KR101630397B1 - Composition for Phototherapy of Cancer Comprising Complex of Liposome, Indocyanine Green And Anti-cancer drug - Google Patents

Abstract

The present invention relates to a composition for treating cancer, containing a liposome composite in which an anticancer drug and indocyanine green are encapsulated. The present invention further relates to a production method thereof. More specifically, the present invention relates to a composition for treating cancer, which contains a composite including liposome, an anticancer drug, and indocyanine green, and has cancer treating efficacy due to the anticancer drug as well as photothermal effects by indocyanine green generated by irradiation of therapeutically effective light. The present invention further relates to a production method thereof.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating cancer including an anticancer agent-indocyanine green-liposome complex,

The present invention relates to a composition for treating cancer, which comprises an anticancer agent and a liposome complex encapsulated with indocyanine green, and a method for preparing the same, and more particularly to a composition for treating cancer, The present invention relates to a composition for treating cancer and a method for preparing the same.

Indocianin green (ICG) is a near-infrared fluorescent dye (NIR) and is approved for use in the US Food and Drug Administration (FDA) for the diagnosis of cardiovascular system as well as lymphatic system. In particular, Indocyanine Green is known as an excellent probe for the imaging of metastatic lymph nodes for the early diagnosis of breast cancer and the mapping of sentinel lymph nodes.

Such indocyanine green has a disadvantage of low hydrophilicity, low light stability, low photon yield and low sensitivity. Also, indocyanine green is vulnerable to nonspecific aggregation, has the disadvantage of being chemically degraded by external light, solvent and temperature change, has a problem of low molecular weight and hydrophobic characteristic, have.

In order to overcome these disadvantages, nanomaterial-based indocyanine green probes have been studied, and the in vivo and in vitro stability of indocyanine green by capturing indocyanine green in nanoparticles or by using polymers There have been increased studies. By capturing indocyanine green into nanoparticles, the physicochemical stability to external light and temperature was significantly increased.

Cancer light is based on light, and it is a method to treat cancer by irradiating light on the tumor site, not by dangerous and costly surgery in cancer treatment.

Phototherapy is nondestructive, simple, and has fewer side effects than surgery. In addition, general anesthesia is unnecessary, the patient has little pain, and the period for stabilization and recovery is short.

On the other hand, the technique of treating cancer with heat by simply heating by inserting a heating device into the tumor site has been attempted a long time ago, but it is impossible to distinguish cancer cells from normal cells, so that normal cells around cancer cells are also destroyed Therefore, it has not been widely applied in clinical practice. In contrast, phototherapy is a new cancer treatment that overcomes both the shortcomings of drug therapy and radiation therapy. The initial phototherapy varies according to the light source, but it has a disadvantage that the treatment period is long and the interface of the treatment site is inaccurate.

For example, phototherapy combining gold-coated nanoshells with silica nanoparticles and near-infrared light, or phototherapy combined with near-infrared light for single-wall carbon nanotubes (SWCNTs) However, since they are not substances approved for application in the body, they are limited in clinical application. In order to obtain the desired heat, very high intensity NIR should be used, but if the intensity of the light source is so high, There is a problem that it may be damaged even if it is not very close to a heat generating agent such as a shell or a SWCNT.

In Korean Patent No. 10-2015-0068674, the present inventors have developed a composition for treating cancer wherein indocyanine green is combined with a liposome applicable to the body and applied to a composition for treating cancer. However, the composition is applicable only to cancer light therapies, so that it is necessary to develop a composition for treating cancer which has a low anticancer effect and a high anticancer effect.

Under these technical backgrounds, the inventors of the present application have found that when the indocyanine green is further encapsulated in a liposome-encapsulated complex, it is possible to induce apoptosis not only by therapeutically effective light irradiation, The inventors of the present invention have found that it is possible to solve the problems of the prior art through the composition for treating cancer which can inhibit the growth of cancer cells more effectively and achieve the desired cancer treatment effect.

An object of the present invention is to provide a composition for cancer treatment which contains, as an active ingredient, an anticancer agent and a liposome complex encapsulated with indocyanine green and simultaneously has a cancer cell phototherapy efficacy and a cancer treatment efficacy in a therapeutically effective light irradiation And a method for producing the same.

Which comprises as an active ingredient a complex prepared by using phosphatidylcholine, a stabilizer, an anticancer agent and indocyanine green as raw materials, wherein the liposome is encapsulated with an anticancer agent and indocyanine green, , A cancer cell phototherapy efficacy, and a cancer treatment efficacy by an anticancer agent.

