CN111920960B - Protoporphyrin liposome for chemotherapy drug cell nucleus delivery and preparation method and application thereof - Google Patents
Protoporphyrin liposome for chemotherapy drug cell nucleus delivery and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a protoporphyrin liposome for cell nucleus delivery of a chemotherapeutic drug, and a preparation method and application thereof, wherein the liposome mainly comprises four components of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin. The liposome can be prepared by a film hydration method and further ultrasonic or extrusion treatment, and can encapsulate various chemotherapeutics to form protoporphyrin liposome loaded with the chemotherapeutics to realize in vivo long circulation and tumor delivery of the drugs. In addition, compared with the traditional liposome, the liposome can increase the nuclear distribution of the chemotherapeutic drugs, realize more efficient nuclear drug delivery and cell killing, has good safety and universality, and is expected to become a universal chemotherapeutic drug nuclear delivery carrier.
Description
Technical Field
The invention belongs to biotechnology, and particularly relates to protoporphyrin liposome for cell nucleus delivery of a chemotherapeutic drug, and a preparation method and application thereof.
Background
Chemotherapy is one of the main means for treating cancer clinically at present, however, the therapeutic effect is limited due to poor targeting of chemotherapeutic drugs, low tumor enrichment efficiency and other reasons, and the therapeutic effect is accompanied by greater systemic toxicity. The delivery of the chemotherapeutic drugs through the nano-carrier can effectively prolong the in vivo circulation time of the chemotherapeutic drugs and improve the tumor enrichment efficiency of the drugs. However, the final target of most common chemotherapeutics (such as doxorubicin, cisplatin and camptothecin) is the cell nucleus, and the encapsulation by the nano-carrier often changes the subcellular localization of the chemotherapeutics, so that the efficiency of entering the cell nucleus is greatly reduced. The process of taking up drugs by cells is very complex, many nano-drugs are inevitably captured by lysosomes after entering cells and are difficult to escape from lysosomes, and nuclear pore complexes exist on the surface of the cell nucleus, which usually only allow substances with small molecular weight to freely enter and exit the cell nucleus, while substances with larger molecular weight are difficult to pass through the nuclear pore complexes. Thus, many nanocarriers have difficulty in allowing the drug to enter the nucleus for action due to the difficulty in releasing the small molecule drug loaded therein in time after delivering the drug to the tumor area. For example, although liposomal Doxorubicin (DOXIL) used clinically at present has reduced cardiotoxicity and higher tumor enrichment efficiency than doxorubicin, the efficiency of doxorubicin loaded therein entering the tumor cell nucleus after reaching the tumor area is very low, so that most of doxorubicin molecules are difficult to exert chemotherapy effect, thereby greatly reducing the anticancer efficacy of doxorubicin. Therefore, how to develop a nano-carrier which can stably wrap a chemotherapeutic drug to realize long circulation and tumor enrichment in vivo and ensure the cell nucleus delivery effect of the drug is a problem to be solved.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the invention provides protoporphyrin liposome for the cell nucleus delivery of chemotherapeutic drugs, which can rapidly release the wrapped drugs after contacting cell membranes, thereby realizing the cell nucleus delivery of various chemotherapeutic drugs.
The invention also provides a preparation method and application of the protoporphyrin liposome for chemotherapy drug cell nucleus delivery.
The technical scheme is as follows: in order to achieve the aim of the invention, the protoporphyrin liposome for cell nucleus delivery of the chemotherapeutic drugs is mainly composed of four components of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin.
Wherein, the mole ratio of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin is 63.3:5:31.7: (1-10).
Preferably, the molar ratio of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin is 63.3:5:31.7:5.
the preparation method of protoporphyrin liposome for chemotherapy drug cell nucleus delivery comprises the following steps:
(1) The hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin are dissolved in a mixed solution of chloroform and methanol, and uniformly mixed;
(2) Blowing the mixed solution by nitrogen and drying in vacuum to form a film;
(3) Phosphate buffer is added into the film for hydration, and liposome is formed by ultrasonic or extrusion.
Wherein, the phosphate buffer solution is added for hydration in the step (3), and the liposome is formed by extrusion through a probe ultrasonic or extrusion instrument under the condition of 55-75 ℃.
