CN111920960A - Protoporphyrin liposome for chemotherapeutics cell nucleus delivery and preparation method and application thereof - Google Patents
Protoporphyrin liposome for chemotherapeutics cell nucleus delivery and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a protoporphyrin liposome for chemotherapeutics cell nucleus delivery, and a preparation method and application thereof. The liposome can be prepared by a film hydration method and further ultrasonic treatment or extrusion treatment, and can wrap various chemotherapeutic drugs to form protoporphyrin liposome loaded with the chemotherapeutic drugs to realize long circulation in vivo and tumor delivery of the drugs. In addition, compared with the traditional liposome, the liposome can increase the nuclear distribution of chemotherapeutic drugs, realizes 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 the biotechnology, and particularly relates to protoporphyrin liposome for chemotherapeutics cell nucleus delivery, and a preparation method and application thereof.
Background
Chemotherapy is one of the main means for treating cancer clinically at present, however, the treatment effect of the chemotherapy is limited and the chemotherapy is accompanied by larger systemic toxicity due to poor targeting and low tumor enrichment efficiency of the chemotherapy drugs. The delivery of the chemotherapeutic drug through the nano-carrier can effectively prolong the in vivo circulation time of the chemotherapeutic drug and improve the tumor enrichment efficiency of the drug. However, the final action target of most common chemotherapeutic drugs (such as adriamycin, cisplatin and camptothecin) is cell nucleus, and the encapsulation of the nanocarrier can change the subcellular localization of the chemotherapeutic drugs, so that the efficiency of the chemotherapeutic drugs entering the cell nucleus is greatly reduced. The process of drug uptake by cells is very complicated, many nano-drugs are inevitably captured by lysosomes after entering the cells and are difficult to escape from the lysosomes, and nuclear pore complexes exist on the surface of the cell nucleus, which generally only allow small molecular weight substances to freely enter and exit the cell nucleus, while substances with larger molecular weight are difficult to pass through the nuclear pore complexes. Therefore, many nano-carriers have difficulty in accessing the nucleus to exert their effects due to the difficulty in releasing the small-molecule drug loaded therein in time after delivering the drug to the tumor region. For example, although liposome adriamycin (DOXIL) used clinically at present has reduced cardiotoxicity and higher tumor enrichment efficiency than adriamycin, the efficiency of adriamycin loaded in the liposome adriamycin after reaching a tumor area to enter the nucleus of tumor cells is very low, so that most adriamycin molecules are difficult to exert chemotherapy effect, and the anticancer efficacy of adriamycin is greatly reduced. Therefore, how to develop a nano carrier which can not only stably wrap chemotherapeutic drugs to realize long circulation in vivo and tumor enrichment, but also ensure the nuclear delivery effect of the drugs is a problem to be solved urgently.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a protoporphyrin liposome for chemotherapeutics nuclear delivery, which can rapidly release the encapsulated drugs after contacting with cell membranes, thereby realizing the nuclear delivery of various chemotherapeutics.
The invention also provides a preparation method and application of the protoporphyrin liposome for the nuclear delivery of chemotherapeutic drugs.
The technical scheme is as follows: in order to achieve the above purpose, the protoporphyrin liposome for chemotherapy drug nuclear delivery is mainly composed of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin.
Wherein the molar ratio of the hydrogenated soybean lecithin, the distearoylphosphatidylethanolamine-polyethylene glycol 2000, the cholesterol and the protoporphyrin is 63.3: 5: 31.7: (1-10).
Preferably, the molar ratio of hydrogenated soy lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol, and protoporphyrin is 63.3: 5: 31.7: 5.
the preparation method of the protoporphyrin liposome for the nuclear delivery of the chemotherapeutic drugs comprises the following steps:
(1) dissolving hydrogenated soybean lecithin, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin in a mixed solution of chloroform and methanol, and uniformly mixing;
(2) drying the mixed solution by nitrogen and vacuum drying to form a film;
(3) adding phosphate buffer solution into the film for hydration, and carrying out ultrasonic treatment or extrusion to form the liposome.
Wherein, the phosphate buffer is added in the step (3) for hydration, and the liposome is formed by extrusion through a probe ultrasonic or an extruder under the condition of 55-75 ℃.