The present invention also provides a method for preparing a complex in which an anticancer agent and indocyanine green are encapsulated in a liposome comprising the steps of:

a) dissolving at least one phosphatidylcholine, a stabilizing agent and indocyanine green in an organic solvent, and removing the organic solvent to prepare a cake;

(b) mixing an anticancer agent in the cake, and then hydrating the dispersion to prepare a dispersion, and extruding the dispersion to form a liposome containing an anticancer agent and indocyanine green; And

(c) separating the complex of the anticancer agent and indocyanine green in the liposome of step (b) encapsulated in the liposome.

The composition for treating cancer according to the present invention shows a significant cytotoxic effect by irradiating a therapeutically effective light to a complex comprising an anticancer agent and an indocyanine green encapsulated liposome, Alternatively, the tumor can be removed without surgery. In addition, it is possible to selectively kill cancer cells while minimizing the effect on normal cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph comparing absorbance and fluorescence intensities according to phosphatidylcholine and ICG ratios in a mixture for the preparation of encapsulated liposome complexes of indocyanine green;
2 is a graph comparing absorbance and fluorescence intensities according to the ratio of phosphatidylcholine and ICG in a mixture for preparing a liposome complex encapsulated with cisplatin and indocyanine green;
Figure 3 shows the content of indocyanine green in the liposomes according to the ratio of phosphatidylcholine and ICG in a mixture of cisplatin and indocyanine green encapsulated liposome complex in a fixed concentration of cisplatin, based on the amount of lipids in the liposome The graphs are compared;
Fig. 4 is a graph showing the content of cisplatin in the liposome according to the ICG ratio, based on the amount of lipids in the liposome, with the ratio of phosphatidylcholine and cisplatin in the mixture for producing the liposome complexes of cisplatin and indocyanine green encapsulated to be;
5 (A) shows the content of indocyanine green in liposomes according to the proportion of cisplatin in a mixture of phosphatidylcholine and ICG in a mixture for preparing a liposome complex encapsulated with cisplatin and indocyanine green, FIG. 5 (B) is a graph comparing the amounts of phosphatidylcholine and ICG in the mixture for preparing the liposome complexes of cisplatin and indocyanine green encapsulated liposomes of cisplatin in the liposome according to the ratio of cisplatin Based on the amount of lipids in the liposome.
Figure 6 compares the physical properties of complexes according to the ratio of phosphatidylcholine and ICG in liposome complexes containing ICG only, cisplatin and ICG encapsulated liposomes, wherein (A) is the measurement of the hydrodynamic mean diameter and B) is a measurement of Polydispersity Index (PDI), (C) is a measurement of zeta potential;
Fig. 7 is a graph showing the photothermal effect of a liposome complex encapsulated in ICG alone, encapsulated in ICG-only liposome complex, cisplatin existing outside liposome, and liposome complex encapsulated with cisplatin and ICG;
8 is a graph showing the release efficiency of cisplatin by photothermal effect in liposome complexes encapsulated with DPPC, cisplatin and indocyanine green, and liposome complexes encapsulating HSPC, cisplatin and indocyanine green;
FIG. 9 is a graph showing the cytotoxic effect of photothermal effect at various concentrations of cisplatin and ICG and the cytotoxic effect of cisplatin due to photothermal effect; FIG.
10 is a graph showing the stability in blood flow according to the ratio of phosphatidylcholine and ICG in liposomal ICG in which liposomal ICG and cisplatin are combined;
Figure 11 compares the amount accumulated in major organs after 24 hours of injection of liposomal ICG and cisplatin coupled liposomal ICG into the body;
FIG. 12 is a graph comparing the photothermal effects of liposomal ICG and cisplatin-conjugated liposomal ICG injected into the body, followed by near-infrared irradiation;
FIG. 13 is a graph comparing effects of cancer treatment after irradiation with liposomal ICG and cisplatin-conjugated liposomal ICG in the body, followed by irradiation with near infrared rays. FIG. 13A shows the survival days and the cancer sizes of mice in various experimental groups and control groups (B) is a photograph of the cancerous areas of various experimental groups and control mice;
FIG. 14 is a graphical illustration of the photothermal effect and the anticancer effect when the chemotherapeutic agent-ICG-liposome complex of the present invention is irradiated with therapeutically effective light.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient a complex prepared from a phosphatidylcholine, a stabilizer, an anti-cancer agent, and indocyanine green as raw materials, wherein the liposome is encapsulated with an anticancer agent and indocyanine green, The present invention relates to a composition for treating cancer, which has both cancer cell phototherapeutic efficacy and cancer treatment efficacy by an anticancer agent when treating a therapeutically effective light.