The protoporphyrin liposome is applied to realizing the cell nucleus delivery of chemotherapeutic drugs, wherein the chemotherapeutic drugs are the chemotherapeutic drugs with cell nuclei as the action sites, such as doxorubicin, daunorubicin, cisplatin, carboplatin, camptothecine, taxol and the like.
The preparation method of the protoporphyrin liposome loaded with the chemotherapeutic drug comprises the following steps:
(1) The hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin are dissolved in a mixed solution of chloroform and methanol, and uniformly mixed;
(2) Blowing the mixed solution by nitrogen and drying in vacuum to form a film;
(3) Dissolving a chemotherapeutic drug in a phosphate buffer solution to prepare a drug solution;
(4) Adding the drug solution prepared in the step (3) into the film prepared in the step (2) for hydration, and performing ultrasonic treatment or extrusion to form liposome;
(5) Dialyzing the liposome obtained in the step (4) in phosphate buffer, and removing the medicine which cannot be wrapped in the liposome to obtain the protoporphyrin liposome loaded with the chemotherapeutic medicine.
Wherein the chemotherapeutic medicine comprises doxorubicin, daunorubicin, cisplatin, carboplatin, camptothecine or taxol and other chemotherapeutic medicines acting on the cell nucleus.
Wherein, in the step (4), the drug solution prepared in the step (3) is added into the film prepared in the step (2) for hydration, and the film is extruded by a probe ultrasonic or an extruder at 55-75 ℃ to form liposome.
Wherein, the liposome obtained in the step (5) is dialyzed for 20-24 hours in phosphate buffer by using a dialysis bag with the molecular weight cutoff of 3500 Da.
The chemotherapeutic medicine is dissolved in phosphate buffer, the film is hydrated by the phosphate buffer dissolved with the chemotherapeutic medicine, and the non-encapsulated chemotherapeutic medicine is removed by dialysis in the phosphate buffer with a dialysis bag with the molecular weight cut-off of 3500Da after ultrasonic treatment or extrusion.
The protoporphyrin liposome loaded with the chemotherapeutic drug, which is prepared by the preparation method, is provided.
The liposome of the present invention consists of three lipid components (hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol) and protoporphyrin. The protoporphyrin liposome is formed by subjecting four components of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin to a film hydration method and further performing ultrasonic or extrusion treatment, so that the coating of various small molecule chemotherapeutic drugs can be realized. The liposome formed by the invention can be respectively loaded with hydrophilic and hydrophobic drugs in the inner water cavity and the hydrophobic tail chain, and is a good drug carrier, and chemotherapeutic drugs such as doxorubicin, cisplatin and the like are loaded in the water cavity.
The protoporphyrin molecule has higher affinity to mammalian cell membrane, can be quickly separated from liposome and anchored on the cell membrane when contacting with the cell membrane, damages the integrity of the liposome and quickly releases the medicament wrapped by the chemotherapeutic medicament loaded by the protoporphyrin liposome, and realizes the nuclear delivery of the medicament. Meanwhile, the preparation method is simple, the safety is good, the universality is strong, the protoporphyrin liposome has the advantages of good stability, longer in vivo circulation time, higher tumor enrichment efficiency and the like, and meanwhile, the efficient nuclear drug delivery can be realized, so that the tumor treatment effect of the chemotherapeutic drug is greatly improved. In addition, compared with the traditional liposome, the liposome can increase the nuclear distribution of the chemotherapeutic drugs, and realize more efficient nuclear drug delivery and cell killing.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) The preparation method is simple: the preparation process of the protoporphyrin liposome does not involve chemical reaction and complex purification process;
(2) Good stability and drug loading effect: the components of the liposome include distearoyl phosphatidylethanolamine-polyethylene glycol 2000, which can impart stable properties to the whole liposome under long circulation and physiological conditions in vivo. The other two components, hydrogenated soybean lecithin and cholesterol, further ensure the stability of the liposome. In addition, the water cavity and the hydrophobic tail chain in the liposome can be respectively loaded with hydrophilic and hydrophobic drugs, so that the liposome becomes a good drug carrier;
(3) Efficient nuclear drug delivery efficiency: due to the incorporation of protoporphyrin, the protoporphyrin liposome of the invention can rapidly disintegrate and release the wrapped medicament when contacting with cell membranes, so that the chemotherapy medicament can enter the cell nucleus to exert anticancer effect with high efficiency, and more efficient cell nucleus medicament delivery and cell killing are realized;
in conclusion, the protoporphyrin liposome has the advantages of good stability, longer in vivo circulation time, higher tumor enrichment efficiency and the like, can realize efficient cell nucleus drug delivery, greatly improves the tumor treatment effect of the chemotherapeutic drug, can be used for preparing the protoporphyrin liposome loaded with the chemotherapeutic drug, and is hopeful to become a universal chemotherapeutic drug cell nucleus delivery carrier.