The protoporphyrin liposome is applied to realizing the delivery of the cell nucleus of a chemotherapeutic drug, wherein the chemotherapeutic drug has an action site of the cell nucleus, such as adriamycin, daunorubicin, cisplatin, carboplatin, camptothecin, taxol and the like.
The preparation method of the protoporphyrin liposome loaded with the chemotherapeutic drug comprises the following steps:
(1) dissolving hydrogenated soybean lecithin, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin in a mixed solution of chloroform and methanol, and uniformly mixing;
(2) drying the mixed solution by nitrogen and vacuum drying to form a film;
(3) dissolving chemotherapeutic medicine in phosphate buffer solution to prepare medicine solution;
(4) adding the prepared drug solution in the step (3) into the film prepared in the step (2) for hydration, and carrying out ultrasonic treatment or extrusion to form liposome;
(5) and (4) dialyzing the liposome obtained in the step (4) in a phosphate buffer solution, and removing the drug which cannot be encapsulated in the liposome to obtain the protoporphyrin liposome loaded with the chemotherapeutic drug.
Wherein the chemotherapy drugs comprise chemotherapy drugs acting on cell nucleus, such as adriamycin, daunorubicin, cisplatin, carboplatin, camptothecin or paclitaxel.
Wherein, in the step (4), the prepared drug solution in the step (3) is added into the film prepared in the step (2) for hydration, and the liposome is formed by extrusion through a probe ultrasonic or an extruder under the condition of 55-75 ℃.
Wherein, the liposome obtained in the step (5) is dialyzed for 20-24h in phosphate buffer solution by using a dialysis bag with the molecular weight cutoff of 3500 Da.
Dissolving the chemotherapeutic drug in phosphate buffer solution, hydrating the film with the phosphate buffer solution in which the chemotherapeutic drug is dissolved, performing ultrasonic treatment or extruding, and dialyzing in the phosphate buffer solution by using a dialysis bag with the molecular weight cutoff of 3500Da to remove the uncoated chemotherapeutic drug.
The protoporphyrin liposome loaded with the chemotherapeutic drug prepared by the preparation method is provided by the invention.
The liposome of the present invention consists of three lipid components (hydrogenated soy lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and cholesterol) and protoporphyrin. The protoporphyrin liposome is formed by further performing ultrasonic treatment or extrusion treatment on four components, namely hydrogenated soybean lecithin, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin, by a film hydration method, and can realize the encapsulation of various small-molecule chemotherapeutic drugs. The internal water cavity and the hydrophobic tail chain of the liposome formed by the invention can be respectively loaded with hydrophilic drugs and hydrophobic drugs, and are good drug carriers, and the water cavity is loaded with therapeutic drugs such as adriamycin, cisplatin and the like.
The protoporphyrin molecules have high affinity to the cell membrane of a mammal, can be quickly separated from the liposome and anchored on the cell membrane when contacting with the cell membrane, damage the integrity of the liposome, quickly release the drug wrapped by the protoporphyrin liposome loading chemotherapeutic drug and realize the nuclear delivery of the drug. Meanwhile, the protoporphyrin liposome has the advantages of good stability, longer in-vivo circulation time, higher tumor enrichment efficiency and the like, can realize efficient nuclear drug delivery, and greatly improves the tumor treatment effect of chemotherapeutic drugs. In addition, compared with the traditional liposome, the liposome can increase the nuclear distribution of chemotherapeutic drugs, and realize more efficient nuclear drug delivery and cell killing.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the preparation method is simple and convenient: the preparation process of the protoporphyrin liposome does not involve chemical reaction and complex purification process;
(2) good stability and drug loading effect: the liposome composition comprises distearoylphosphatidylethanolamine-polyethylene glycol 2000, which can impart the whole liposome with stable properties in vivo under long circulation and physiological conditions. The other two components, hydrogenated soy lecithin and cholesterol, further ensure the stability of the liposomes. In addition, the internal water cavity and the hydrophobic tail chain of 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 doping of protoporphyrin, the protoporphyrin liposome loaded chemotherapeutic drug can be rapidly disintegrated and release the wrapped drug when contacting with a cell membrane, so that the chemotherapeutic drug can efficiently enter a cell nucleus to play an anticancer curative effect, and more efficient cell nucleus drug delivery and cell killing are realized;
in conclusion, the protoporphyrin liposome disclosed by the invention has the advantages of good stability, longer in vivo circulation time, higher tumor enrichment efficiency and the like, can realize efficient nuclear drug delivery, greatly improves the tumor treatment effect of a chemotherapeutic drug, can be used for preparing the protoporphyrin liposome loaded with the chemotherapeutic drug, and is expected to become a universal chemotherapeutic drug nuclear delivery carrier.
