CN112451487A - Curcumin active drug-loaded liposome and preparation method thereof - Google Patents
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Abstract
The invention relates to the field of liposome drug delivery, in particular to curcumin active drug-loaded liposome and a preparation method thereof. The curcumin active drug-loaded liposome is prepared from the following components: curcumin, phospholipid, Cholesterol (Chol), metal ion salt solution and external water phase buffer solution. The weight ratio of curcumin to total phospholipids (sum of phospholipids and cholesterol) is 0.5-1: 10. The metal ion salt solution can be sulfate, gluconate or acetate solution of copper ions or zinc ions; the concentration of copper ions or zinc ions in the metal ion salt solution is 50-300 mM. The invention is characterized in that the curcumin active drug-carrying liposome preparation with petal-like conformation is successfully prepared for the first time, and cell experiments show that the curcumin active drug-carrying liposome preparation has stronger active oxygen generation capacity and stronger tumor cell inhibition activity; tumor-bearing mouse experiments show that the preparation has obvious effects of inhibiting subcutaneous tumor growth and relieving metastatic tumor progress.
Description
Technical Field
The invention relates to the field of liposome drug delivery, in particular to curcumin active drug-loaded liposome and a preparation method thereof.
Background
Curcumin (Curcumin, Cur) is a chemical component extracted from rhizomes of some plants in Zingiberaceae and Araceae, is a diketone compound, is orange yellow crystalline powder, slightly bitter in taste, insoluble in water, soluble in ethanol, and soluble in DMSO, DMF, PEG400, etc. A large number of researches prove that the curcumin has a plurality of important biological effects including anti-inflammatory, anticoagulation, antioxidation, antiangiogenesis and antitumor activities, the effects of promoting wound healing, reducing blood fat, preventing and treating cardiovascular diseases and nervous system degenerative diseases and the like, and is used for treating liver diseases, digestive systems, infections, rheumatoid arthritis and the like. Research shows that the composition can prevent the occurrence of chemically and radioactively induced skin cancer, liver cancer, stomach cancer, prostatic cancer, colon cancer, breast cancer and the like of rats, can inhibit the growth, invasion and metastasis of tumors, and has the effect of enhancing the treatment effect of radiotherapy and chemotherapy. However, curcumin cannot be administered by intravenous injection due to its unstable structure and poor water solubility, and has extremely poor bioavailability due to its rapid in vivo metabolism and extremely short half-life when administered orally. Therefore, there is a need to develop curcumin formulations and suitable drug delivery systems to ameliorate the current dilemma.
Liposome (liposome) is a drug carrier with a phospholipid bilayer structure, and is widely researched and applied because the liposome can be used for coating hydrophobic drugs and loading water-soluble drugs. Generally, poorly soluble drugs are passively loaded into liposomal phospholipid bilayers to improve drug solubility and bioavailability. Curcumin is difficult to realize active drug loading due to the extremely low water solubility, and curcumin is usually prepared into a liposome preparation in a passive drug loading mode in the existing research. The passive drug-loaded liposome has the defects of low entrapment rate and drug-loading rate, poor stability and the like; and after the curcumin is loaded by the nano-carrier, the cell activity of the drug-loaded preparation is reduced, so that the drug effect in animals is general.
Disclosure of Invention
In view of the background, the present invention aims to provide a curcumin active drug-loaded liposome and a preparation method thereof, so as to realize effective drug delivery for cancer and metastatic cancer treatment. Because the structure of curcumin has a diketone structure, the structure can be complexed with metal ions to form a stable insoluble complex. Therefore, the invention takes metal ions as an internal water phase, and prepares the liposome with transmembrane metal ion gradient to actively load curcumin. The research unexpectedly finds that the prepared curcumin liposome has stronger capability of inducing the generation of Reactive Oxygen Species (ROS) in tumor cells compared with curcumin, thereby generating stronger cell activity; and the lipid carrier is used as a microreactor with a stable structure, so that the effective delivery of the curcumin-metal ion compound is realized, the tumor accumulation of the medicine is improved, and a better tumor inhibition effect is generated.
