CN110882218A

CN110882218A - Liposome composition and preparation and application thereof

Info

Publication number: CN110882218A
Application number: CN201810937361.7A
Authority: CN
Inventors: 汪贻广; 张强; 杨林洁; 莫玉霖; 万方劼
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2018-08-16
Filing date: 2018-08-16
Publication date: 2020-03-17
Anticipated expiration: 2038-08-16
Also published as: CN110882218B

Abstract

The present invention provides liposome compositions comprising liposomes and a photosensitizer, which is present in the internal aqueous phase of the liposomes, methods of their preparation and uses thereof. The liposome composition has high drug loading and encapsulation efficiency, the photosensitizer encapsulated in the liposome internal water phase is stable, the half-life period is prolonged, and the phototoxicity is obviously reduced.

Description

Liposome composition and preparation and application thereof

Technical Field

The invention relates to the field of pharmaceutical preparations and photodynamic therapy, in particular to photosensitizer liposome and preparation and application thereof.

Background

In the 80 s of the 20 th century, photodynamic therapy (PDT) emerged from a number of tumor therapies. PDT is the generation of singlet oxygen (singlet oxygen,¹O₂) And Reactive Oxygen Species (ROS), thereby killing tumor cells.

Among these photosensitizers, Chlorin e6(Chlorin e6, hereinafter abbreviated as Ce6), Protoporphyrin (Protoporphyrin IX, hereinafter abbreviated as PpIX), Hematoporphyrin monomethyl ether (Hematoporphyrin monomethylether, hereinafter abbreviated as HMME), Pyropheophorbide a hexyl ether [2- (1-hexyloxylethyl) -2-decylpyrophyllophosphorbide a, hereinafter abbreviated as HPPH ], Pyropheophorbide a (Pyropheophorbide a, hereinafter abbreviated as PPA), Benzoporphyrin derivative monoacid a (benzoylporphyrin derivative monoacid a, hereinafter abbreviated as BPD-MA), and the like are most widely used.

The common photosensitizer has porphyrin mother ring and carboxyl in the structure, and shows weak acidity. However, the above photosensitizers are all hydrophobic, and from the viewpoint of tumor, the lipophilicity admits the photosensitizers to enter cells through membranes, but clinical requirements for the photosensitizers have certain hydrophilicity so as to be convenient for intravenous injection to disperse into blood stream. Furthermore, most photosensitizers are not capable of binding selectively to tumor tissue and cannot be enriched more in tumors than normal tissues.

Since the 20 th 60 th century, liposome has been used as a nano-drug delivery carrier for a plurality of anticancer drugs in clinic, and the passive drug loading technology in the prior art is to encapsulate hydrophobic drugs on a phospholipid bimolecular membrane, however, the encapsulation efficiency and drug loading capacity of the passive drug loading technology are low, and the delivery requirement cannot be met.

Because of the hydrophobicity of the photosensitizer, all photosensitizer formulations reported in the clinical practice previously utilize the hydrophobicity of the photosensitizer to entrap it in the phospholipid bilayer membrane of the liposome. The photosensitizer liposome prepared by the method has low drug-loading rate (usually less than 1 percent), unsatisfactory in vivo stability, quick release, and phototoxicity on blood, skin, eyes and the like due to the physical properties of the photosensitizer and the space limitation of a liposome membrane. How to deliver the photosensitizer to tumor tissues in a sufficient amount to exert efficient tumor killing and turn off phototoxicity during delivery to reduce toxicity to normal tissues is a central importance in exerting the antitumor effect of the photosensitizer, and the technology is not realized in the prior art.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a photosensitizer-encapsulated liposome composition in an internal water phase, which realizes high stability of a photosensitizer liposome, reduces the generation amount of photosensitizer fluorescence signals and singlet oxygen in blood and non-target tissues, and reduces phototoxicity.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

[1] a liposome composition comprising a liposome and a photosensitizer, said photosensitizer being present in the internal aqueous phase of the liposome.