Drawings
FIG. 1 is a schematic representation of the structure of protoporphyrin liposomes and doxorubicin-loaded protoporphyrin liposomes (protoporphyrin/doxorubicin liposomes) of the present invention;
FIG. 2 is a graphical representation of the one-week stability of protoporphyrin liposomes and blank liposomes of the present invention;
FIG. 3 is a schematic representation of the intracellular distribution of the protoporphyrin liposomes encapsulated doxorubicin of the present invention;
FIG. 4 is a schematic representation of the cytotoxicity of protoporphyrin liposomes of the present invention, encapsulated in doxorubicin, against human breast cancer cells;
FIG. 5 is a schematic representation of the cytotoxicity of protoporphyrin liposomes of the present invention encapsulated cisplatin towards human lung carcinoma cells;
FIG. 6 is a photograph of an image of a mouse after doxorubicin has been encapsulated in protoporphyrin liposomes of the present invention;
FIG. 7 is a graph showing the effect of the protoporphyrin liposome encapsulated with doxorubicin on the tumor.
Detailed Description
The invention is further illustrated by the following figures and examples.
Materials, reagents and the like used in examples are commercially available unless otherwise specified.
Hydrogenated soybean lecithin and distearoylphosphatidylethanolamine-polyethylene glycol 2000 were purchased from Shanghai Everet pharmaceutical science, Inc., cholesterol was purchased from Shanghai Meclin Biochemical science, Inc., protoporphyrin, cisplatin and carboplatin were purchased from Aladdin reagent (Shanghai), doxorubicin hydrochloride was purchased from Beijing Huavong Bibock, Inc., and paclitaxel was purchased from Beijing Sorbox Tech, Inc. The phosphate buffers in the examples were all phosphate buffers for cells, pH 7.4, and ionic strength 150 mM.
Example 1
Step 1: dissolving 13.07mg of hydrogenated soybean lecithin, 3.70mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, 3.23mg of cholesterol and 0.74mg of protoporphyrin in 1mL of a mixed organic solvent of chloroform and methanol (chloroform: methanol 65: 35 by volume), and mixing uniformly;
step 2: blowing the mixed solution obtained in the step 1 by using nitrogen, and performing vacuum drying for 12 hours to form a film;
and step 3: the film formed in step 2 was hydrated with 5mL of phosphate buffer, and subjected to probe sonication (sonication power: 163W, total sonication time: 40s (sonication 4s, stop 2s, repeat 10 times)) at 60 ℃ to form a protoporphyrin liposome dispersion.
The structure of the protoporphyrin liposomes prepared in this example is shown in figure 1. The liposome is integrally formed into a vesicular structure, the inside is a water cavity, and both phospholipid molecules (HSPC) and DSPE-PEG can provide a hydrophobic tail chain.
Example 2
Evaluation of the stability of the protoporphyrin liposomes prepared in example 1:
the protoporphyrin liposome dispersion obtained in example 1 and the liposome dispersion containing no protoporphyrin (no protoporphyrin was added in the preparation process of example 1) were mixed with a phosphate buffer containing 10% fetal bovine serum at a volume ratio of 1:9, respectively, and placed at 37 ℃. Particle size was monitored over one week for both liposomes. The particle size of the liposomes was measured by a potential-particle size analyzer.
The results are shown in fig. 2, and the particle sizes of both liposomes do not change significantly within one week, indicating that both protoporphyrin liposomes and blank liposomes have good stability.