The technical scheme of the invention is as follows:
a curcumin active drug-loaded liposome is prepared from the following components: curcumin, phospholipid, Cholesterol (Chol), metal ion salt solution and external water phase buffer solution.
Wherein the weight ratio of curcumin to total phospholipids (sum of phospholipids and cholesterol) is 0.5-1: 10.
The phospholipid can be natural phospholipid egg yolk lecithin (EPC), hydrogenated soybean lecithin (1-palmityl-2-stearyl-sn-glycerol-3-phosphatydilcholine, HSPC); commonly used synthetic phospholipids such as: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (1,2-Dioleoyl-sn-glycero-3-phosphocholine, DOPC), Dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylethanolamine (DSPE), Dioleoylphosphatidylethanolamine (DOPE), 1, 2-palmitoylphosphatidylglycerol (1, 2-dioleoylphosphatidylethanolamine, 1- (1, 2-dioleoylphosphatidylethanolamine, DPC), Dioleoylphosphatidylethanolamine (DPPC), dioleoylphosphatidylethanolamine (1, 2-dioleoylphosphatidylethanolamine, 1-3-phospholethanolamine, DPC), Dioleoylphosphatidylethanolamine (DPC), di-2-phospholethanolamine, DPC), and DPC glycol) -2000], DSPE-PEG 2000).
Further preferred phospholipids include one or more of hydrogenated soy lecithin (HSPC), Distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000);
furthermore, the cholesterol content in the liposome is 10 to 45 percent in mol percentage;
further, the optimal content of the cholesterol is 30-45% in mole percentage;
further, the selected metal ions can be copper ions or zinc ions;
further, the optional metal ion salt solution may be a sulfate, gluconate, or acetate solution of copper ions or zinc ions;
further, acetate solutions of copper ions or zinc ions are preferable;
further, the concentration of copper ions or zinc ions in the metal ion salt solution is 50-300 mM;
furthermore, the concentration of copper ions or zinc ions in the metal ion salt solution is 150-200 mM.
Further, the external aqueous phase buffer may be Phosphate Buffered Saline (PBS) containing sodium chloride or sucrose, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer;
further, the external aqueous phase is preferably a HEPES buffer containing sodium chloride or sucrose in isotonic concentration;
further, the pH of the external aqueous phase buffer solution is 5-8;
further, when the inner aqueous phase is an acetate solution of copper ions, the pH of the outer aqueous phase is 5 to 6.
Further, when the inner aqueous phase is an acetate solution of zinc ions, the pH of the outer aqueous phase is 6 to 7.
The active drug-loaded liposome comprises the following steps:
(1) preparation of large unilamellar liposomes
Weighing phospholipid and cholesterol according to the prescription amount, dissolving the phospholipid and the cholesterol by using an organic solvent, fully removing the organic solvent to form a phospholipid membrane, adding an inner aqueous phase metal ion salt solution for hydration until the phospholipid membrane completely falls off, and reducing the particle size of the liposome by extruding or probe ultrasound and the like to obtain the large single-chamber liposome with uniform particle size.
(2) Establishment of a gradient of metal ions across a membrane
And (2) fully exchanging the liposome obtained in the step (1) with an external aqueous phase buffer solution containing disodium ethylene diamine tetraacetate (EDTA-2Na), fully removing external metal ions of the liposome, and fully exchanging with a solution containing sodium chloride or sucrose and HEPES to obtain the blank liposome.
(3) Drug loading
Adding the concentrated curcumin solution into the liposome solution obtained in the step (2) for incubation, terminating the drug loading process after a certain time, and storing.