[2] The liposome composition according to [1], wherein the internal aqueous phase further contains: acetate or hydrochloride.

[3] The liposome composition according to [1] or [2], wherein the liposome comprises a lipid carrier, and the liposome is a unilamellar or a multilamellar liposome.

[4] The liposome composition according to any one of [1] to [3], wherein the liposome carrier contains phospholipids, cholesterol and pegylated phospholipids in a molar ratio range of (10-80): (0.1-50): (0.1-40).

[5] The liposome composition according to any one of [1] to [4], wherein the photosensitizer is a weakly acidic photosensitizer or a photosensitizer having an electron-withdrawing group in the structure.

[6] The liposome composition according to any one of [1] to [5], wherein the photosensitizer has a structure comprising a group selected from a carboxyl group, a phenolic hydroxyl group, an alcoholic hydroxyl group and a sulfonic acid group.

[7] The liposome composition as described in any one of [1] to [6], wherein the photosensitizer is selected from one or more of chlorin e6, protoporphyrin, hematoporphyrin monomethyl ether, pyropheophorbide a-hexyl ether, pyropheophorbide-a, benzoporphyrin derivative monoacids.

[8] The liposome composition according to any one of [1] to [7], wherein the mass ratio of the photosensitizer to the lipid carrier is 1: 100-100: 1.

[9] the liposome composition according to any one of [1] to [8], wherein the acetate salt is calcium acetate and/or sodium acetate.

[10] The liposome composition according to any one of [1] to [9], wherein the liposome has a particle size of nanometer or micrometer scale, preferably a particle size of nanometer scale, such as 10-200nm, preferably around 100nm, such as 80-120 nm.

[11] The liposome composition according to any one of [1] to [10], wherein the drug loading amount of the photosensitizer in the liposome is 30% or more.

[12] A method of preparing the liposome composition of any one of [1] to [11], the method comprising the steps of:

(1) preparing blank liposome;

(2) forming a salt gradient in the internal and external aqueous phases of the blank liposome;

(3) feeding a photosensitizer into the liposome with the salt gradient for entrapment; and

(4) separating to remove free photosensitizer and obtaining the liposome with photosensitizer in the inner water phase.

[13] The production method according to [11], wherein an entrapment rate of the photosensitizer in the liposome is 85% or more.

[14] A liposome preparation comprising the liposome composition of any one of [1] to [11 ].

[15] The liposome preparation according to [14], which is an injection or a lyophilized powder preparation for injection.

[16] Use of a liposome composition according to any one of [1] to [10] for the preparation of a medicament for the treatment of a tumor, a vascular disease, a skin disease or an autoimmune disease.

[17] The use according to [16], wherein the vascular disease is an ophthalmic disease.

[18] Use of a liposome composition according to any one of [1] to [11] in combination with other drugs for the preparation of a medicament for the treatment of a tumor, a vascular disease (e.g. an ophthalmic disease), a skin disease or an autoimmune disease.

Drawings

FIG. 1 is a photograph showing the appearance of liposomes of 6 photosensitizers in example 1

FIG. 2 is a graph showing the encapsulation efficiency, drug loading and fluorescence change before and after liposome destruction of 6 photosensitizer liposomes in example 1

FIG. 3 is a graph showing the particle sizes of 6 photosensitizers in example 1

FIG. 4 is a transmission electron micrograph of the photosensitizer liposome in example 1 (X19000)

FIG. 5 shows the change of fluorescence values (after rupture of membrane/before rupture of membrane) of Ce6 and SOSG in example 2 with the drug loading

FIG. 6 is a linear correlation curve of fluorescence values (after rupture of membrane/before rupture of membrane) of Ce6 and SOSG in example 2

FIG. 7 shows the particle size of the Ce6 liposome with different formulations in example 3

FIG. 8 shows the results of confocal laser microscopy in example 4

FIG. 9 shows the leakage rate curve of Ce6 liposome in example 5 (n-3)

FIG. 10 is the release curve of Ce6 liposome in example 6 (n ═ 3)