Drawings
FIG. 1 is a schematic diagram of the structure of protoporphyrin liposome and doxorubicin-loaded protoporphyrin liposome (protoporphyrin/doxorubicin liposome) of the present invention;
FIG. 2 is a schematic representation of one week stability of protoporphyrin liposomes and blank liposomes of the present invention;
FIG. 3 is a schematic diagram showing intracellular distribution of protoporphyrin liposome coated with doxorubicin according to the present invention;
FIG. 4 is a schematic representation of cytotoxicity of protoporphyrin liposome of the present invention on human breast cancer cells after encapsulation of doxorubicin;
FIG. 5 is a schematic of cytotoxicity of human lung cancer cells after encapsulation of cisplatin with protoporphyrin liposome of the present invention;
FIG. 6 is an in vivo image of mice after encapsulation of doxorubicin with protoporphyrin liposomes of the present invention;
FIG. 7 is a graph showing the effect of protoporphyrin liposome on tumor therapy after encapsulation of doxorubicin according to the present invention.
Detailed Description
The invention is further described below with reference to the drawings and examples.
Materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Hydrogenated soybean lecithin and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 were purchased from Shanghai Ai Weite pharmaceutical technologies, cholesterol was purchased from Shanghai Michelin Biochemical technologies, protoporphyrin, cisplatin and carboplatin were purchased from Ara Ding Shiji (Shanghai) Inc., doxorubicin hydrochloride was purchased from Beijing Huavone Bibo technologies, and paclitaxel was purchased from Beijing Soy Bao technologies, inc. The phosphate buffer in the examples was phosphate buffer for cells, pH 7.4, and ionic strength 150mM.
Example 1
Step 1: 13.07mg of hydrogenated soybean lecithin, 3.70mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000, 3.23mg of cholesterol and 0.74mg of protoporphyrin are dissolved in 1mL of chloroform-methanol mixed organic solvent (chloroform: methanol=65:35, volume ratio) and mixed uniformly;
step 2: drying the mixed solution in the step 1 by nitrogen and drying in vacuum for 12 hours to form a film;
step 3: the film formed in step 2 was hydrated with 5mL of phosphate buffer and probe-sonicated (ultrasonic power: 163W, total time of sonication: 40s (ultrasonic 4s, stop 2s, repeat 10 times)) at 60℃to form protoporphyrin liposome dispersion.
The structure of protoporphyrin liposome prepared in this example is shown in FIG. 1. The liposome integrally forms a vesicle structure, the inside of which is a water cavity, and both phospholipid molecules (HSPC) and DSPE-PEG can provide hydrophobic tail chains.
Example 2
Stability evaluation of protoporphyrin liposome prepared in example 1:
the protoporphyrin liposome dispersion obtained in example 1 and the protoporphyrin-free liposome dispersion (no protoporphyrin was added during the preparation of example 1) were mixed with a phosphate buffer solution containing 10% fetal bovine serum at a volume ratio of 1:9, respectively, and placed at 37 ℃. Particle size was monitored for both liposomes over a week. The particle size of the liposomes was measured by a potential-particle size analyzer.
The results are shown in figure 2, and the particle size of the two liposomes is not changed obviously within one week, which indicates that the protoporphyrin liposome and the blank liposome have good stability.
