WO2016186429A1

WO2016186429A1 - Composition for cancer treatment comprising indocyanine green-liposome composite

Info

Publication number: WO2016186429A1
Application number: PCT/KR2016/005209
Authority: WO
Inventors: 박지호; 윤환준; 이혜성; 김현구
Original assignee: 고려대학교산학협력단
Priority date: 2015-05-18
Filing date: 2016-05-17
Publication date: 2016-11-24
Also published as: KR101686145B1; KR20160135424A

Abstract

The present invention relates to a composition for cancer treatment comprising an indocyanine green-capsulated liposome composite, a cancer treatment method using the same, and a method for preparing the same and, specifically, to a composition for cancer treatment, which comprises a composite containing indocyanine green and a liposome and treats caner using the light heat generated from the irradiation of therapeutically effective light, to a cancer treatment method using the same, and a method capable of preparing the same.

Description

Cancer treatment composition comprising indocyanine green-liposomal complex

The present invention relates to a composition for treating cancer comprising a liposome complex encapsulated with indocyanine green, a method for treating cancer using the same, and a method for preparing the same, wherein the induction of apoptosis by therapeutically effective light irradiation is provided. It relates to a composition for treating cancer, a method for treating cancer using the same, and a method for producing the same.

Indocianin green (ICG) is a near-infrared fluorescent die (NIR) that is licensed by the US Food and Drug Administration (FDA) for the diagnosis of the heart and liver vascular systems as well as the lymphatic system. In particular, indocyanine green is known as an excellent probe for imaging metastatic lymph nodes and mapping of lymph nodes for early diagnosis of breast cancer.

Such indocyanine green has low hydrophilicity, low light stability, low photon yield, and low sensitivity. In addition, indocyanine green is susceptible to nonspecific aggregation, has a disadvantage of being chemically decomposed by external light, solvent and temperature change, and has a problem of being rapidly absorbed into serum proteins and rapidly removed through the liver due to low molecular weight and hydrophobic characteristics. have.

In order to overcome this drawback, nanomaterial-based indocyanine green probes have been studied, and indocyanine green is trapped in nanoparticles, or polymers are used to examine in vivo and in vitro stability of indocyanine green. There has been increased research. By capturing indocyanine green into nanoparticles, the physicochemical stability against external light and temperature was significantly increased.

Nevertheless, there is currently no example in which indocyanine green is combined with liposomes applicable in the body and applied to the composition for treating cancer. Cancer phototherapy is based on light, and is a method of treating cancer by radiating light at a tumor site, rather than by dangerous and expensive surgery.

Phototherapy is nondestructive, simple and has fewer side effects than surgical procedures. In addition, there is no need for general anesthesia, little pain of the patient, a short period of stability and recovery as well as the advantage that can be repeated several times.

On the other hand, cancer treatment technology by heat having a method of simply heating by inserting a heating device in the tumor site has been tried a long time ago, but since it is impossible to identify cancer cells and normal cells, normal cells around cancer cells are also destroyed. Because of this, it is not widely applied in actual clinical practice. In contrast, phototherapy is a new cancer treatment that overcomes the shortcomings of both drug and radiation therapies. Early phototherapy has a variety of types depending on the light source, but most of them have a disadvantage that the treatment period is long, and the interface of the treatment site is inaccurate.

For example, phototherapy technology that combines silica nanoparticles with gold-coated nanoshells and near infrared light, or phototherapy technology that combines single wall carbon nanotubes (SWCNT) with near infrared light, is a method for selective cancer treatment. It has been suggested that they can be used, but they are not approved for in vivo application and thus have limitations in clinical applications, and very high intensity NIR should be used to obtain the desired heat. The problem is that they can be damaged even if they are not very close to a heating agent such as a shell or SWCNT.

Under this technical background, the inventors of the present application include a complex in which indocyanine green is encapsulated in liposomes, and solve the conventional problems through a composition for treating cancer that can induce apoptosis by therapeutically effective light irradiation. It was confirmed that the desired cancer treatment effect can be achieved, and completed the present invention.