The inventors of the present application not only can improve the stability of indocyanine green by overcoming the disadvantages associated with low stability of indocyanine green through an anticancer agent and a liposome complex encapsulated with indocyanine green, It was confirmed that cancer treatment is possible by the cytotoxic effect of cancer drug and indocyanine green which are released during irradiation.

In the present specification, cell death by light irradiation is achieved by phototherapy, which is one of the widely used clinical methods for cancer treatment because it is less adverse, noninvasive, and specific to light of a specific wavelength. Phototherapy includes photodynamic therapy (PDT) and photothermal therapy (PTT), which include reactive oxygen species (ROS) and light and light photosensitizers to generate heat energy, respectively. The formulation is needed.

In the present invention, the photothermal therapy efficacy induces the death of cancer cells by indocyanine green upon irradiation with therapeutically effective light.

In particular, the inventors of the present application have confirmed that an excellent phototherapeutic effect can be obtained in light irradiation of an anticancer agent and a liposome complex encapsulating indocyanine green according to the present invention. It was confirmed that this effect can enhance the cytotoxicity against cancer cells and thus, the cancer treatment effect can be exhibited.

In one embodiment, the liposomes are prepared comprising phosphatidylcholine and a stabilizing agent, and liposomes in the form of a unilamellar or complex lamellar may be used. The phosphatidylcholine is a kind of phospholipid introduced with choline, and includes a hydrophobic tail of saturated or unsaturated fatty acid and a hydrophilic head. For example, L-α-phosphatidylcholine (Egg, Chicken), L phosphatidylcholine, hydrogenated (Egg, Chicken), L- alpha -phosphatidylcholine (Soy), L-alpha-phosphatidylcholine, hydrogenated (Soy) 1,2-didecanoyl-sn- glycero-3- , 1,2-diundecanoyl-sn-glycero-3-phosphocholine (11: 0 PC), 1,2-dilauroyl-sn-glycero-3-phosphocholine glycero-3-phosphocholine (13: 0 PC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (15: 0 PC), 1,2-diphytanoyl- : 0 PC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17: 0 PC), 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine glycero-3-phosphocholine (14: 1 (Δ9-Cis) PC), 1,2-dimyristoyl-sn-glycero-3- glycero-3-phosphocholine (16: 1 (Δ9-Cis) PC), 1,2-dipalmitoyl- glycero-3-phosphocholine (18: 1 (Δ6-Cis) PC), 1,2-dipeptroselenoyl-sn-glycero-3-phosphocholine (16: 1-dioleoyl-sn-glycero-3-phosphocholine (18: 1 (Δ9-Cis) PC (DOPC) , 2-dilinoleoyl-sn-glycero-3-phosphocholine (18: 2 (Cis) PC), 1,2-dilinolenoyl-sn- glycero-3-phosphocholine glycero-3-phosphocholine (20: 1 (Cis) PC), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl- ), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), HSPC (Hydrogenated soybean phosphatidylcholine) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) But is not limited thereto.

The inventors of the present application have confirmed that an enhanced photothermal effect is exhibited in ICG encapsulated with liposomes compared to ICG bound to liposome surface and to free ICG (known indocyanine green alone) without liposome binding. Based on this, the encapsulation used herein means that the indocyanine green and the anti-cancer agent are substantially present only in the liposome space to form the liposome-ICG complex, and the free ICG (free ICG) or the liposome The ICG bound to the surface only means that it is removed and is not substantially present.

In the present invention, the phosphatidylcholine may preferably be DPPC.

In the present invention, the stabilizer may be a lipid, a lipid derivative, a protein or a peptide, and specifically, a phosphatidylethanolamine conjugated with polyethylene glycol. The polyethylene glycol may be used without limitation as long as it is suitable for stabilization. For example, the polyethylene glycol may have a molecular weight of about 1, 000 to 10,000, preferably 2,000 to 8,000, more preferably 2,000 to 6,000.

In the present invention, the phosphatidylethanolamine may be a mono- or di-unsaturated fatty acid, and examples thereof include DMPE (dimyristoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), DSPE (distearoylphosphatidyl-ethanolamine) and DSPE-PEG2000 (distearoylphosphatidyl- ethanolamine-polyethylene glycole-2000), but the present invention is not limited thereto.