In the step (1), the organic solution is: one or more of absolute ethyl alcohol, chloroform and dichloromethane;
the metal ion salt solution can be sulfate, gluconate or acetate solution of copper ions or zinc ions;
the concentration of copper ions or zinc ions in the metal ion salt solution is 50-300 mM;
in the step (2), the external aqueous phase buffer solution comprises: a 20mM HEPES solution containing 15mM EDTA-2Na and a HEPES buffer containing isotonic sodium chloride or sucrose, at a pH which depends on the metal ion salt solution selected for use, the pH of the external aqueous phase being between 5 and 6 in the case of a copper ion salt solution; when the inner water phase is zinc ion salt solution, the pH value of the outer water phase is 6-7.
In the step (3), curcumin is dissolved in a proper amount of dimethyl sulfoxide (DMSO) or N, N-Dimethylformamide (DMF) to prepare a concentrated solution. The adding volume of the curcumin solution is 5 percent of the total volume, and the mass ratio of the medicine to the total phospholipid is 0.5-1: 10.
The invention has the advantages that:
firstly, the preparation of curcumin active drug-loaded liposome is realized by metal ion gradient, under the preferable condition, HSPC or DSPC (55-70 mol percent), cholesterol (30-45 mol percent) and DSPE-PEG (0-0.5 mol percent) are selected, copper acetate or zinc acetate is used as an inner water phase, HEPES containing sodium chloride or sucrose is used as an outer water phase, and when the ratio of curcumin to total phospholipid is 1:10, the curcumin encapsulation rate is more than 95%;
secondly, the metal ions are used as the internal water phase, so that the copper ions and the zinc ions can form a stable compound with curcumin, the leakage is not easy to occur, and the stability is good;
the prepared curcumin liposome has a unique petal-shaped structure, is beneficial to the cell uptake of the curcumin liposome, and improves the accumulation of tumor tissues;
compared with curcumin, the prepared curcumin liposome has stronger capability of inducing the generation of Reactive Oxygen Species (ROS) in tumor cells, so that stronger tumor cell inhibition activity is generated, and particularly, the anti-tumor activity of a compound formed by curcumin and copper ions is stronger;
compared with the curcumin solution, the prepared curcumin liposome has stronger tumor cell inhibition activity;
the curcumin active drug-loaded liposome preparation has obvious anti-tumor and anti-metastatic invasion characteristics in vivo and in vitro, can effectively inhibit tumor growth, and effectively prolongs the life cycle of tumor-bearing mice.
Drawings
FIG. 1 is a schematic diagram of active drug-loaded liposome of curcumin, wherein M represents a metal ion capable of being complexed with curcumin;
FIG. 2 is a graph showing the effect of aqueous salt of the same concentration on curcumin particle size and encapsulation efficiency in example 1;
wherein, 1 is copper sulfate solution, 2 is copper gluconate solution, 3 is zinc sulfate, 4 is copper acetate, and 5 is zinc acetate;
FIG. 3 is the effect of the concentration of the internal aqueous phase on curcumin encapsulation efficiency and particle size in example 2;
FIG. 4 is a graph showing the effect of lipid ratio on encapsulation efficiency and particle size in example 3;
FIG. 5 is the effect of pH of the external aqueous phase on curcumin encapsulation efficiency in example 4;
FIG. 6 is the effect of cholesterol content on curcumin encapsulation efficiency and particle size in example 5;
FIG. 7 shows the results of the stability of two curcumin liposomes in example 6;
figure 8 cryo-electron micrograph of curcumin liposomes: (a) a zinc ion liposome; (b) a copper ion liposome; (c) zinc-curcumin liposomes; (d) copper-curcumin liposomes;
figure 9 tissue distribution profiles (upper) 1 hour (middle) 4 hours (lower) 24 hours at different time points for curcumin and its liposomal formulations; (1) heart, (2) liver, (3) spleen, (4) lung, (5) kidney, (6) tumor;
FIG. 10 is a graph of relative content of active oxygen induced by curcumin liposome in tumor cells, with the abscissa showing drug action time divided into 2h and 8h, and the ordinate showing fluorescence intensity reflecting the amount of active oxygen generated;
FIG. 11 is the tumor curve of the drug effect experiment of curcumin on subcutaneous tumor in example 7;
FIG. 12 is a graph showing the weight change of the preparations of example 10;
fig. 13 is a graph showing the survival of the curcumin liposome preparation against metastatic tumors in example 10.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the claims.