발명의 요약Summary of the Invention

Disclosure of Invention An object of the present invention is to provide a composition for treating cancer comprising a liposome complex in which indocyanine green is encapsulated by inducing apoptosis by therapeutically effective light irradiation and exhibiting a desired cancer therapeutic effect, and a method for preparing the same. There is.

In order to achieve the above object, the present invention contains indocyanine green-liposomal complex prepared by mixing phosphatidylcholine, stabilizer and indocyanine green, the indocyanine green is encapsulated in liposomes, It provides a composition for treating cancer, which induces apoptosis by therapeutically effective light irradiation.

The present invention also provides a method for treating cancer, comprising the following steps:

(a) administering to the patient an indocyanine green-liposomal complex in which the indocyanine green is encapsulated in the liposome; And

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

The present invention also provides a method of preparing a composition for treating cancer, comprising the following steps:

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

(b) hydrating the cake to prepare a dispersion, and extruding the porous polymer membrane to prepare liposomes containing indocyanine green; And

(c) isolating a complex in which indocyanine green is encapsulated in the liposome in the liposome of step (b).

1 is a graph comparing the degree of exotherm upon light irradiation according to the ratio of phosphatidylcholine and ICG in a mixture for preparing an indocyanine green encapsulated liposome complex;

Figure 2 is a graph comparing the degree of exotherm upon light irradiation according to the DSPC and ICG ratio in the mixture for preparing a liposome complex when preparing a liposome containing DSPC;

3 is a graph comparing the exothermic degree upon light irradiation according to the ratio of DPPC and ICG in a mixture for preparing a liposome complex when preparing a liposome including a DPPC;

Figure 4 is a graph comparing the exothermic degree upon light irradiation according to the ratio of DMPC and ICG in the mixture for preparing a liposome complex when preparing a liposome containing a DMPC;

5 is a graph comparing the degree of exotherm upon light irradiation according to the ratio of DOPC and ICG in a mixture for preparing a liposome complex when preparing a liposome including DOPC;

Figure 6 is a graph comparing the degree of exotherm when irradiated with light to free ICG (lip ICo) free ICG;

7 is a graph comparing the absorbance according to the DSPC and ICG ratio in the mixture for preparing a liposome complex when preparing a liposome comprising a DSPC;

8 is a graph comparing the absorbance according to the ratio of DPPC and ICG in a mixture for preparing a liposome complex when preparing liposomes including DPPC;

9 is a graph comparing the absorbance according to the DMPC and ICG ratio in the mixture for preparing liposome complex when preparing liposomes including DMPC;

10 is a graph comparing the absorbance according to the ratio of DOPC and ICG in a mixture for preparing a liposome complex when preparing a liposome including DOPC;

FIG. 11 is a graph comparing relative fluorescence intensities according to DSPC, DPPC, DMPC and DOPC and ICG ratios in liposomes including DSPC, DPPC, DMPC and DOPC, respectively; FIG.

FIG. 12 is a graph comparing the passive mechanical average diameter according to DSPC, DPPC, DMPC and DOPC and ICG ratios in liposomes including DSPC, DPPC, DMPC and DOPC, respectively; FIG.

FIG. 13 is a graph comparing zeta potential according to DSPC, DPPC, DMPC and DOPC and ICG ratios in liposomes including DSPC, DPPC, DMPC and DOPC, respectively; FIG.

FIG. 14 is a graph comparing polydispersity index (PDI) according to DSPC, DPPC, DMPC and DOPC and ICG ratio in liposomes including DSPC, DPPC, DMPC and DOPC, respectively; FIG.

15 is a graph showing the photothermal effect according to the DSPC, DPPC, DMPC and DOPC and ICG ratios; And

16 is a graph showing cell viability according to light irradiation in a liposome complex containing DMPC.