FIG. 11 shows the results of MTT cytotoxicity of Ce6 liposome in example 7 (n ═ 6)

FIG. 12 shows the results of MTT cell dark toxicity of Ce6 liposome in example 8 (n ═ 6)

FIG. 13 shows the pharmacokinetic profile of the drugs in example 9 (n ═ 5)

FIG. 14 is the ratio of AUC of Ce6 entrapped in the aqueous phase and phospholipid membrane in liposome in example 9

FIG. 15 is a graph showing the fold change in fluorescence before and after rupture of the membrane of the in vivo plasma sample containing the Ce6 liposome supported on the internal aqueous phase and the phospholipid membrane in example 9.

Detailed Description

In the present invention, as the carrier for forming the liposome of the present invention, there are included, but not limited to, lipid carriers such as phospholipids (neutral phospholipids, negatively charged phospholipids, positively charged phospholipids), phospholipid derivatives, phospholipids modified with functional groups, cholesterol derivatives, cholesterol modified with functional groups, gangliosides and derivatives thereof, long-circulating lipids modified with polyethylene glycol and derivatives thereof, and various other materials for regulating the function of liposomes, or various complexes of the above lipids. Further preferably, the liposome carrier is, for example: hydrogenated soybean lecithin (HSPC), Cholesterol (CHOL), distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoyl phosphatidyl choline (DPPC), lysolecithin (lyso-PC), distearoyl phosphatidyl choline (DSPC) and the like. Further preferably, the molecular weight of PEG in the DSPE-PEG is in a range of 50-10000, and more preferably, the molecular weight of PEG in the DSPE-PEG is 2000, which is expressed as PEG 2000.

The liposome carrier of the present invention may include one, two, three, or more of the above-mentioned carrier components, and the ratio of the components is not particularly limited, and for example, when the liposome carrier contains phospholipid, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol, the molar ratio of the three components may be (10 to 98): (0.1-50): (0.1-40). Further preferred ranges are: (40-94): (5-30): (1-30), more preferably, when the liposome carrier contains phospholipid, cholesterol and distearoyl phosphatidyl ethanolamine-polyethylene glycol, the molar ratio of the three in the liposome carrier is 65:25: 10; when the phospholipid and the distearoyl phosphatidyl ethanolamine-polyethylene glycol are contained, the molar ratio of the phospholipid to the distearoyl phosphatidyl ethanolamine-polyethylene glycol can be (10-98): (0.1-40), preferably (40-94): (1-30), more preferably 25: 1; when the phospholipid and the cholesterol are contained, the molar ratio of the phospholipid to the cholesterol can be (10-98): (0.1 to 50), preferably (40 to 94): (5-30), more preferably 70: 30.

The photosensitizer according to the present invention includes, but is not limited to, weakly acidic photosensitizers, photosensitizers having an electron-withdrawing group in the structure, and the like, such as photosensitizers having a structure containing a carboxyl group, a phenolic hydroxyl group, an alcoholic hydroxyl group, a sulfonic acid group, and the like. Further preferably, the photosensitizer may be Chlorin e6(Chlorin e6, Ce6), protoporphyrin (PpIX), hematoporphyrin monomethyl ether (HMME), pyropheophorbide a hexyl ether (HPPH), pyropheophorbide-a (ppa), benzoporphyrin derivative monoacid (BPD-MA), the specific structure of which is as follows.

In the liposome composition of the present invention, the mass ratio of the photosensitizer to the lipid carrier (the mass ratio of the photosensitizer to the lipid carrier upon administration) includes, but is not limited to, 1: 100-100: 1, preferably in the range of 1: 100-50: 100.

in the liposome composition of the present invention, the liposome further contains a salt, such as acetate, hydrochloride, etc., in the internal aqueous phase. The acetate salt is, for example, calcium acetate, sodium acetate, or the like. The hydrochloride is, for example, calcium chloride or the like. The concentration of the salt is not particularly limited, and for example, the concentration of the acetate salt is, for example: it is about 200mM, for example 240mM, in terms of acetate.