In the present invention, the phosphatidylcholine and the stabilizer may be included to a degree suitable for liposome formation suitable for photothermal effect together with indocyanine green, for example, a molar ratio of phosphatidylcholine and stabilizer is about 85:15 to about 98: 2, preferably about 90: 10 to 95: 5. Within the above molar ratio range, the in vivo liposome is most stable and the life can be maximized.

In the present invention, the content of the phosphatidylcholine, the indocyanine green and the anticancer agent in the mixture is not limited as long as it is suitable for achieving the photothermal effect for the treatment. For example, it is included in the molar ratio of 250: 0.1-32: 1-200 . Preferably, the content of phosphatidylcholine, indocyanine green and anticancer agent in the mixture may be in a molar ratio of 250: 0.5-16: 5-50, and most preferably the content of phosphatidylcholine, indocyanine green and anticancer agent Can be included as a molar ratio of 250: 7: 11 = total lipid: ICG: Cisplatin. And can exhibit the desired photothermal effect throughout the range.

In particular, the inventors of the present application have found that when a phosphatidylcholine and an indocyanine green and an anticancer agent are contained in the molar ratio within the range, the indocyanine green and the anticancer agent can be optimally dispersed within the liposome to form an encapsulated complex, It was confirmed that it has a significant photothermal effect by having the optimum absorbance to exhibit the effective photothermal effect.

When the content of indocyanine green and anticancer agent is smaller than the range described above, the desired photoreaction effect and cancer treatment effect can not be expected, and when considering the cost and time required for liposome production due to a relatively large amount of liposome If the amount of indocyanine green and the amount of the anticancer drug contained is larger than the range described above, the synergistic photothermal effect can not be expected because aggregation occurs in the liposome or reaches a threshold value to the extent that it no longer reacts to light. In particular, it was confirmed that phosphatidylcholine and indocyanine green exhibited the best light heat effect with a small amount of liposome when the molar ratio was 250: 7 (FIG. 3). This is probably due to encapsulation of indocyanine green in liposome complexes prepared from phosphatidylcholine and indocyanine green at a molar ratio of 250: 7. In addition, when the liposome complex prepared from phosphatidylcholine and indocyanine green contained at a molar ratio of 250: 7 was synthesized, the initial concentration of cisplatin was 250: 7: 11 = 4). When the concentration of cisplatin is less than 0.25 mg / ml at the time of synthesis, for example, when the concentration of cisplatin is 0.1 mg / ml at the time of liposome synthesis, encapsulation is performed in the liposome (Fig. 5). ≪ tb > < TABLE >

INDUSTRIAL APPLICABILITY The anticancer agent-indocyanine green-liposome complex of the present invention not only induces cell death by inducing a photothermal effect of indocyanine green upon treatment of therapeutically effective light, but also changes the bilayer permeability of the liposome Thereby promoting the release of the anticancer drug contained in the liposome. In this case, the released anticancer drug exhibits the cancer cell treatment effect in a selective region irradiated with the laser. As a result, it was confirmed that the anticancer effect is superior to that of the conventional liposome therapeutic agent and that side effects caused by the anticancer agent can be minimized.

In one embodiment, heat may be generated when the complex is irradiated with near-infrared rays, and the near-infrared rays may be irradiated at a power of 100 mW or more at 700-900 nm for 3 minutes or more. At this time, the power for generating heat may be, for example, 100 mW or more, 1 W or less, and preferably 400 mW or more and 800 mW or less. The light irradiation time may be from 3 minutes to 30 minutes or less. Under the light irradiation condition lower than the above-mentioned range, the lead-free green is not effectively consumed in the generation of the photothermal effect, and in the high light irradiation condition, the peripheral normal cells are exposed to the light toxicity and may be damaged.

The heat generated at this time may be, for example, a temperature of at least about 45 캜, preferably at least 50 캜, more preferably at least 60 캜, at which more than 90% of cell death, preferably more than 95% .

Among the heat-generating complexes, the indocyanine green may be contained at a concentration of, for example, 1-30 ug / ml, preferably 5-10 ug / ml, and the anticancer agent may contain, for example, 1-50ug / ml, Preferably at a concentration of 1-20ug / ml. The inventors of the present application have confirmed that heat can be generated in a liposome complex containing an indocyanine green and an anticancer agent in the concentration range described above to such an extent that a temperature rise of 10 ° C or more can occur at least 95% .

Particularly, it has been confirmed that as the accumulation amount of the liposome complex including the indocyanine green and the anticancer agent is higher among the lymph nodes in which the tumor has occurred, the heat generation is also increased and the cell death may occur due to the heat generated above the level sufficient for the main cell destruction. This confirms that liposome complexes, including indocyanine green and anticancer agents, developed on the basis of FDA licensed substances, can have great clinical potential.