발명의 상세한 설명 및 바람직한 Detailed description of the invention and preferred 구현예Embodiment

In one aspect, the present invention contains an indocyanine green-liposomal complex prepared by mixing phosphatidylcholine, a stabilizer, and an indocyanine green, wherein the indocyanine green is encapsulated in liposomes and is therapeutic It relates to a composition for treating cancer, which induces cell death by effective light irradiation.

The inventors of the present application can improve the stability of indocyanine green by overcoming the disadvantages associated with low stability of indocyanine green through liposome complexes in which indocyanine green is encapsulated, It was confirmed that cancer treatment by apoptosis is possible.

In the present specification, apoptosis by light irradiation is achieved by phototherapy, and phototherapy is one of clinical methods widely used in cancer treatment because it has fewer side effects, is non-invasive, and is specific for light of a specific wavelength. Phototherapy includes photodynamic therapy (PDT) and photothermal therapy (PTT), which include reactive oxygen species (ROS) and light and photosensitizers for thermal energy generation, respectively. Formulation is required.

In particular, the inventors of the present application confirmed that the indocyanine green according to the present invention may exhibit an excellent photothermal treatment effect upon light irradiation to the encapsulated liposome complex. This effect was confirmed to increase the cytotoxicity to cancer cells can exhibit a cancer treatment effect.

In one embodiment, the liposomes are prepared including phosphatidylcholine and stabilizers, and liposomes in the form of a single lamellar or a multilamellar may be used. The phosphatidylcholine is a kind of phospholipid with choline, and includes a hydrophobic tail and a choline hydrophilic head of saturated or unsaturated fatty acids, for example, L-α-phosphatidylcholine (Egg, Chicken), L -α-phosphatidylcholine, hydrogenated (Egg, Chicken), L-α-phosphatidylcholine (Soy), L-α-phosphatidylcholine, hydrogenated (Soy) 1,2-didecanoyl-sn-glycero-3-phosphocholine (10: 0 PC) , 1,2-diundecanoyl-sn-glycero-3-phosphocholine (11: 0 PC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (12: 0 PC (DLPC)), 1,2-ditridecanoyl- sn-glycero-3-phosphocholine (13: 0 PC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (15: 0 PC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16 : 0 PC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17: 0 PC), 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (19: 0 PC), 1,2-diarachidoyl -sn-glycero-3-phosphocholine (20: 0 PC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (14: 1 (Δ9-Cis) PC), 1,2-dimyr istelaidoyl-sn-glycero-3-phosphocholine (14: 1 (Δ9-Trans) PC), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16: 1 (Δ9-Cis) PC), 1,2- dipalmitelaidoyl-sn-glycero-3-phosphocholine (16: 1 (Δ9-Trans) PC), 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (18: 1 (Δ6-Cis) PC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (18: 1 (Δ9-Cis) PC (DOPC)), 1,2-dielaidoyl-sn-glycero-3-phosphocholine (18: 1 (Δ9-Trans) PC), 1 , 2-dilinoleoyl-sn-glycero-3-phosphocholine (18: 2 (Cis) PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18: 3 (Cis) PC), 1,2-dieicosenoyl -sn-glycero-3-phosphocholine (20: 1 (Cis) PC), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine ), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) may be one or more selected from, but is not limited thereto. .

The inventors of the present application confirmed that the liposome-encapsulated ICG exhibited an improved photothermal effect as compared to free ICG with liposomes unbound (known indocyanine green alone) and ICG bound to liposome surfaces. Based on this, encapsulation as used herein means that indocyanine green is substantially present only in the space in the liposome to form a liposome-ICG complex, and only on the free ICG or liposome surface to which the liposome is not bound. Bound ICG is removed to mean that it is substantially nonexistent.

In addition, the inventors of the present application confirmed that the photothermal effect is improved, especially as the hydrophobic tail length of the phospholipid is shortened, and that the unsaturated fatty acid is included in the hydrophobic tail.