The photosensitizer is encapsulated in the liposome water phase, which means that the photosensitizer can be encapsulated in the liposome water phase by liposome phospholipid bilayers in the forms of dissolution, photosensitizer monomolecular or photosensitizer salt precipitation and the like.

In the liposome of the present invention, the drug loading of the photosensitizer includes, but is not limited to, 1% to 90%, preferably 5% to 50%, more preferably 30% or more. The calculation method of the drug loading rate comprises the following steps: the drug loading was defined as the mass of photosensitizer encapsulated in liposome/(mass of photosensitizer encapsulated in liposome + mass of total lipid) × 100%.

The present invention also provides a method for preparing the liposome composition of the present invention, which comprises the following steps:

(1) preparing blank liposome;

Optionally, the liposome obtained in step (4) may be further solidified by freeze drying, spray freeze drying or spray drying. The lyoprotectant may be one or a combination of two or more of PVP, glucose, trehalose, maltose, galactose, mannitol, etc. According to the requirement, the freeze-drying protective agent can be directly used for freeze-drying without any freeze-drying protective agent, and the mixture can be reconstituted by adding an aqueous medium.

The photosensitizer can be entrapped in the inner water phase of the liposome by the method. The preparation method of the invention can be an active drug loading method, such as a pH gradient method, an ammonium sulfate gradient method and a calcium acetate gradient method, or other acetate gradient methods such as sodium acetate and the like, a hydrochloride gradient method and the like.

Taking the calcium acetate gradient method as an example, the liposome composition of the present invention can be prepared as follows:

(1) the blank liposome is prepared by conventional liposome preparation method, such as hand shaking method, ultrasonic method, freeze-drying hydration method, extrusion method, high pressure homogenization method, reverse phase evaporation method, thin film hydration method, ethanol injection method, etc., preferably ethanol injection method and thin film hydration method, and the water phase is calcium acetate solution. If necessary, homogenizing under high pressure or extruding the liposome vesicles through the polycarbonate membranes with different pore diameters in sequence to obtain blank liposomes with uniform particle size. The particle size of the liposome can be controlled at nanometer or micrometer level, preferably the particle size is nanometer level, such as 10-200nm, preferably about 100nm, such as 80-120 nm.

(2) Replacing or partially replacing the external water phase with a non-calcium acetate aqueous solution by a gel column separation, dialysis or high-speed centrifugation method to prepare the liposome with the internal and external water phases with calcium acetate gradient. The external water phase is non-calcium acetate aqueous solution, such as PBS solution, 0.9% sodium chloride solution or pure water solution, and preferably 0.9% sodium chloride solution.

(3) Putting photosensitizer powder or solution (preferably solution) into the step (2) for active drug loading, wherein the drug loading temperature comprises but is not limited to 0-100 ℃, and the further preferred drug loading temperature is 55-80 ℃; the drug loading time includes but is not limited to 0-72 h, and the further preferable drug loading time is 30 min-3 h.

(4) Removing free photosensitizer by gel column separation, dialysis or high speed centrifugation to obtain photosensitizer-entrapped liposome, preferably gel column separation.

In the above preparation method, the calcium acetate solution in step (1) is prepared by dissolving calcium acetate in an aqueous medium, the aqueous medium includes but is not limited to a PBS solution, a 0.9% sodium chloride solution, pure water or a 5% glucose solution, etc., the pH includes but is not limited to 4.0-9.0, the concentration of calcium acetate includes but is not limited to 0-10000 mM, and the further preferred concentration of calcium acetate is 20-500 mM.

The preparation method, in particular to the calcium acetate gradient active entrapment technology, has wide applicability to different types of liposomes and different types of photosensitizers.

In the method for producing the liposome composition of the present invention, the liposome encapsulation efficiency of the photosensitizer is preferably 85% or more, and preferably 90% or more. The calculation method of the encapsulation efficiency comprises the following steps: the entrapment rate is the mass of photosensitizer entrapped in the liposome/the mass of photosensitizer put in the liposome at the time of administration × 100%.