Example 3
Encapsulation of chemotherapeutic drug doxorubicin by protoporphyrin liposome prepared in example 1:
step 1: 13.07mg of hydrogenated soybean lecithin, 3.70mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000, 3.23mg of cholesterol and 0.74mg of protoporphyrin are dissolved in 1mL of chloroform-methanol mixed organic solvent (chloroform: methanol=65:35, volume ratio) and mixed uniformly;
step 2: drying the mixed solution in the step 1 by nitrogen and drying in vacuum for 12 hours to form a film;
step 3: dissolving 5mg of doxorubicin hydrochloride in 5mL of phosphate buffer to obtain 1mg/mL of doxorubicin solution;
step 4: the film formed in step 2 was mixed with the entire doxorubicin solution in step 3, and probe ultrasound (ultrasound power: 163W, total ultrasound time: 40s (ultrasound 4s, 2s stopped, 10 repetitions)) was performed at 60 ℃ to form a liposome dispersion.
Step 5: dialyzing the liposome dispersion liquid formed in the step 4 in a phosphate buffer solution for 24 hours by a dialysis bag with the molecular weight cut-off of 3500Da to obtain purified protoporphyrin liposome loaded with the chemotherapeutic drug, namely protoporphyrin liposome dispersion liquid (marked as protoporphyrin/doxorubicin liposome) coated with doxorubicin.
The specific structure of protoporphyrin/doxorubicin liposome is shown in figure 1. The liposome integrally forms a vesicle structure, the inside of the liposome is a water cavity, and doxorubicin is loaded in the water cavity. In addition, both phospholipid molecules (HSPC) and DSPE-PEG can provide hydrophobic tail chains.
Example 4
The product protoporphyrin/doxorubicin liposome obtained in example 3 was observed for intracellular distribution upon interaction with human breast cancer cells:
after culturing human breast cancer cells in 8-well plates for 24 hours, protoporphyrin/doxorubicin liposomes were dispersed at doxorubicin concentration of 2. Mu.M in DMEM complete medium and added to 8-well plates (200. Mu.L/well) at 37deg.C, 5% CO 2 The original medium was discarded after incubation in the environment for 2 hours. The nuclear dye Hoechst 33342 was dispersed in phosphate buffer at 10. Mu.g/mL and added to an 8-well plate (200. Mu.L/well), incubated for 10 minutes, washed twice with phosphate buffer, and then supplemented with fresh DMEM complete medium, and observed by confocal fluorescence microscopy.
Confocal fluorescence microscopy imaging observations: doxorubicin fluoresces orange-red under excitation light of 488nm and the nuclear dye Hoechst 33342 fluoresces blue under excitation light of 405nm, the result is shown in fig. 3. From the figure, it can be seen that protoporphyrin/doxorubicin liposome can enter the nucleus rapidly and largely after incubation with human breast cancer cells, indicating that the liposome can effectively deliver doxorubicin into the nucleus of cancer cells.
Example 5
Toxicity of the product protoporphyrin/doxorubicin liposome obtained in example 3 was evaluated on human breast cancer cells:
human breast cancer cells were cultured in 96-well plates for 24 hours, and then the original medium was discarded, and 100. Mu.L of doxorubicin hydrochloride (labeled "doxorubicin"), liposome-encapsulated doxorubicin free of protoporphyrin (no protoporphyrin added during the preparation of example 3, labeled "doxorubicin liposome"), and complete DMEM medium of protoporphyrin/doxorubicin liposome were added at 37℃and 5% CO, respectively, at 0.2, 0.5, 1.0, 1.5, 2.0, and 4.0. Mu.M concentrations of doxorubicin hydrochloride, respectively 2 Is incubated for 24 hours. Finally, the cytotoxicity of these materials was evaluated by MTT assay, and the results are shown in FIG. 4.
The experimental results in fig. 3 show that protoporphyrin/doxorubicin lipids can cause efficient killing of cancer cells, and compared with free doxorubicin and doxorubicin liposomes, cytotoxicity of protoporphyrin/doxorubicin liposomes is significantly improved. It has thus been demonstrated that the liposomes achieve efficient killing of tumor cells by efficient delivery of doxorubicin into the tumor cell nucleus, since liposomes containing protoporphyrins have a greater affinity for cell membranes and transfer to the cell membranes when they enter the cell, resulting in liposome disintegration to release doxorubicin, which released free doxorubicin can enter the cell nucleus, but liposomes without protoporphyrins cannot do so.