The related nouns are:
entrapment Efficiency (EE), which refers to the ratio of the drug loading in the liposome to the drug dosage during drug loading;
calculating the formula: EE ═ Winside ofl iposome/Wtotal of input X100％
Drug Loading (DL), which refers to the ratio of the mass of Drug loaded in the liposome to the total mass of the liposome preparation;
calculating the formula: DL ═ Winside ofl iposome/Wtotal X100％
Drug-to-Lipid ratio (Drug: Lipid, D: L), which refers to the ratio of Drug to total Lipid component (phospholipid plus cholesterol) mass example 1 Effect of aqueous salt in the Drug on the encapsulation efficiency of curcumin liposome particle size
68.1mg HSPC, 22.8mg Chol and 1.2mg DSPE-PEG2000 are weighed and dissolved in chloroform, the organic solvent is removed by rotary evaporation at 37 ℃ under reduced pressure to form a phospholipid membrane, various metal ion salt solutions (200mM) in the following table are used for hydration for 30min at 65 ℃, and the obtained crude liposome solution is sequentially passed through 0.4, 0.2 and 0.1 mu m polycarbonate membranes in an extrusion device to measure the particle size. Removing metal ions outside the liposome by sephadex chromatographic column exchange, and exchanging an external water phase to obtain HEPES buffer solution to obtain the blank liposome. Dissolving curcumin in DMSO to prepare 20mg/mL curcumin solution, adding 50 μ L curcumin solution into 1mL10mg/mL blank liposome, incubating at 65 deg.C for 30min, stopping drug loading in ice bath, and determining particle size and encapsulation efficiency, wherein the results are shown in FIG. 2.
The results show that: different internal water phase salts have small influence on the encapsulation efficiency of curcumin and large influence on the particle size, wherein acetate is superior to other anion salt solutions.
Example 2 Effect of aqueous phase concentration on curcumin liposome particle size and encapsulation efficiency
Blank liposomes were prepared according to the method of example 1 and with copper acetate as the internal aqueous phase, the internal aqueous phase concentrations being set as in the table below. The prepared blank liposome and curcumin are mixed according to the medicine-lipid ratio of 1:10 at 65 ℃ for 30 minutes, stopping the drug loading in ice bath after the drug loading is finished, and measuring the particle size and the entrapment rate of the liposome, wherein the results are shown in figure 3.
The results show that the curcumin encapsulation efficiency is increased along with the increase of the concentration of the internal aqueous phase, and when the concentration is more than 200mM, the liposome encapsulation efficiency is reduced, and the particle size is also obviously increased.
Example 3 Effect of drug lipid ratio on liposome particle size encapsulation efficiency
Weighing 68.1mg HSPC, 22.8mg Chol and 1.2mg DSPE-PEG2000, dissolving in chloroform, performing rotary evaporation at 37 ℃ under reduced pressure to remove an organic solvent to form a phospholipid membrane, performing hydration for 30min at 65 ℃ by using 200mM copper acetate solution, sequentially passing the obtained crude liposome solution through 0.4, 0.2 and 0.1 mu m polycarbonate membranes in an extrusion device, and determining the particle size. Removing metal ions outside the liposome by sephadex chromatographic column exchange, and exchanging an external water phase to obtain HEPES buffer solution to obtain the blank liposome. Dissolving curcumin in DMSO to prepare 20mg/mL curcumin solution, adding 50 μ L curcumin solution into 1mL10mg/mL blank liposome, incubating at 65 deg.C for 30min, stopping drug loading in ice bath, and measuring particle diameter and encapsulation efficiency, with the result shown in FIG. 4.
The results show that: when the medicine-fat ratio is more than 1:10, the encapsulation efficiency of the curcumin is reduced, and the particle size is obviously increased.