Example 3
Encapsulation of chemotherapeutic drug doxorubicin by protoporphyrin liposomes prepared in example 1:
step 1: dissolving 13.07mg of hydrogenated soybean lecithin, 3.70mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, 3.23mg of cholesterol and 0.74mg of protoporphyrin in 1mL of a mixed organic solvent of chloroform and methanol (chloroform: methanol 65: 35 by volume), and mixing uniformly;
step 2: blowing the mixed solution obtained in the step 1 by using nitrogen, and performing vacuum drying for 12 hours to form a film;
and step 3: dissolving 5mg of doxorubicin hydrochloride in 5mL of phosphate buffer solution to obtain a 1mg/mL doxorubicin solution;
and 4, step 4: the film formed in step 2 was mixed with the whole of the doxorubicin solution in step 3, and subjected to probe sonication (sonication power: 163W, total sonication time: 40s (sonication 4s, stop 2s, repeat 10 times)) at 60 ℃ to form a liposome dispersion.
And 5: and (3) 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 cutoff of 3500Da to obtain the purified protoporphyrin liposome loaded with the chemotherapeutic drug, namely the protoporphyrin liposome dispersion liquid (marked as protoporphyrin/adriamycin liposome) wrapped with adriamycin.
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 adriamycin is loaded in the water cavity. In addition, both phospholipid molecules (HSPC) and DSPE-PEG can provide hydrophobic tails.
Example 4
The intracellular distribution of the product protoporphyrin/doxorubicin liposome obtained in example 3 after interaction with human breast cancer cells was observed:
after culturing human breast cancer cells in an 8-well plate for 24 hours, protoporphyrin/doxorubicin liposome was dispersed in DMEM complete medium at an doxorubicin concentration of 2. mu.M and added to an 8-well plate (200. mu.L/well) at 37 ℃ with 5% CO2After incubation for 2 hours in the environment, the original medium was discarded. The nuclear dye Hoechst 33342 was dispersed in phosphate buffer at 10. mu.g/mL and added to 8-well plates (200. mu.L/well), incubated for 10 minutes, washed twice with phosphate buffer, supplemented with fresh DMEM complete medium, and observed by confocal fluorescence microscopy.
And (3) confocal fluorescence microscope imaging observation: doxorubicin fluoresces orange-red under 488nm excitation and the nuclear dye Hoechst 33342 fluoresces blue under 405nm excitation, as shown in figure 3. As can be seen, the protoporphyrin/doxorubicin liposome can rapidly enter the nucleus in large quantities after being incubated with human breast cancer cells, indicating that the liposome can effectively deliver doxorubicin into the nucleus of cancer cells.
Example 5
The product protoporphyrin/doxorubicin liposomes obtained in example 3 were evaluated for toxicity to human breast cancer cells:
human breast cancer cells were cultured in a 96-well plate for 24 hours, the original medium was discarded, and then 100. mu.L of doxorubicin hydrochloride (designated "doxorubicin") having doxorubicin concentrations of 0.2, 0.5, 1.0, 1.5, 2.0, and 4.0. mu.M, liposome-encapsulated doxorubicin containing no protoporphyrin (designated "doxorubicin liposome" in the preparation of example 3 without protoporphyrin), and DMEM complete medium containing protoporphyrin/doxorubicin liposome were added thereto, and the mixture was incubated at 37 ℃ and 5% CO2And incubated for 24 hours. The cytotoxicity of these materials was finally evaluated using the MTT assay, and the results are shown in fig. 4.
The experimental result of fig. 3 shows that the protoporphyrin/doxorubicin liposome can kill cancer cells efficiently, and compared with free doxorubicin and doxorubicin liposome, the cytotoxicity of the protoporphyrin/doxorubicin liposome is improved significantly. It is thus also demonstrated that the liposome achieves efficient killing of tumor cells by efficiently delivering doxorubicin into the tumor cell nucleus, because the stronger affinity of protoporphyrin for the cell membrane when the protoporphyrin-containing liposome enters the cell transfers to the cell membrane, which results in the liposome disintegrating to release doxorubicin, and the released free doxorubicin can enter the cell nucleus, but the liposome not containing protoporphyrin cannot achieve this process.