Based on this, considering liposomes including DSPC, DPPC, DMPC or DOPC, it was confirmed that the liposomes containing DMPC showed the best photothermal effect at the same indocyanine green concentration. DMPC has 14 carbon atoms in hydrophobic tail, shorter hydrophobic tail length than 16 DPPC, 18 DSPC or DOPC, and showed excellent photothermal effect compared to DOPC containing unsaturated fatty acid in hydrophobic tail (Test Example 3 ). Thus, the phosphatidylcholine may preferably be DMPC.

The stabilizer may be a lipid, a lipid derivative, a protein or a peptide, and the like, and specifically, may be phosphatidylethanolamine conjugated with polyethylene glycol. The polyethylene glycol may be used without limitation as long as it is a size suitable for stabilization, for example, 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.

The phosphatidylethanolamine may be mono or diunsaturated fatty acid, and for example, may be one or more selected from the group consisting of dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and DSPE (distearoylphosphatidyl-ethanolamine). It is not limited.

The phosphatidylcholine and stabilizer may be included with indocyanine green to an extent suitable for liposome formation suitable for photothermal effect, for example, the molar ratio of phosphatidylcholine and stabilizer is from about 85:15 to about 98: 2, preferably about 90:10 to 95: 5. Within this molar ratio range, liposomes in vivo are most stable to maximize lifespan.

Phosphatidylcholine and indocyanine green in the mixture are not limited so long as they are suitable for producing a photothermal effect for cancer treatment, but may be included, for example, in a molar ratio of 250: 0.1-32. Preferably, phosphatidylcholine and indocyanine green in the mixture may be included in a molar ratio of 250: 0.5-16. The desired photothermal effect can be exhibited throughout this range, and the inventors of the present application find that indocyanine green is optimally dispersed and encapsulated inside liposomes, particularly when phosphatidylcholine and indocyanine green are included in the molar ratio of the range. Complexes can be formed, and having an optimal absorbance that can exhibit an effective photothermal effect in the treatment of cancer, it was confirmed that it exhibits a significant photothermal effect.

When the amount of indocyanine green is smaller than the range described, the desired degree of photoreaction effect cannot be expected, and the amount of liposomes is relatively high, which is very inefficient considering the cost and time required for preparing liposomes. If the amount of indocyanine green is higher, agglomeration may occur in liposomes, or a threshold level that no longer reacts to light may be reached, and a synergistic photothermal effect may not be expected. In particular, it was confirmed that phosphatidylcholine and indocyanine green had the best photothermal effect when they had a molar ratio of 250: 1. This may be due to the highest dispersion of indocyanine green among liposome complexes prepared from phosphatidylcholine and indocyanine green in a molar ratio of 250: 1.

In one embodiment, heat may be generated when the composite is irradiated with near infrared rays, and the near infrared rays may be irradiated for 3 minutes or more with a power of 100 mW or more at 700-900 nm. At this time, the power for generating heat may be, for example, 100 mW or more, 1 W or less, preferably 400 mW or more and 800 mW or less. The light irradiation time may be 3 minutes or more and 30 minutes or less. In the light irradiation conditions lower than the above range, indocyanine green is not effectively consumed in generating the photothermal effect, and in high light irradiation conditions, surrounding normal cells may be exposed to phototoxicity and damage may be caused.

The heat generated at this time may be, for example, a temperature of about 45 ° C. or higher, preferably 50 ° C. or higher, more preferably 60 ° C. or higher, at which 90% or more apoptosis, preferably 95% or more, may occur. .

Indocyanine green in the heat generating complex may include, for example, at a concentration of 0.1-30 ug / ml, preferably 1-10 ug / ml. The inventors of the present application confirmed that heat can be generated to a temperature rise of 10 ° C. or more, in which near-infrared irradiation may cause 95% or more apoptosis of liposome complexes including indocyanine green in the aforementioned concentration range.