In the liposome composition of the present invention, the photosensitizer is present in the internal aqueous phase of the liposome, rather than on the phospholipid membrane of the liposome. Therefore, the liposome system of the invention is more stable in vivo, the half-life period is prolonged, the fluorescence value (after membrane rupture/before membrane rupture) and the singlet oxygen generation amount (after membrane rupture/before membrane rupture) are increased, and the phototoxicity is obviously reduced. The fluorescence value and the singlet oxygen generation amount of the liposome after membrane rupture are obviously increased.

The liposome composition of the present invention can be prepared into preparations, such as parenteral preparations for oral administration, injection, etc., according to conventional methods in the art, optionally with appropriate addition of suitable excipients. Preferably, the preparation can be administered by injection, such as intravenous injection, intramuscular injection, subcutaneous injection or spray gun injection. The injection comprises injection and sterile powder injection. The photosensitizer liposome injection dispersion medium can be prepared from 5 percent glucose, normal saline or other isotonic systems, and is suitable for clinical application. The photosensitizer liposome freeze-dried powder can be added with a proper amount of 5 percent glucose aqueous solution, normal saline or other isotonic solution to reconstruct a dispersion system for injection administration, and is suitable for clinical use.

The liposome composition of the present invention can be used for photodynamic therapy of diseases including tumor therapy, treatment of benign vascular diseases such as ophthalmic diseases, skin diseases or autoimmune diseases, etc. Treatment may also be carried out in combination with other drugs, for example in combination with chemotherapeutic agents and immunotherapeutic agents, and may be formed as a combination formulation, or as two or more formulations for simultaneous or spaced administration.

The photosensitizer liposome provided by the invention keeps high stability in vivo, and reduces the whole body phototoxicity of the photosensitizer. As shown in the following examples, the photosensitizer liposome of the present invention can significantly inhibit the proliferation of human non-small cell lung cancer cells, and the pharmacokinetic and fluorescence analysis of mice shows that: the photosensitizer reduces the amount of fluorescence signal and singlet oxygen production in blood and non-target tissues, thereby reducing phototoxicity.

Compared with the prior art, the invention has the following advantages: the invention firstly obtains the liposome composition in which the photosensitizer is entrapped in the liposome water phase. The photosensitizer is encapsulated in the water phase in the liposome, so that the fluorescence signal and the singlet oxygen generation amount are obviously reduced; when the photosensitizer is completely released, a significant increase (e.g., 1-to hundreds-fold) in the fluorescence signal and singlet oxygen production can be induced. Compared with the liposome with photosensitizer encapsulated on phospholipid membrane, the liposome system of the invention has the advantages of more stable in vivo, prolonged half-life, increased fluorescence value (after/before membrane rupture) and reduced phototoxicity.

Examples

The present invention is explained in detail below with reference to examples, which are only for illustration and should not be construed as limiting, but can be varied within the scope of the invention.

Example 1

Preparing blank gradient liposome by a thin film hydration method: HSPC/CHOL/DSPE-PEG2000(65:25:10, mol%, 10mg, 2mg and 5mg, respectively) was weighed as a lipid material into a bottle shaped like a eggplant, 10mL of chloroform was added to dissolve the lipid material, chloroform was removed by rotary evaporation at 37 ℃ under reduced pressure, and a thin lipid film was formed on the wall of the bottle. Adding 120mM calcium acetate water solution into eggplant-shaped bottle, hydrating for 20min at 60 deg.C, and performing ultrasonic treatment until light blue opalescence appears. And (3) finishing the prepared blank liposome by a liposome extrusion device, and removing the calcium acetate of the external water phase by a Sephadex G-50 gel column to form a concentration gradient of the calcium acetate of the internal water phase and the external water phase.