Example 6
Encapsulation of chemotherapeutic cisplatin by liposome:
step 1: 13.07mg of hydrogenated soybean lecithin, 3.70mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000, 3.23mg of cholesterol, and 0.74mg of protoporphyrin were co-dissolved in 1mL of a mixed organic solvent of chloroform and methanol (chloroform: methanol 65: 35, volume ratio);
step 2: blowing the mixed solution obtained in the step 1 by using nitrogen, and performing vacuum drying for 12 hours to form a film;
and step 3: 2mg of cisplatin is dissolved in 5mL of phosphate buffer solution to obtain a cisplatin solution with the concentration of 400 mug/mL;
and 4, step 4: mixing the film formed in the step 2 with the whole cisplatin solution in the step 3, and performing probe ultrasound (ultrasound power: 163W, total ultrasound time: 40s (ultrasound 4s, stop 2s, repeat 10 times)) at 60 ℃ to form a liposome dispersion liquid;
and 5: and (3) 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 cutoff of 3500Da to obtain the purified cisplatin-coated protoporphyrin liposome (marked as "protoporphyrin/cisplatin liposome").
Example 7
The product obtained in example 6 was evaluated for toxicity to human lung cancer cells:
human lung cancer cells were cultured in 96-well plates for 24 hours, the original medium was discarded, and 100. mu.L of a complete medium containing cisplatin at a concentration of 0.5, 1.0, 1.5, 2.0, 2.5. mu.M, cisplatin encapsulated in a liposome containing no protoporphyrin (no protoporphyrin was added in the preparation of example 6, and referred to as "cisplatin liposome") and protoporphyrin/cisplatin liposome was added thereto, respectively, in a DMEM complete medium at 37 ℃ with 5% CO2And incubated for 24 hours. Finally, the material was evaluated for fineness using the MTT assayCytotoxicity, results are shown in figure 5.
The experimental result of fig. 5 shows that the protoporphyrin/cisplatin liposome can kill cancer cells efficiently, and compared with free cisplatin and cisplatin liposome, the cytotoxicity of the protoporphyrin/cisplatin liposome is improved significantly. It is also demonstrated that the liposome achieves high killing efficiency on tumor cells by delivering cisplatin into the tumor cell nucleus with high efficiency.
Example 8
The product protoporphyrin/doxorubicin liposomes obtained in example 3 were evaluated for their tumor enrichment in vivo:
several healthy 4-week-old BALA/c athymic female nude mice were collected and injected with human breast cancer cells subcutaneously. When the tumor volume reaches 50mm3At the left and right, the body weight of the mice was measured, and the required dose of doxorubicin (5mg/kg) was calculated from the actual body weight of the mice. Protoporphyrin/doxorubicin liposome obtained in example 3 containing the above doxorubicin dose was diluted with phosphate buffer to a final volume of 200 μ L and injected into nude mice by tail vein injection, and the in vivo fluorescence distribution thereof was observed over three days, and the results are shown in fig. 6.
The experimental result of fig. 6 shows that the protoporphyrin/adriamycin liposome has better tumor enrichment effect and more ideal tumor retention time.
Example 9
The product protoporphyrin/doxorubicin liposomes obtained in example 3 were evaluated for their in vivo anticancer effects:
several healthy 4-week-old BALA/c athymic female nude mice were collected and injected with human breast cancer cells subcutaneously. When the tumor volume reaches 50mm3At the left and right, the body weight of the mice was measured, and the required dose of doxorubicin (5mg/kg) was calculated from the actual body weight of the mice. Protoporphyrin/doxorubicin liposome, free doxorubicin and doxorubicin liposome obtained in example 3 containing the doxorubicin dose were diluted with phosphate buffer solution to a final volume of 200. mu.L, and injected into nude mice by tail vein injection, while tumor-bearing mice injected with 200. mu.L of phosphate buffer solution in tail vein were used as control group and injected with the control groupThe tumor size was observed on day 14 after the injection, and the results are shown in FIG. 7.
The experimental result of figure 7 shows that the protoporphyrin/adriamycin liposome has effective in vivo anticancer effect, compared with free adriamycin and adriamycin liposome, the protoporphyrin/adriamycin liposome has more obvious tumor inhibition effect, and can basically and completely inhibit the growth of tumor within 14 days, thus proving that the protoporphyrin liposome has better application prospect of drug delivery.