In particular, the higher the accumulation amount of liposome complex containing indocyanine green in the tumor-induced lymph nodes, the higher the heat generation, it was confirmed that more than enough heat to generate a major cell destruction can cause cell death. Through this, it was confirmed that the liposome complex including indocyanine green, which was developed based on the FDA approved substance, may have great clinical potential.

In one embodiment, the complex may be determined in size (diameter) according to the type and size of liposomes in which indocyanine green is encapsulated, and the average hydrodynamic diameter (HD) of the complex is, for example, It may be 50-200 nm, preferably 100-150 nm.

In yet another embodiment, the composite exhibits a low degree of polydispersity index to confirm that the molecular weight and structure is uniform, wherein the polydispersity index may be 0.2 or less.

In some cases, the composition for treating cancer according to the present invention may further include an antibody effective for treating cancer or an immunotherapeutic fragment thereof, a cytotoxic agent or a chemotherapeutic agent thereof.

Antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from two or more intact antibodies, and antibody fragments exhibiting the desired biological cancer therapeutic activity, wherein immunotherapeutic antibody fragments include antigen binding or variable regions thereof For example, Fab, Fab ', F (ab') 2, Fv and Fc fragments, diabodies, linear antibodies, single chain antibody molecules; And multispecific antibodies formed from antibody fragments, including portions of intact antibodies.

Cytotoxic agents are substances that inhibit or prevent the function of a cell and / or cause cell disruption, and include radioisotopes, chemotherapeutic agents and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or And fragments thereof.

Chemotherapeutic agents include chemical agents that prevent the development, maturation or proliferation of new cells directly on tumor cells, such as by cytostatic or cytotoxic effects and not indirectly through mechanisms such as changes in biological response. Chemotherapeutic agents suitable for the present invention preferably include natural or synthetic chemical compounds.

'Cancer' and 'tumor' refer to or describe the physiological condition in mammals, typical of uncontrolled cell growth, and according to the compositions of the present invention, breast, heart, lung, small intestine, large intestine, spleen Tumors or metastatic tumors, such as tumors of the kidneys, bladder, head and neck, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, cervix and liver, can be treated.

Based on this, the present invention provides a cancer treatment method comprising the following steps:

In another aspect, the present invention relates to a method for preparing a composition for treating cancer, comprising the following steps:

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

In the manufacturing method according to the present invention, step (a) is a step of preparing a cake by dissolving at least one phosphatidylcholine, a stabilizer, and indocyanine green in an organic solvent, and removing the organic solvent. Compositions comprising complexes in which indocyanine green is encapsulated in liposomes can be prepared via film hydration / extrusion.

The phosphatidylcholine and stabilizer, which are the materials of the liposomes, are equally applicable to the above-mentioned invention related to the preparation method. Phosphatidylcholine and a stabilizer may be dissolved in the organic solvent.

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

Thereafter, (b) hydrating the cake prepared by removing the organic solvent and drying to prepare a dispersion, and is extruded to a porous polymer membrane to prepare a liposome containing indocyanine green. The dispersion may be prepared by hydration with an isotonic solution, the isotonic solution may include, for example, glucose, trehalose, etc., the isotonic agent may be, for example, 1-10% (w / v), preferably To 3-7% (w / v).

The dispersion may be prepared by liposomes in the form of single or multi-lamellar vesicles by extruding the porous polymer membrane, wherein the porous polymer membrane may be, for example, a polycarbonate polymer membrane, and the pore diameter of the porous polymer membrane may be, for example. For example 10-1000 nm, preferably 50-200 nm.

In the next step (c), the indocyanine green separates the complex encapsulated in the liposome. The method used for removal can be used without limitation as long as indocyanine green can separate complexes encapsulated within liposomes and free indocyanine green and liposome surfaces, or indocyanine green that has been exposed or exposed. For example, only the complexes in which the desired indocyanine green is encapsulated in liposomes can be separated by passing through a dialysis membrane or using chromatography (eg, size exclusion chromatography, etc.).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Liposomal ICG

ICG (IR-25, laser grade pure) was obtained from Acros Organics. The main component lipids are DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3 -phosphocholine) or DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] ) Mixed in a solution of chloroform in a ratio of 95: 5, and mixed indocyanine green (total molar concentration: ICG = 250: X (M)) dissolved in methanol at 1 mg / ml in an amount of the required organic solvent. After fully evaporating the resulting lipid cake at the bottom of the flask was hydrated with 2 ml of 5% aqueous glucose solution.