In one embodiment, the complex can have a size (diameter) determined by the type and size of the liposome encapsulated with the indocyanine green and the anti-cancer agent, and the hydrodynamic diameter (HD) For example, 50-200 nm, preferably 100-150 nm.

In another embodiment, the complex has a low degree of polydispersity index, confirming that the molecular weight and structure are uniform, but the polydispersity index may be less than or equal to 0.2, but is not limited thereto.

In the present invention, the zeta potential of the complex may be -50-0 mv.

In the present invention, the anticancer agent may be an alkylating agent capable of inhibiting the synthesis of DNA by forming a covalent bond with a nucleic acid, although the anticancer agent may be a water-soluble small molecule.

In the present invention, the alkylating agent is selected from the group consisting of Mechlorethamine, Cyclophosphamide, Ifosfamide, Melphalan, Chlorambucil, Busulfan, May be at least one member selected from the group consisting of Thiotepa, nitrosourea and platinum compounds, and most preferably may be cisplatin or carboplatin.

In the present invention, 'cancer' and 'tumor' mean or describe a physiological condition in a mammal, which is characteristic of uncontrolled cell growth. By the composition of the present invention, breast, heart, lung, Tumors or metastatic tumors such as small intestine, large intestine, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, cervix and liver can be treated .

Based on this, the present invention provides a method of treating cancer comprising the steps of:

(a) administering to the patient an indocyanine green-liposome complex wherein the anti-cancer agent and the indocyanine green are encapsulated in a liposome; And

(b) irradiating therapeutically effective light to the complex administered in step (a), which is accumulated in the tumor site.

In another aspect, the present invention relates to a method for producing a complex in which liposome is encapsulated with an anticancer agent and indocyanine green.

The production process according to the present invention may preferably be characterized by including, but not limited to, the following steps:

(a) dissolving at least one phosphatidylcholine, a stabilizer and indocyanine green in an organic solvent to prepare a mixture thereof, and removing the organic solvent to prepare a cake;

(b) mixing an anticancer agent in the cake, and then hydrating the dispersion to prepare a dispersion, and extruding the dispersion to form a liposome containing an anticancer agent and indocyanine green; And

(c) separating the complex of the anticancer agent and indocyanine green in the liposome of step (b) encapsulated in the liposome.

In the production method according to the present invention, step (a) is a step of dissolving at least one phosphatidylcholine, a stabilizing agent and indocyanine green in an organic solvent and removing the organic solvent to prepare a cake. Compositions comprising complexes in which anticancer agents and indocyanine green are encapsulated in liposomes can be prepared via film hydration / extrusion methods.

In the present invention, the phosphatidylcholine and the stabilizer which are the materials of the liposome are similarly applied to the above-mentioned invention related to the production method. Phosphatidylcholine and a stabilizer can be dissolved in the organic solvent.

In the present invention, in the step (a), the organic solvent may be, for example, methanol, ethanol, propanol, isopropanol, butanol, acetone, may be at least one member selected from the group consisting of ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane, Chloroform can be used.

Thereafter, (b) an organic solvent is removed and dried, and then an anticancer agent is mixed with the prepared cakes, followed by hydration to prepare a dispersion, and extruding the mixture into a porous polymer membrane to prepare an anticancer agent and a liposome containing indocyanine green. The dispersion may be prepared by hydration with an isotonic solution, which may include, for example, glucose, trehalose and the like as an isotonic agent, and the isotonicity agent may be, for example, 1-10% (w / v) (W / v). ≪ / RTI >

The dispersion liquid may be extruded into a porous polymer membrane to produce a liposome in the form of a single or multi-lamellar vesicle. The porous polymer membrane used herein may be, for example, a polycarbonate polymer membrane, and the pore diameter of the porous polymer membrane may be, For example, 10 to 1000 nm, preferably 50 to 200 nm.

In the next step (c), the complex encapsulated within the liposome is separated from the anticancer agent and the indocyanine green. The method used for removal can be used without limitation as long as the anticancer agent and indocyanine green can be separated from the complex encapsulated in the liposome and the indocyanine green simply bonded to or exposed to the surface of the free indocyanine green and liposome, For example, only the complex encapsulated within the liposome can be isolated by passing the desired indocyanine green through a dialysis membrane or using chromatography (e.g., size exclusion chromatography, etc.).