Example 4 Effect of external Water on encapsulation efficiency of curcumin liposomes
Weighing 68.1mg HSPC, 22.8mg Chol and 1.2mg DSPE-PEG2000, dissolving in chloroform, performing rotary evaporation at 37 ℃ under reduced pressure to remove organic solvent to form a phospholipid membrane, performing hydration for 30min at 65 ℃ by using 200mM copper acetate or zinc acetate solution, sequentially passing the obtained crude liposome solution through 0.4, 0.2 and 0.1 mu m polycarbonate membranes in an extrusion device, and determining the particle size. Removing metal ions outside the liposome by Sephadex column exchange, and exchanging external water phase with HEPES buffer solution (pH 5.0), HEPES buffer solution (pH 6.0), HEPES buffer solution (pH 7.0), and HEPES buffer solution (pH 8.0) to obtain blank liposome. Dissolving curcumin in DMSO to prepare a 20mg/mL curcumin solution, adding 50 μ L curcumin solution into 1mL10mg/mL blank liposome, incubating at 65 ℃ for 30 minutes, stopping drug loading in an ice bath, and determining the particle size and encapsulation efficiency.
The results show that: when the metal ions of the inner water phase are different, the influence of the pH of the outer water phase on the encapsulation efficiency and the particle size is different. When the inner water phase is copper acetate, the pH value of the outer water phase is 5-6; when the inner aqueous phase is zinc acetate, the outer aqueous phase preferably has a pH of 7.
Example 5 Effect of Cholesterol content on encapsulation efficiency of curcumin liposomes
Weighing the preparation components according to the formula in the following table, dissolving in chloroform, rotary evaporating at 37 deg.C under reduced pressure to remove organic solvent to obtain phospholipid membrane, hydrating with 200mM copper acetate solution at 65 deg.C for 30min, sequentially passing the obtained crude liposome solution through 0.4, 0.2, and 0.1 μm polycarbonate membrane in an extrusion device, and measuring the particle size. Removing metal ions outside the liposome by Sephadex column exchange, and exchanging the external water phase to HEPES buffer solution (pH 5.0) to obtain blank liposome. Dissolving curcumin in an organic solvent, and mixing the following raw materials: the mass ratio of the phospholipid is 1:10 are added into blank liposome, after 30 minutes of incubation at 65 ℃, the ice bath stops the drug loading, and the particle size and the encapsulation efficiency are measured.
The results show that: as the content of cholesterol is increased, the encapsulation efficiency of curcumin is increased, and the particle size is gradually reduced. When the cholesterol molar content is more than 30%, the encapsulation efficiency is not increased any more.
Example 6 curcumin liposome stability study
68.1mg HSPC, 22.8mg Chol and 1.2mg DSPE-PEG2000 are weighed and dissolved in chloroform, the organic solvent is removed by rotary evaporation at 37 ℃ under reduced pressure to form a phospholipid membrane, 200mM copper acetate and 200mM zinc acetate solutions are respectively used for hydration for 30min at 65 ℃, and the obtained crude liposome solution is sequentially passed through 0.4, 0.2 and 0.1 mu m polycarbonate membranes in an extrusion device to measure the particle size. Removing metal ions outside the liposome by sephadex chromatographic column exchange, and exchanging an external water phase to obtain HEPES buffer solution to obtain the blank liposome. Dissolving curcumin in DMSO to prepare a 20mg/mL curcumin solution, adding 50 μ L curcumin solution into 1mL10mg/mL blank liposome, incubating at 65 ℃ for 30 minutes, and stopping drug loading in ice bath. The liposome is respectively placed at 4 ℃ and 25 ℃ for a long time, and the stability of the preparation is inspected.
The results show that: the liposome has slightly increased particle size and better stability when placed at different temperatures for 6 months without obvious change.