At this time, the composition of the hydration solution was DSPC, DPPC, DMPC, or the ratio of DOPC and ICG was 250: 0.5, 250: 1, 250: 4, 250: 8, 250: 16 and 250: 32.

Using a 0.1 um polycarbonate membrane, the hydrated liposome solution was extruded to an average of 100 nm single or multi lamellar liposomes. A 100 nm single or multi lamellar liposome solution, complete with extrusion, was passed through a column filled with Sephadex G-50 to separate ICG and ICG-containing liposomes, which were not included in liposomes, using a size-exclusion chromatography technique. do. Dialysis membranes with a 100 kDa MWCO (molecular weight cut-off) were used for dialysis to remove trace amounts of free ICG and ICG adhering to the liposome surface, thereby preparing an ICG encapsulated liposome complex.

The increase in fluorescence intensity and absorbance due to the dispersion effect of ICG encapsulated in liposomes were used to determine the presence of free ICG and liposomes exposed to liposome surface in liposome ICG solution.

The concentration of phosphatidylcholine was determined in the liposome-ICG complex from which free ICG was removed by Stewart assay, which shows the concentration of phosphatidylcholine and phosphatidylcholine in DSPE-PEG200. In addition, when 80% methanol and 20% liposome solution are mixed, the liposome structure is all collapsed, and the ICG shows the original absorbance, and the absorbance has a maximum at 785 nm. Since the absorbance of ICG increases in proportion to the concentration in methanol of 80%, the concentration of ICG contained in the liposome complex was obtained through the absorbance of 785 nm. {(Total Phosphatidylcholine Concentration) / (ICG Concentration)} can determine the ratio between phosphatidylcholine and ICG in the liposome-ICG complex. This allows comparing the amount of ICG included in the finally synthesized liposome-ICG complex, compared to the amount of ICG added when preparing a phosphatidylcholine-containing cake to synthesize each liposome-ICG complex.

As a result, when the amount of ICG added during cake making was in the range of phosphatidylcholine: ICG in the range of 250: 0.5 to 16 (M), the amount of ICG contained in liposomes increased as the amount of ICG added increased. . However, if the molar ratio of phosphatidylcholine: ICG exceeds 250: 16, the amount of ICG contained in the liposomes does not increase.

Test Example 1 Photothermal Effect of Liposomal ICG

In the Liposomal ICG complex prepared in Example 1, all the Liposomal ICG and Free ICG ICG concentrations were adjusted to 5 ug / ml, and the Liposomal ICG complex prepared in Example 1 was irradiated with a laser of 808 nm at 650 mW for 3 minutes (81 J, Joule), the thermal imaging camera (FLIR) to measure the photothermal effect caused by the total volume of liposome solution 100ul.

The measured photothermal effects are shown in FIGS. 1 to 6. Comparing FIGS. 1 and 6, in the case of free ICG not bound to liposomes, the temperature rise is only 5 ° C. despite light irradiation of 150 seconds or more, and is relatively weaker than that of ICG encapsulated in liposomes. It was confirmed that the light heat effect.

The photothermal effects occurring in each of the complexes including DSPC, DPPC, DMPC, or DOPC in FIG. 1 are shown in FIGS. 2 to 5, respectively. As the hydrophobic tail length of the phospholipid constituting the complex becomes shorter, the photothermal effect is improved, and when the unsaturated lipid chain is included in the hydrophobic tail, the photothermal effect is reduced. Among the DSPC, DPPC, DMPC and DOPC, DMPC (FIG. 4) shows that the best photothermal effect occurs. When the molar ratio of DSPC, DPPC, DMPC, or DOPC: ICG was 250: 0.5 to 250: 16, the desired photothermal effect was observed. In particular, when the molar ratio of phosphatidylcholine: ICG was 250: 1, it was related to the type of phosphatidylcholine. Excellent photothermal effect.