Example 6
Encapsulation of chemotherapy drug cisplatin by liposomes:
step 1: 13.07mg of hydrogenated soybean lecithin, 3.70mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000, 3.23mg of cholesterol and 0.74mg of protoporphyrin were co-dissolved in 1mL of a chloroform-methanol mixed organic solvent (chloroform: methanol=65:35, volume ratio);
step 2: drying the mixed solution in the step 1 by nitrogen and drying in vacuum for 12 hours to form a film;
step 3: 2mg of cisplatin was dissolved in 5mL of phosphate buffer to obtain 400. Mu.g/mL of cisplatin solution;
step 4: mixing the film formed in the step 2 with the whole cisplatin solution in the step 3, and performing probe ultrasound (ultrasonic power: 163W, total ultrasonic time: 40s (ultrasonic 4s, stop 2s, repeat 10 times)) at 60 ℃ to form liposome dispersion;
step 5: the liposome dispersion formed in the step 4 is dialyzed for 24 hours in a phosphate buffer solution by a dialysis bag with the molecular weight cut-off of 3500Da, and purified protoporphyrin liposome (marked as 'protoporphyrin/cisplatin liposome') wrapped with cisplatin is obtained.
Example 7
Toxicity of the product obtained in example 6 on human lung cancer cells was evaluated:
human lung cancer cells were cultured in 96-well plates for 24 hours, after which the original medium was discarded, 100. Mu.L of cisplatin containing liposomes coated with cisplatin at cisplatin concentrations of 0.5, 1.0, 1.5, 2.0, 2.5. Mu.M (no protoporphyrin added during the preparation of example 6, labeled "cisplatin liposome") and the complete DMEM medium of protoporphyrin/cisplatin liposome were added, respectively, at 37℃and 5% CO 2 Is incubated for 24 hours. Finally, the cytotoxicity of these materials was evaluated by MTT assay, and the results are shown in FIG. 5.
The experimental results of fig. 5 show that protoporphyrin/cisplatin lipids can cause efficient killing of cancer cells, and compared with free cisplatin and cisplatin liposomes, cytotoxicity of protoporphyrin/cisplatin liposomes is significantly improved. It was also demonstrated that the liposomes achieve efficient killing of tumor cells by efficient delivery of cisplatin into the tumor cell nuclei.
Example 8
The product protoporphyrin/doxorubicin liposome obtained in example 3 was evaluated for in vivo tumor enrichment effect:
several female BALA/c athymic nude mice of 4 weeks old with good health condition are taken, and human breast cancer cells are subcutaneously injected therein. When the tumor volume reaches 50mm 3 When left and right, the mouse body weight was measured, and the required doxorubicin dose (5 mg/kg) was calculated from the actual mouse body weight. The protoporphyrin/doxorubicin liposome obtained in example 3 containing the above doxorubicin dose was diluted with phosphate buffer to a final volume of 200. Mu.L and injected into nude mice by tail vein injection, and its in vivo fluorescence distribution was observed over three days, and the results wereSee fig. 6.
The experimental result in FIG. 6 shows that protoporphyrin/doxorubicin liposome has better tumor enrichment effect and more ideal tumor retention time.
Example 9
The product protoporphyrin/doxorubicin liposome obtained in example 3 was evaluated for in vivo anticancer effect:
several female BALA/c athymic nude mice of 4 weeks old with good health condition are taken, and human breast cancer cells are subcutaneously injected therein. When the tumor volume reaches 50mm 3 When left and right, the mouse body weight was measured, and the required doxorubicin dose (5 mg/kg) was calculated from the actual mouse body weight. Protoporphyrin/doxorubicin liposomes obtained in example 3, free doxorubicin and doxorubicin liposomes containing the above doxorubicin doses were respectively diluted appropriately with phosphate buffer to a final volume of 200 μl and injected into nude mice by tail vein injection, tumor-bearing mice injected with 200 μl phosphate buffer as a control group, and their tumor sizes were observed on day 14 after injection, and the results are shown in fig. 7.