Example 7 curcumin liposome morphology
Curcumin liposomes were prepared as in example 6, and the surface and internal crystal morphology of the liposomes was observed by cryo-transmission electron microscopy, 3.5 μ l of liposomes were precisely weighed onto an R1.2/1.3100-pore carbon membrane grid (Cu 200 mesh), excess liposome suspension was rapidly aspirated off and the carbon membrane grid was frozen in a mixture of liquid ethane and methane, all in a Vitrobot sampling robot cell. Data were acquired using a FEI Talos F200C electron microscope at an operating voltage of 200kV and images were collected on a charge coupled device at 36,000 times magnification. Finally, the collected data was processed using SerialEM software.
The observation of a cryo-transmission electron microscope shows that the curcumin liposome prepared by copper ion or zinc ion gradient presents double-chamber liposome conformation, two-sheet or three-sheet petal-shaped conformation, and the inside of the liposome presents an electron cloud structure with different densities, so that the curcumin and metal ions form a compound precipitation structure in the liposome.
Example 8 curcumin liposome cytotoxicity study
Adding 4T1 cells (breast cancer cells), RM-1 cells (prostate cancer cells), Panc-1 cells (pancreatic cancer cells) and B16F10 (melanoma cells) in a logarithmic growth phase into a 96-well plate according to the number of 1000, 3000 and 5000 cells in each well, adding a curcumin solution, a curcumin-copper liposome and a curcumin-zinc liposome into the well plate according to a set concentration (n is 6) after 24h of cells adhere to the wall in each well volume of 100 microliter, continuously culturing for 48h and 72h in a carbon dioxide incubator respectively, adding MTT, generating crystal violet in the well plate after 4h of culture, adding DMSO to dissolve the crystal violet, measuring absorbance on a microplate reader, setting the excitation wavelength to be 490nm, and measuring the absorbance to calculate the cell survival rate according to the following formula:
cell survival rate (OD control group-OD blank)/(OD administration group-OD blank) 100%
Half lethal dose IC50 was calculated by Graphpad software fitting according to cell viability
The results show that: curcumin liposomes prepared by different ion gradients are more cytotoxic than curcumin, and curcumin liposomes prepared with different metal ion drug loadings exhibit differences in toxicity among the cells tested.
Example 9 curcumin liposomes induce reactive oxygen species ROS effects
105Spreading the cells in a 24-pore plate, culturing for 24h, discarding the old culture medium, diluting free curcumin, copper ion liposome, zinc ion liposome, curcumin-copper liposome and curcumin-zinc liposome with the culture medium, and treating the cells for 2h and 8h respectively. After incubation, the drug-containing medium was discarded, and the cells were incubated with 20. mu. mol of DCFH-DA for 30 minutes and then the active oxygen content was measured by flow cytometry, the experimental results are shown in FIG. 10.
The results show that over time to 8 hours, curcumin liposomes, particularly curcumin-copper liposomes, are able to induce the production of more ROS compared to curcumin solutions, which is beneficial for inhibiting the activity of tumor cells.
Example 10 curcumin Liposome tissue distribution experiment
4T1 cells in logarithmic growth phase were treated at 5X 10 cells per mouse6Inoculating 0.2ml of each cell/0.2 ml of the vaccine to the right back side of about 20g of female balb/c mice, observing and measuring the tumor volume every day until the tumor volume reaches 200-3When mice were randomly divided into 3 groups (n ═ 3): the administration dosage of the curcumin solution group, the copper-curcumin liposome group and the zinc-curcumin liposome group is 20mg/kg, mice are killed at 1, 4 and 24 hours after administration, and tissues are taken to determine the curcumin concentration in each tissue.
The result shows that the accumulation amount of the curcumin liposome tumor tissue is obviously higher than that of the curcumin solution, and the accumulation amount of the copper-curcumin liposome at the tumor part is higher than that of the zinc-curcumin liposome.
Example 11 curcumin liposome subcutaneous tumor pharmacodynamic examination
Will be in logarithmic growthLong-term 4T1 cells were measured at 5X 10 cells/mouse6Inoculating 0.2ml of each cell/0.2 ml into the right back side of female balb/c mice with the volume of about 20g, observing and measuring the tumor volume every day until the tumor volume reaches 100mm3Mice were randomly divided into 6 groups (n ═ 6): the administration dosage is 20mg/kg, once every three days, and four times of administration. The body weight was weighed every two days, the major and minor diameters of the tumor were measured, and the tumor volume was calculated.