Adding photosensitizer Ce6, PpIX, HMME, HPPH, PPA and BPD-MA respectively according to 35%, 30%, 20%, 25%, 15% and 20% of the mass of the lipid material, carrying the drug for 30min at 60 ℃, passing through a Sephadex G-50 gel column, removing free photosensitizer, and obtaining the liposome encapsulating the photosensitizer.

Measuring the particle size at 25 ℃ by using a Malvern particle sizer; and (3) quantifying the amount of the photosensitizer before and after passing through the gel column by adopting an ultraviolet spectrophotometer, and calculating the encapsulation efficiency and the drug-loading rate.

The column-separated liposomes were diluted 100-fold with 200. mu.L each of 0.9% sodium chloride (before rupture of the membrane) and TX100 (after rupture of the membrane), and the fluorescence values before and after rupture of the membrane were measured by a fluorometer to calculate the change in fluorescence.

The forms of the liposomes of Ce6, PpIX and HPPH containing 3, 2 and 1 carboxyl groups in the structure are selected and observed by a transmission electron microscope.

As a result: the 6 photosensitizer liposomes are clear and transparent (see figure 1), the entrapment rate is over 85 percent, the drug-loading rate is over 20-30 percent, the fluorescence intensity of the liposomes after membrane rupture is 30-254 times higher than that before membrane rupture (see figure 2), the particle sizes are all about 100nm (see figure 3), the particle size distribution is narrow, and the vesicle-shaped structure can be clearly seen by a transmission electron microscope (see figure 5).

Example 2

Ce6 is used as a model photosensitizer, Ce6 liposome with drug loading of 5-35% is prepared according to the thin film hydration method in the example 1, and the fluorescence value of Ce6 before and after the rupture of the membrane of the liposome is measured according to the measuring method in the example 1. Taking 200 mu L of liposome separated by a column, diluting the liposome by 100 times respectively with 0.9% sodium chloride (before membrane rupture) and TX100 (after membrane rupture), respectively adding a novel singlet oxygen fluorescent probe SOSG (Singlet oxygen sensor green) reagent with the final concentration of 10 mu M, irradiating for 10min by 660nm laser of 50mW, measuring the fluorescence of the SOSG by a fluorescence photometer, and measuring the change of the fluorescence value of the SOSG before and after the membrane rupture of the liposome.

As a result: with the increase of the drug loading, the fluorescence value (after rupture/before rupture) of Ce6 is increased, and the fluorescence value (after rupture/before rupture) of the generated active oxygen (SOSG label) is also increased and can reach hundreds of times (see figure 6), and the two are in linear positive correlation (see figure 7).

Example 3

Preparation of formulation No. 1: HSPC/CHOL/DSPE-PEG2000(65:25:10, mol%).

Lipid materials were weighed in the above ratio, liposomes encapsulating Ce6 were prepared by the thin film hydration method in example 1, the encapsulation efficiency, drug loading amount, and particle size were measured, fluorescence values before and after rupture of membranes were measured by a fluorometer, and fluorescence values (after rupture of membranes/before rupture of membranes) were calculated.

Preparation of formulation No. 2: HSPC/CHOL (70:30, mol%).

Preparation of formulation No. 3: dipalmitoylphosphatidylcholine (DPPC)/HSPC/CHOL/DSPE-PEG2000(50:25:15:3, mol%).

Preparation of formulation No. 4: DPPC/lysolecithin (lyso-PC)/DSPE-PEG2000(90:10:4, mol%).

Preparation of formulation No. 5: DPPC/Distearoylphosphatidylcholine (DSPC)/DSPE-PEG2000(80:15:5, mol%).

As a result: the entrapment rate of the 5 liposome preparations is more than 90%, the drug-loading rate is more than 15%, the particle sizes are all less than 100nm (see figure 8), the particle size distribution is narrow, and the fluorescence value after membrane rupture is remarkably increased to 89-308 times compared with the fluorescence value before membrane rupture.