The composition for treating cancer of the present invention may further comprise suitable excipients and diluents conventionally used in the production of pharmaceutical compositions. The composition for treating cancer according to the present invention can be administered orally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, external preparations, suppositories, And can be used as formulations.

Examples of carriers, excipients and diluents that can be contained in the composition including the anticancer drug-indocyanine green-liposome complex include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil have.

In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives are included . Formulations for non-oral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous preparation and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Examples of suppository bases include witepsol, macrogol, Tween 61, cacao paper, laurel jersey, glycerogelatin and the like. As the coloring agent, it is allowed to add it to medicines. Examples of the mating agent include cocoa powder, peppermint, aromatic acid, peppermint oil, cerebrospinal fluid, cinnamon powder and the like. These tablets do not exclude that they are coated appropriately with sugar, gelatin, and other necessities in the case of granules.

When the composition of the present invention is prepared by injection, a pH adjusting agent, a buffering agent, a stabilizer, a preservative, and the like are added as needed, and subcutaneous, intramuscular, and intravenous injections are prepared by a conventional method.

The amount of the composition containing the anticancer agent-indocyanine green-liposome complex of the present invention may vary depending on the age, sex and body weight of the patient, but is generally in the range of 5 to 500 mg / kg, preferably 100 to 250 mg / kg May be administered one to three times per day, and the dose may be increased or decreased depending on the route of administration, disease severity, sex, weight, age, and the like. Accordingly, the dosage amounts do not in any way limit the scope of the invention

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One: Liposomal ICG's  Produce

ICG (IR-25, laser grade pure) was obtained from Acros Organics. DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (ICG = 250: X (M)) dissolved in methanol at a concentration of 1 mg / ml was dissolved in chloroform at a molar ratio of 95: 5, and the organic solvent was completely dissolved After evaporation, the resulting lipid cake on the bottom of the flask was hydrated with 2 ml of a 5% glucose aqueous solution per 0.672 mg of DSPE-PEG2000.

The composition of the hydration solution was 250: 1, 250: 4, 250: 8, 250: 16, 250: 32 and 250: 64, respectively.

The liposome solution that had been hydrated using a 0.1 um polycarbonate membrane was extruded to an average 100 nm single or multi-lamella liposome. The extruded 100 nm single or multi-lamellar liposome solution was passed through a column filled with Sephadex G-50 to separate ICG and ICG-containing liposomes not contained in the liposome by size-exclusion chromatography do. The free ICG (free ICG) and the ICG attached to the surface of the liposome were removed by dialysis using a dialysis membrane having a molecular weight cut-off of 100 kDa to prepare an ICG-encapsulated liposome complex.

The presence of free ICG present in the liposome ICG solution and the ICG exposed on the surface of the liposome were confirmed using the increase in fluorescence intensity and the change in absorbance due to the dispersion effect caused by the encapsulation of ICG in the liposome.

As a result, it was confirmed that the amount of ICG encapsulated (i. E., Ultimately, a phosphatidylcholine: ICG ratio of 250: 7) inside the liposome when the phosphatidylcholine: ICG ratio at the production stage was 250: 4 (FIGS. 1 and 2) .

Example  2: Cisplatin  loaded Liposomal ICG (Cis-liposomal ICG)  Produce

In the method of Example 1, cis-liposomal ICG was prepared by mixing cisplatin 1 mg / ml aqueous solution when hydrating lipid cake and hydration to obtain free ICG present in cis-liposomal ICG solution The presence of ICG exposed on the surface of the liposome was confirmed, and it was confirmed that ICG could be encapsulated in the cis-liposomal ICG to the maximum when the initial phosphatidylcholine: ICG ratio was 250: 4 (FIG. 2).

Then, in order to obtain an optimized molar ratio of phosphatidylcholine: ICG: cisplatin in the production of Cis-liposomal ICG by the above-mentioned method, the concentration of cisplatin at the time of hydration was fixed to 1 mg / ml and the phosphatidylcholine and ICG (ICG concentration) / (total phosphatidylcholine) obtained by determining the concentration of phosphatidylcholine in the liposome-ICG complex from which free ICG was removed through the Stewart assay, in which the concentration of phosphatidylcholine constituting the liposome and the phosphatidylcholine in the DSPE- (ICG) ratio of 250: 4, the loading efficiency of ICG was the maximum (FIG. 3).

When the ratio of phosphatidylcholine and cisplatin was fixed and the ratio of ICG was varied, the concentration of phosphatidylcholine in the final product was calculated through stewart awway and the concentration of cisplatin in the final product was measured by ICP- MS, it was confirmed that a certain ratio of cisplatin was encapsulated in the liposome regardless of the ratio of ICG, at least within the range of total lipid (ICG): 250: 0 to 250: 8 4).