Example 10
Example 10 the same procedure as in example 1 was conducted, except that the molar ratio of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol, and protoporphyrin was 63.3: 5: 31.7: 1; and (3) adding phosphate buffer solution for hydration, and extruding by an extruder at the temperature of 55 ℃ to form the liposome.
Example 11
Example 11 the same procedure as in example 1 was conducted, except that the molar ratio of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin was 63.3: 5: 31.7: 10; and (3) adding phosphate buffer solution for hydration, and forming the liposome by ultrasonic treatment at the temperature of 75 ℃.
Example 12
Example 12 was prepared according to the same manner as in example 3, except that the molar ratio of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol, and protoporphyrin was 63.3: 5: 31.7: 1; 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 at 55 ℃ by using an extruder to form protoporphyrin liposome dispersion liquid; and (4) 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 chemotherapy drug-loaded protoporphyrin liposome.
Example 13
Example 13 was prepared according to the same manner as in example 3, except that the molar ratio of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-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 using an extruder at 75 ℃ to form protoporphyrin liposome dispersion liquid; and (4) dialyzing the protoporphyrin liposome dispersion liquid formed in the step (4) in a phosphate buffer solution for 22h by a dialysis bag with the molecular weight cutoff of 3500Da to obtain the chemotherapy drug-loaded protoporphyrin liposome.
Claims (10)
1. A protoporphyrin liposome for nuclear delivery of chemotherapeutic drugs, which consists essentially of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin.
2. The protoporphyrin liposome of claim 1, wherein the molar ratio of hydrogenated soybean lecithin, distearoylphosphatidylethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin is 63.3: 5: 31.7: (1-10).
3. A method of preparing protoporphyrin liposomes for nuclear delivery of chemotherapeutic agents as defined in claim 1 or 2, comprising the steps of:
(1) dissolving hydrogenated soybean lecithin, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin in a mixed solution of chloroform and methanol, and uniformly mixing;
(2) drying the mixed solution by nitrogen and vacuum drying to form a film;
(3) adding phosphate buffer solution into the film for hydration, and carrying out ultrasonic treatment or extrusion to form the liposome.
4. The method according to claim 3, wherein the step (3) is performed by adding phosphate buffer for hydration and extruding the hydrated solution at 55-75 ℃ by means of probe ultrasound or an extruder to form the liposome.
5. Use of a protoporphyrin liposome for nuclear delivery of chemotherapeutic agents as defined in claim 1 or 2 for effecting nuclear delivery of chemotherapeutic agents.
6. A preparation method of protoporphyrin liposome loaded with chemotherapeutic drugs is characterized by comprising the following steps:
(1) dissolving hydrogenated soybean lecithin, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, cholesterol and protoporphyrin in a mixed solution of chloroform and methanol, and uniformly mixing;
(2) drying the mixed solution by nitrogen and vacuum drying to form a film;
(3) dissolving chemotherapeutic medicine in phosphate buffer solution to prepare medicine solution;
(4) adding the prepared drug solution in the step (3) into the film prepared in the step (2) for hydration, and carrying out ultrasonic treatment or extrusion to form liposome;
(5) and (4) dialyzing the liposome obtained in the step (4) in a phosphate buffer solution, and removing the drug which cannot be wrapped in the liposome to obtain the protoporphyrin liposome loaded with the chemotherapeutic drug.
7. A process for the preparation of chemotherapeutic drug loaded protoporphyrin liposomes according to claim 6, wherein the chemotherapeutic drug preferably comprises doxorubicin, daunorubicin, cisplatin, carboplatin, camptothecin or paclitaxel.
8. A process for preparing chemotherapy drug-loaded protoporphyrin liposomes according to claim 6, wherein the drug solution prepared in step (3) is added to the film prepared in step (2) in step (4) to hydrate and the liposomes are formed by extrusion through a probe ultrasound or an extruder at 55-75 ℃.
9. The method for preparing protoporphyrin liposome carrying chemotherapeutic drugs according to claim 6, wherein the liposome obtained in step (5) is dialyzed in phosphate buffer for 20-24h using dialysis bag with molecular weight cutoff of 3500 Da.
10. A chemotherapeutic drug loaded protoporphyrin liposome prepared by the method of claim 6.
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