Example 4

Verification of photosensitizer Encapsulated in the internal aqueous phase of liposomes

Ce6, HMME and PPA containing 3, 2 and 1 carboxyl groups in the molecular structure of the photosensitizer are selected, and the liposome is prepared by adopting a calcium acetate gradient method according to the proportion of the lipid material (the photosensitizer is dosed according to 5 percent of the mass of the lipid material) and the preparation method in the embodiment 1.

In addition, photosensitizer liposomes prepared by passive entrapment were used as controls. The passive entrapment method comprises the following preparation processes:

the lipid material was weighed according to the recipe in example 1, the photosensitizer was weighed in an amount of 5% by mass of the lipid material, the lipid material and the photosensitizer were added together in an eggplant-shaped bottle, 10mL of chloroform was added to dissolve the lipid material, chloroform was removed by rotary evaporation at 37 ℃ under reduced pressure, and a thin lipid film was formed on the wall of the bottle. Adding 0.9% sodium chloride solution into eggplant-shaped bottle, hydrating at 60 deg.C for 20min, and performing ultrasonic treatment until light blue opalescence appears. And (3) finishing the prepared liposome by a liposome extrusion device, and removing free photosensitizer by a Sephadex G-50 gel column to obtain the liposome with the photosensitizer passively coated on the membrane.

The distribution of the photosensitizer in the liposomes was verified using a laser confocal microscope (TCS SP8 laser confocal microscope, Leica, germany). As a result: according to the calcium acetate gradient active drug loading technology, a photosensitizer is encapsulated in an internal water phase, and imaging is displayed as a solid red ball; and the photosensitizer is loaded on the phospholipid membrane, so that a hollow red ring is formed (see figure 8).

In addition, the above-mentioned method of encapsulating Ce6 in liposomes by calcium acetate gradient method and the Ce6 liposomes prepared by passive encapsulation method were compared as controls, and the drug loading, encapsulation efficiency, and fold change of fluorescence value before and after rupture of membranes of liposomes were compared.

As a result: compared with the Ce6 liposome prepared by a passive drug loading method, the drug loading rate and the entrapment rate of the Ce6 liposome prepared by the calcium acetate gradient method and the change multiple of the fluorescence value of the liposome before and after membrane rupture are obviously improved.

Example 5

Examination of Liposome leakage Rate

The scheme is as follows: the formulations No. 3, 4 and 5 of example 3 were selected to evaluate the leakage rate of liposomes: the three kinds of liposome are respectively placed in a water bath at 37 ℃, and samples are taken at different time points to measure the fluorescence value.

As a result: the three liposome formulations selected were stable well until 48h with almost no leakage of Ce6 (see fig. 9).

Example 6

In vitro Release assay

The scheme is as follows: the preparation No. 1 in the example 3 is selected as a subject to be examined, the proper concentration is selected to ensure that the concentration of Ce6 meets the condition of a leak tank, the sample is placed in a water bath at 37 ℃, the rotating speed of 100rpm is kept for stirring, samples are taken at different time points, free Ce6 is separated through ultrafiltration, and the release percentage is calculated.

As a result: the selected liposome preparation has no obvious burst release, and the Ce6 can be slowly released for 24h (see figure 10).

Example 7

Evaluation of cytotoxicity

The scheme is as follows: the preparations No. 1 to 5 in example 3 were selected as subjects to be examined, a549 cells were selected, and cytotoxicity was evaluated by MTT method (thiazole blue method): a549 cells (purchased from cell bank of basic medical college of beijing cougho medical college) were cultured in a 24-well plate, the preparations were diluted in a gradient according to the concentration of Ce6 and added to the cells for incubation for 4h, excess preparations were washed off with PBS, the cells were irradiated with a 660nm laser at a power of 50mW for 5min and incubated overnight at 37 ℃, MTT was added for incubation for 2h, MTT was washed off, formazan was dissolved with DMSO, absorbance was measured with a microplate reader, and the cell survival rate was calculated.

As a result: the selected preparations No. 1 to No. 5 all showed strong killing effect on a549 cells (see fig. 11).