Experiments were performed according to the above method. When the ratio of phosphatidylcholine and ICG was fixed and the ratio of cisplatin was varied, the concentration of cisplatin in the final product was determined by ICP-MS. As a result, when the concentration of cisplatin was 0.25 mg / ml or more It was confirmed that cisplatin and ICG could be loaded with maximum efficiency (FIG. 5).

As a result, it was confirmed that the most optimal mole ratio in the production of Cis-liposomal ICG was phosphatidylcholine: ICG: cisplatin = 250: 4: 11 (or more).

In addition, liposomal ICG and cis-liposomal ICG with phosphatidylcholine: ICG ratios of 250: 1, 250: 4 or 250: 8 were prepared and their hydrodynamic diameter, polydispersity index, As a result of measuring the zeta potential, it was confirmed that the physical properties of the composite did not change due to the presence or absence of cisplatin (FIG. 6).

Test Example 1: Cis - liposomal ICG's Photothermal effect

ICG concentration of all Liposomal ICG and Free ICG among the Liposomal ICG complex prepared in Example 1 and the Cis-liposomal ICG complex prepared in Example 2 was adjusted to 5 μg / ml, and the Liposomal ICG complex prepared in Examples 1 and 2 And Cis-liposomal ICG complexes were irradiated with laser at 808 nm for 3 minutes at 650 mW (81 J, Joule), and photothermal effects were measured using a thermal imaging camera (FLIR) with a total volume of 100 μl of the liposome solution.

The measured photothermal effect is shown in Fig. As shown in Fig. 7, it was confirmed that the Cis-liposomal ICG complex maximizes the photothermal effect with the same amount of ICG.

In addition, Cis-liposomal ICG was prepared by the method of Example 2, and Cis-liposomal ICG (HSPC) prepared by using HSPC and phosphatidylcholine were used in Cis-liposomal ICG using DPPC. The amount of cisplatin released outside the liposome due to the effect was measured by the following equation.

Cisplatin emission amount = {(LC-C) / (O-C)} * 100

LC = concentration of cisplatin in solution after removal of liposomes using a centrifugal filter after laser irradiation

C = concentration of cisplatin in the liposome-removed solution using a centrifugal filter prior to laser irradiation

O = concentration of cisplatin in whole solution containing liposome before laser irradiation

As a result, it was confirmed that thermosensitive release can be realized when using DPPC (FIG. 8).

Test Example  2: Cis - liposomal ICG's  Cell killing effect

HeLa cells (ATCC® CCL-2 ™) were prepared in an amount of 5000 Cells / 100 μl 1 Well. Liposomal ICG complex with lipid: ICG ratio of 250: 1 or 250: 4, lipid species of DPPC or HSPC, and cisplatin concentration of 11 uM were treated with 5 μg / ml (based on ICG concentration) After that, the laser of 808 nm and 650 mW was irradiated for 3 minutes. After the irradiation with the laser, the liposomes containing the indocyanine green remaining in the cell culture medium were removed after 30 minutes, and the cells were cultured for 24 hours, and then the cell death effect was measured by MTT assay.

The results are shown in Fig. As shown in Fig. 9, the most improved apoptosis effect was observed in the case of the cisplatin-containing complex (final lipid: ICG: cisplatin molar ratio of 250: 7: 11) containing DPPC and having a lipid: ICG ratio of 250: Respectively.

Test Example  3: Cis - liposomal ICG's  Identify in vivo stability and storage location

Liposomal ICG and Cis-liposomal ICG were prepared in the same manner as in Examples 1 and 2, except that the complexes prepared with the initial lipid: ICG: cisplatin ratios of 250: 1: 11, 250: 4: 11 and 250: 8: / ml, and 200 μl was intravenously injected intravenously into C57BL / 6 mice. Venous blood samples were taken at 30 minutes, 2 hours, 6 hours, 12 hours and 24 hours after the injection, and the fluorescence intensity was measured to determine the amount of ICG Respectively.

As a result, as shown in FIG. 10, it was confirmed that the stability in blood flow of Cis-liposomal ICG having an initial lipid: ICG ratio of 250: 4 was the highest.