If the molar ratio of phosphatidylcholine: ICG is less than 250: 0.5, for example 250: 0.1, in order to obtain a similar degree of photothermal effect, the amount of liposomes added is too high and the cost and time required for the preparation of liposomes is increased. It is very inefficient when considered and is not suitable when considering industrial applications. Thus, the molar ratio of phosphatidylcholine: ICG in the composition for treating cancer through the photothermal effect can be optimized from the range of 250: 0.5. In addition, if the phosphatidylcholine: ICG molar ratio is greater than 250: 16, for example 250: 32, the addition of ICG does not increase the ratio of ICG in the liposome-ICG complex, but rather aggregation occurs. Not suitable (FIG. 15).

Test Example 2: Characteristics of Liposomal ICG

Dynamic light scattering was used to determine the average hydrodynamic diameter (HD), polydispersity index, and zeta potential of each composite. In addition, absorbance was measured at intervals of 5 nm at a wavelength of 400 nm to 1000 nm, and fluorescence intensity was measured by Excitation 785 nm and Emission 820 nm. The concentration used at this time is 5 ug / ml based on the ICG.

The results are shown in FIGS. 7 to 14. 7 to 10, it can be seen that the composite including DSPC, DPPC, DMPC, or DOPC has a maximum absorbance at 800 nm and shows a low peak at the early 700 nm. At the same ICG concentration, the higher the molar ratio of DSPC, DPPC, DMPC, or DOPC (M): ICG (M) to ICG, the lower the maximum absorbance at 800 nm and the lower peak in the early 700 nm range. This means that if DSPC, DPPC, DMPC or DOPC: ICG exceeds 250: 16, the likelihood of ICG aggregation inside the liposome complex is high.

Referring to FIG. 11, it can be seen that the fluorescence intensity decreases as the ratio of total lipid (M) to ICG (M) in the same ICG concentration increases, as shown in FIGS. 12 to 14. (hydrodynamic diameter, HD), zeta potential, and polydispersity index were not significantly different depending on the ratio of total lipid (M): ICG (M) or lipid type.

Test Example 3: Apoptosis of Liposomal ICG

HeLa cells (ATCC® CCL-2 ™) were prepared in an amount of 5000 Cells / 100ul 1 Well. Hela cells were treated with lipids (phosphatidylcholine) (M): ICG (M) prepared in 250: 1 ratio, respectively, at 5 ug / ml (based on the concentration of ICG), respectively, at 808 nm. , 650mW laser was irradiated for 3 minutes. After irradiating the laser, the liposomes including indocyanine green remaining in the cell culture were removed after 30 minutes, and the cells were cultured for 24 hours, and then apoptosis was measured by MTT assay.

The results are shown in FIG. Referring to FIG. 16, it was confirmed that the liposome-ICG complex including DMPC exhibited the most improved apoptosis effect.

The composition for treating cancer according to the present invention exhibits a significant apoptosis effect by irradiating therapeutically effective light to a complex comprising liposomes encapsulated with indocyanine green, thereby showing an excellent cancer treatment effect, Tumors can be removed without surgical intervention. In addition, while minimizing the effect on normal cells, it may selectively exhibit apoptosis effect against cancer cells.