Tumor volume is long diameter x short diameter2/2
The results show that: the curcumin liposome has better anti-tumor effect than curcumin free drug, and the copper-curcumin liposome has the best treatment effect on a breast cancer tumor model and is better than a zinc-curcumin liposome preparation.
Example 12 pharmacodynamic study of curcumin liposomes against pulmonary metastasis of breast cancer
4T1 in the logarithmic growth phase was administered to each mouse by tail vein injection of 50 ten thousand tumor cells 48 hours later, and was divided into 6 groups (n-6): the administration dosage is 20mg/kg, once every three days, and four times of administration. Body weight and survival of the mice were recorded.
The results show that: the curcumin liposome preparation can remarkably prolong the survival period of cancer metastasis mice and has better anti-metastasis treatment effect.
Claims (10)
1. The active drug-carrying curcumin liposome is characterized by being prepared from the following components: curcumin, phospholipid, cholesterol, metal ion salt solution and external water phase buffer solution; the metal ion salt solution is a sulfate, gluconate or acetate solution of copper ions or zinc ions, and the external water phase buffer solution is a phosphate buffer solution containing sodium chloride or sucrose or a 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer solution; the weight ratio of curcumin to total phospholipids is 0.5-1: 10; the total phospholipid is the sum of phospholipid and cholesterol. .
2. The curcumin actively loaded liposome of claim 1, wherein the molar percentage of cholesterol is 10% to 45%, preferably 30-45%.
3. The curcumin actively loaded liposome as claimed in claim 1 or 2, wherein the concentration of copper ions or zinc ions in the metal ion salt solution is 50-300mM, preferably 150-200 mM.
4. The curcumin actively loaded liposome as claimed in claim 1, wherein when the metal ion salt solution is an acetate solution of copper ions, the pH of the external aqueous phase is 5 to 6; when the metal ion salt solution is an acetate solution of zinc ions, the pH value of the external water phase is 6-7.
5. A curcumin liposome according to any one of claims 1 to 4, wherein said phospholipid is one or more selected from the group consisting of egg yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, dipalmitoyl lecithin, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, 1, 2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and DSPE-PEG 2000.
6. A curcumin actively loaded liposome as claimed in any one of claims 1 to 5, wherein said curcumin liposome has a petal-shaped structure.
7. The preparation method of curcumin active drug-loaded liposome according to claim 1,
(1) preparation of large unilamellar liposomes
Weighing phospholipid and cholesterol in a prescription amount, dissolving the phospholipid and the cholesterol in an organic solvent, fully removing the organic solvent to form a phospholipid membrane, adding an inner aqueous phase metal ion salt solution for hydration until the phospholipid membrane completely falls off, and reducing the particle size of the liposome in an extrusion or probe ultrasonic mode to change the liposome into a large single-chamber liposome with uniform particle size;
(2) establishment of a gradient of metal ions across a membrane
Fully exchanging the liposome obtained in the step (1) with an external aqueous phase buffer solution containing disodium ethylene diamine tetraacetate (EDTA-2Na), fully removing external metal ions of the liposome, and fully exchanging with a solution containing sodium chloride or sucrose and HEPES to obtain a blank liposome;
(3) drug loading
Adding the concentrated curcumin solution into the liposome solution obtained in the step (2) for incubation, terminating the drug loading process after a certain time, and storing.
8. A curcumin liposome according to any one of claims 1 to 7, for use in the preparation of a medicament having the ability to induce the production of Reactive Oxygen Species (ROS).
9. A curcumin liposome as claimed in any one of claims 1-7, for use in the preparation of an antitumor agent.
10. A curcumin liposome as claimed in any one of claims 1-7, for use in the preparation of a medicament for the treatment of breast cancer, prostate cancer, pancreatic cancer, melanoma.
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