Liposomal ICG and Cis-liposomal ICG were prepared to have a lipid: ICG ratio of 250: 4 by the methods of Examples 1 and 2. The complex was concentrated to an ICG concentration of 400 μg / ml, and then 200 μl of C57BL / 6 mice were euthanized 24 hours later and the mice were euthanized. In order to determine the distribution of ICG in the organ, the main organs were extracted and mass was measured. The tissue was disrupted and 100 ul of the disrupted mixture was added to a 96-well plate The fluorescence intensity of the ICG contained in the tissue was measured using a fluorescence meter, and the mass of the tissue was divided, and the ratios of the fluorescence intensities were compared.

As a result, as shown in Fig. 11, Free ICG accumulates in the order of kidney, liver, lung, tumor, spleen, heart, brain, and in the case of Liposomal ICG, spleen, liver, tumor, lung, And accumulation in the order of liver = spleen, tumor, kidney, lung, heart, brain in the case of Cis-liposomal ICG.

Test Example  4: Cis - liposomal ICG's  In vivo Photothermal effect  And tumor treatment effects

Liposomal ICG and Cis-liposomal ICG were prepared to have a lipid: ICG ratio of 250: 4 by the methods of Examples 1 and 2. The complex was concentrated to an ICG concentration of 400 μg / ml, and 200 μl of the mixture was added to C57BL / 6 mouse The concentration of cisplatin was about 240 μg / ml. Free ICG was dissolved at a concentration of 400 μg / ml. Then, 200 μl was injected intravenously and 200 μl of 5% glucose was injected intravenously. After 30 minutes, the 808 nm laser was irradiated for 20 minutes at an intensity of 0.6 W / cm < 2 >, visualized the temperature distribution varying with time, and measured the maximum temperature in the cancer tissue.

As a result, it was confirmed that Cis-liposomal ICG maintained a high photothermal effect for a long time when the same amount was intravenously injected based on ICG (FIG. 12).

In addition, the mice treated with the above method were monitored for tumor size and viability at intervals of 2 days, and the mice were photographed at 3, 7, and 20 days after laser irradiation, irradiation, and after irradiation.

As a result, in the case of Cis-liposomal ICG, the cancer tissue was denatured three days after the laser irradiation due to the photothermal effect and the effect of the chemotherapy, and it was confirmed that it was cured after 20 days (FIG. 13).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

Phosphatidylcholine, lipids conjugated to polyethylene glycol, alkylating agents having anticancer effects, and complexes which are prepared from indocyanine green and which are encapsulated with an alkylating agent having anti-cancer effects on liposomes and indocyanine green As an active ingredient,
A composition for treating cancer, which has both cancer cell phototherapy efficacy and cytotoxic effect of indocyanine green upon therapeutically effective light irradiation.
delete The composition for treating cancer according to claim 1, wherein the polyethylene glycol has a molecular weight of 1, 000 to 10, 000.
The composition for treating cancer according to claim 1, wherein the indocyanine green is contained at a concentration of 1-50 ug / ml.
The composition for treating cancer according to claim 1, wherein the content of the phosphatidylcholine, indocyanine green and alkylating agent having an anticancer effect is 250: 0.1-32: 1-200 (molar ratio).
The composition for treating cancer according to claim 1, wherein the therapeutically effective light is irradiated at a power of 100 mW or more and 1 W or less at 700-900 nm for 3 minutes to 30 minutes or less.
The composition for treating cancer according to claim 1, wherein the composite has a hydrodynamic diameter (HD) of 50-200 nm.
The composition for treating cancer according to claim 1, wherein the polydispersity index of the complex is 0.2 or less.
The composition for treating cancer according to claim 1, wherein the zeta potential of the complex is -50 -0 mV.
The composition for treating cancer according to claim 1, wherein the photo-thermal treatment effect induces the death of cancer cells by indocyanine green upon irradiation with therapeutically effective light.
delete The composition for treating cancer according to claim 1, wherein the alkylating agent is cisplatin or carboplatin.
CLAIMS What is claimed is: 1. A method of preparing an alkylating agent having an anticancer effect on liposomes comprising the following steps and a complex encapsulated with indocyanine green:
(a) dissolving lipid and indocyanine green conjugated to at least one phosphatidylcholine and polyethylene glycol in an organic solvent, and removing the organic solvent to prepare a cake;
(b) mixing an alkylating agent having an anticancer effect on the cake, preparing a dispersion by hydration, and extruding the dispersion into a porous polymer membrane to prepare an alkylating agent having an anti-cancer effect and a liposome containing indocyanine green; And
(c) separating an alkylating agent having an anticancer effect among the liposomes of the step (b) and a complex encapsulated in the liposome with indocyanine green.
14. The method of claim 13, wherein the porous polymer membrane comprises a polycarbonate having a pore diameter of 50-200 nm.

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