The experimental result of fig. 7 shows that the protoporphyrin/doxorubicin liposome has an effective in vivo anticancer effect, compared with free doxorubicin and doxorubicin liposome, the protoporphyrin/doxorubicin liposome has more obvious tumor inhibition effect, can basically and completely inhibit the growth of tumors within 14 days, and proves that the protoporphyrin liposome has a better application prospect of drug delivery.
Example 10
Example 10 was prepared in the same manner as in example 1, except that the molar ratio of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin was 63.3:5:31.7:1, a step of; and (3) adding phosphate buffer solution for hydration, and extruding the mixture by an extruder at 55 ℃ to form the liposome.
Example 11
Example 11 the same procedure as in example 1 was followed except that hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin were used in a molar ratio of 63.3:5:31.7:10; and (3) adding phosphate buffer solution for hydration, and forming liposome by ultrasonic under the condition of 75 ℃.
Example 12
Example 12 was prepared in the same manner as in example 3 except that the molar ratio of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin was 63.3:5:31.7:1, a step of; replacing doxorubicin hydrochloride in the step (3) with carboplatin, mixing the film formed in the step (2) with all carboplatin solution in the step (3), and extruding by an extruder at 55 ℃ to form protoporphyrin liposome dispersion; and (3) dialyzing the protoporphyrin liposome dispersion liquid formed in the step (4) in a phosphate buffer solution for 20 hours by a dialysis bag with the molecular weight cutoff of 3500Da to obtain the protoporphyrin liposome loaded with the chemotherapeutic drugs.
Example 13
Example 13 was prepared in the same manner as in example 3 except that the molar ratio of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin was 63.3:5:31.7:10; replacing doxorubicin hydrochloride in the step (3) with paclitaxel, mixing the film formed in the step (2) with all the paclitaxel solution in the step (3), and extruding by an extruder at 75 ℃ to form protoporphyrin liposome dispersion liquid; and (3) dialyzing the protoporphyrin liposome dispersion liquid formed in the step (4) in a phosphate buffer solution for 22 hours by using a dialysis bag with the molecular weight cutoff of 3500Da to obtain the protoporphyrin liposome loaded with the chemotherapeutic drugs.
Claims (4)
1. A method for preparing protoporphyrin liposome for rapidly disintegrating and releasing a chemotherapeutic drug loaded on a chemotherapeutic drug cell nucleus delivery, comprising the following steps:
(1) The hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin are dissolved in a mixed solution of chloroform and methanol, and uniformly mixed;
(2) Blowing the mixed solution by nitrogen and drying in vacuum to form a film;
(3) Dissolving a chemotherapeutic drug in a phosphate buffer solution to prepare a drug solution;
(4) Adding the drug solution prepared in the step (3) into the film prepared in the step (2) for hydration, and performing ultrasonic treatment or extrusion to form liposome;
(5) Dialyzing the liposome obtained in the step (4) in phosphate buffer to remove the drugs which cannot be wrapped in the liposome, thereby obtaining protoporphyrin liposome loaded with chemotherapeutic drugs;
the mole ratio of hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin is 63.3:5:31.7: (1-10); the chemotherapeutic drug is doxorubicin.
2. The method for preparing protoporphyrin liposome for rapid disintegration and release of chemotherapeutic drug loaded thereon according to claim 1, wherein the drug solution prepared in step (3) is added to the film prepared in step (2) for hydration in step (4), and is extruded by probe ultrasound or an extruder at 55-75 ℃ to form liposome.
3. The method for preparing protoporphyrin liposome for rapid disintegration and release of chemotherapeutic drug loaded thereon according to claim 1, wherein the liposome obtained in step (5) is dialyzed in phosphate buffer for 20-24 hours using a dialysis bag having a molecular weight cut-off of 3500 Da.
4. A protoporphyrin liposome prepared by the preparation method of claim 1, which rapidly disintegrates and releases chemotherapeutic drug loaded thereon.
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