Those skilled in the art to which the present invention pertains will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

Claims

Contains indocyanine green-liposomal complex prepared by mixing phosphatidylcholine, stabilizer and indocyanine green,

The indocyanine green is encapsulated in liposomes,

A composition for treating cancer, which induces apoptosis by therapeutically effective light irradiation.
According to claim 1, wherein the phosphatidylcholine is L-α-phosphatidylcholine (Egg, Chicken), L-α-phosphatidylcholine, hydrogenated (Egg, Chicken), L-α-phosphatidylcholine (Soy), L-α-phosphatidylcholine, hydrogenated ( Soy) 1,2-didecanoyl-sn-glycero-3-phosphocholine (10: 0 PC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (11: 0 PC), 1,2-dilauroyl-sn- glycero-3-phosphocholine (12: 0 PC (DLPC)), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (13: 0 PC), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (15 : 0 PC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16: 0 PC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17: 0 PC), 1,2- dinonadecanoyl-sn-glycero-3-phosphocholine (19: 0 PC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (20: 0 PC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine ( 14: 1 (Δ9-Cis) PC), 1,2-dimyristelaidoyl-sn-glycero-3-phosphocholine (14: 1 (Δ9-Trans) PC), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine ( 16: 1 (Δ9-Cis) PC), 1,2-dipalmitelaidoyl-sn-glycero-3-phosphochol ine (16: 1 (Δ9-Trans) PC), 1,2-dipetroselenoyl-sn-glycero-3-phosphocholine (18: 1 (Δ6-Cis) PC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (18: 1 (Δ9-Cis) PC (DOPC)), 1,2-dielaidoyl-sn-glycero-3-phosphocholine (18: 1 (Δ9-Trans) PC), 1,2-dilinoleoyl-sn-glycero -3-phosphocholine (18: 2 (Cis) PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18: 3 (Cis) PC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (20: 1 (Cis) PC), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2- Dimyristoyl-sn-glycero-3-phosphocholine) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) The composition for treatment of cancer, characterized in that at least one selected from the group consisting of.
According to claim 1, wherein the stabilizer is a composition for treating cancer, characterized in that the phosphatidylethanolamine conjugated to polyethylene glycol (phosphatidylethanolamine).
The composition of claim 3, wherein the polyethylene glycol has a molecular weight of 1, 000 to 10, 000.
According to claim 3, wherein the phosphatidyl ethanolamine is DMPE (dimyristoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) and DSPE (distearoylphosphatidyl-ethanolamine) composition for cancer treatment, characterized in that at least one.
The method of claim 1, wherein the indocyanine green is a cancer treatment composition, characterized in that it comprises a concentration of 0.1-30 ug / ml.
According to claim 1, wherein the phosphatidylcholine and indocyanine green is a cancer treatment composition, characterized in that contained in a molar ratio of 250: 0.1-32.
8. The composition of claim 7, wherein the phosphatidylcholine and indocyanine green are included in a molar ratio of 250: 0.5-16.
The composition for treating cancer according to claim 1, wherein the therapeutically effective light is irradiated for 3 minutes to 30 minutes at a power of 100 mW or more and 1 W or less at 700-900 nm.
The composition of claim 1, wherein the complex has an average hydrodynamic diameter (HD) of 50-200 nm.
According to claim 1, wherein the polydispersity index (polydispersity index) of the complex is a cancer treatment composition, characterized in that less than 0.2.
The composition for treating cancer of claim 1, further comprising an antibody effective for treating cancer or an immunotherapeutic fragment thereof, a cytotoxic agent, or a chemotherapeutic agent.
A method for preparing a composition for treating cancer according to any one of claims 1 to 12, comprising the following steps:

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

(b) hydrating the cake to prepare a dispersion, and extruding the porous polymer membrane to prepare liposomes containing indocyanine green; And

(c) isolating a complex in which indocyanine green is encapsulated in the liposome in the liposome of step (b).
The method of claim 13, wherein the organic solvent is methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, ether, benzene, Method for producing a composition for treating cancer, characterized in that at least one selected from the group consisting of chloroform, ethyl acetate, ethyl acetate, methylene chloride, hexane, and cyclohexane.
The method of claim 13, wherein the porous polymer membrane is made of a polycarbonate having a pore diameter of 50-200 nm.