensitive liposomes of violacein solution still have a certain inhibitory effect on bacteria.

[0106] Test Example 6

[0107] This test example is used to verify the inhibitory effect of the pH-sensitive liposomes of violacein of the present invention on the growth of HeLa cervical cancer cells.

[0108] HeLa cells in the logarithmic growth phase with good growth status were inoculated into a 12-well culture plate and cultured in an incubator at 37°C with 5% CO2. After 24 hours, the 12-well culture plate of HeLa cells was taken out and placed under an inverted microscope to observe the growth status and cell distribution. On a sterile ultra-clean workbench, after discarding the liquid, the cells were washed twice with PBS buffer solution and then dried by suction. Solutions of violacein and pH-sensitive liposomes of violacein at different concentrations of 0, 2.5, 5, 10, 15, and 30 μg / mL were added, and the volume was made up to 1 mL with culture medium. After culturing in an incubator at 37°C with 5% CO2 for 24 hours, the solution in the 12-well plate was first aspirated dry with a pipette gun, taking care that the tip of the pipette did not touch the cells at the bottom of the well. Preparation of the working solution: The stock solutions of calcein AM and PI reagents were taken out and equilibrated at room temperature for 30 minutes. 5 μL of the 16 mM PI stock solution was added to 10 mL of PBS buffer solution. 20 μL of the 1 mM calcein AM was added to 10 mL of the PI solution and mixed well to prepare the staining agent. 500 μL of the staining agent was added to the above-mentioned washed 12-well plate and incubated at room temperature in the dark for 30 minutes. After the cells were stained, the staining agent was discarded, 500 μL of PBS was added, and the cells were observed and photographed under an inverted fluorescence microscope. The results are as Figure 12 shown.

[0109] As Figure 12 shown by the results, in the case of 0 μg / mL, that is, without adding the drug, it can be seen that the cells are basically completely alive, and there are almost no red light spots, that is, there are no dead cells. With the increase in the drug concentration of violacein and violacein liposomes, the number of dead cells increased significantly, indicating that the proliferation of HeLa cervical cancer cells was inhibited by violacein and violacein liposomes. Moreover, with the increase in the drug concentration, the inhibitory effect was more obvious, and the mortality rate also increased significantly, proving that violacein has an obvious effect on the proliferation and growth of HeLa cervical cancer cells. In addition, after violacein was encapsulated by liposomes, it did not affect the release of the drug, and improved the dispersion of violacein in water, thereby increasing its biological utilization rate.

[0110] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A violacein pH-sensitive liposome, characterized in that, It includes liposomes and violacein encapsulated within the liposomes; The liposomes are prepared from phosphatidylethanolamine, cholesteryl succinate monoesters, and cholesterol; among them, phosphatidylethanolamine is a pH-sensitive material with a pKa of 5.0 to 6.0; cholesteryl succinate monoesters are emulsifiers and liposome carrier materials; cholesterol is a biomembrane material; in the composition of the liposomes: the mass ratio of phosphatidylethanolamine, cholesteryl succinate monoesters, and cholesterol is (6 to 8):(2 to 4):(1 to 2); As the encapsulated bioactive ingredient, the addition amount of violacein is 2% to 5% of the total mass of phosphatidylethanolamine, cholesteryl succinate monoesters, and cholesterol.

2. A method for preparing the violacein pH-sensitive liposome as described in claim 1, characterized in that, It includes the following steps: (1) Weigh violacein and dissolve it in chloroform until completely dissolved; (2) Respectively take phosphatidylethanolamine, cholesteryl succinate monoesters, and cholesterol and dissolve them in the solution obtained in step (1); (3) Place the mixture obtained in step (2) in a rotary evaporator under vacuum to remove the organic solvent until a transparent film is formed on the bottle wall; (4) Place it in a vacuum drying oven for a certain period of time to remove the residual organic solvent; (5) Add a buffer solution and hydrate it at room temperature until the transparent film can fall off; then, use probe ultrasound for 15 to 30 min to obtain the desired violacein pH-sensitive liposomes.

3. The preparation method of the violacein pH-sensitive liposome according to claim 2, wherein, In step (1), the weighed amount of violacein is 0.9 to 3.5 mg, and the volume of chloroform for dissolving violacein is 5 to 8 mL.

4. The preparation method of the violacein pH-sensitive liposome according to claim 2, wherein In step (2), the addition amounts of phosphatidylethanolamine, cholesteryl succinate monoesters, and cholesterol are 30 to 40 mg, 10 to 20 mg, and 5 to 10 mg respectively.

5. The preparation method of the violacein pH-sensitive liposome according to claim 2, characterized in that, In step (3), the temperature of the rotary evaporator under vacuum is 30 to 45 °C; In step (4), the temperature of the vacuum drying oven is 30 to 45 °C, and the placement time is 8 to 15 h.

6. The preparation method of the violacein pH-sensitive liposome according to claim 2, wherein In step (5), the buffer solution is ultrapure water, and the addition amount is 5 to 10 mL.

7. Use of the violacein pH-sensitive liposome as described in claim 1, characterized in that, Use the violacein pH-sensitive liposomes to prepare a therapeutic drug for HeLa cervical cancer.

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