Example 8

Evaluation of cellular dark toxicity

The scheme is as follows: the MTT method evaluates dark toxicity of the formulations No. 1 to 5 in example 3: the operation was the same as in example 6 except that the 660nm laser was not used for irradiating the cells.

As a result: the selected preparation No. 1 to 5 have no obvious cytotoxicity, which indicates that the obtained liposome can keep low toxicity under natural light (see figure 12).

Example 9

In vivo pharmacokinetic evaluation

The scheme is as follows: ce6, HMME and PPA with 3, 2 and 1 carboxyl groups in the molecular structure are respectively selected as photosensitizers, and the liposome with the photosensitizer encapsulated in the liposome internal water phase is prepared according to the lipid material in example 1 and the active drug loading method in example 1 (the photosensitizer is dosed by 5 percent of the mass of the lipid material). In addition, liposomes in which the photosensitizer was entrapped in the phospholipid membrane were prepared as a control according to the passive entrapment method described in example 4.

Injecting the above preparation into mouse tail vein, collecting blood from orbital posterior vein of mouse at 2min,15min,30min,1, 3, 6, 12, and 24h, centrifuging at 2000rpm for 3min, collecting supernatant, adding methanol to precipitate protein to extract photosensitizer, centrifuging at 10000rpm for 5min to remove protein, measuring absorbance with fluorescence spectrophotometer, drawing pharmacokinetic curve, and calculating pharmacokinetic parameters with Das2 software.

As a result: the pharmacokinetic profile is shown in figure 13. The results show that the area under the curve (AUC) for photosensitizer-encapsulated liposomal drug in the internal aqueous phase is as much as 5 times the AUC for photosensitizer-encapsulated liposomes on phospholipid membranes (see figure 14); the change of fluorescence values of the photosensitizer encapsulated in the liposome of the internal water phase before and after rupture of the membrane is more obvious, and taking a 2min sampling point as an example, the fluorescence value is 30 times that of the photosensitizer encapsulated in the phospholipid membrane. Indicating that the photosensitizer is highly stable in vivo when entrapped in the internal aqueous phase, greatly reducing phototoxicity (see figure 15).

The present invention is described above. The present invention includes variations in various ways within its scope, which do not depart from the scope of the invention. Moreover, all such variations, which would be obvious to one skilled in the art to which the invention pertains, are intended to be included within the scope of the following claims.

Claims

1. A liposome composition comprising liposomes and a photosensitizer, said photosensitizer being present in the internal aqueous phase of the liposomes.

2. The liposomal composition of claim 1, wherein the internal aqueous phase further comprises: acetate or hydrochloride.

3. Liposome composition according to claim 1 or 2, wherein the liposomes comprise a lipid carrier and the liposomes are unilamellar or multilamellar liposomes.

4. The liposome composition according to any one of claims 1 to 3, wherein the photosensitizer has a structure comprising a group selected from a carboxyl group, a phenolic hydroxyl group, an alcoholic hydroxyl group and a sulfonic acid group.

5. Liposome composition according to any one of claims 1 to 4, wherein the photosensitizer is selected from one or more of chlorin e6, protoporphyrin, hematoporphyrin monomethyl ether, pyropheophorbide a hexyl ether, pyropheophorbide-a, benzoporphyrin derivative monoacids.

6. The liposome composition according to any one of claims 1 to 5, wherein the mass ratio of photosensitizer to lipid carrier is 1: 100-100: 1.

7. liposome composition according to any one of claims 1 to 6, wherein the acetate salt is calcium acetate and/or sodium acetate.

8. A method of preparing a liposome composition of any one of claims 1 to 7, comprising the steps of:

(1) preparing blank liposome;

(3) feeding a photosensitizer into the liposome with the salt gradient for entrapment;

9. A liposome formulation comprising the liposome composition of any one of claims 1 to 7.

10. Use of a liposome composition according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment of a tumor, a vascular disease, a skin disease or an autoimmune disease.