KR20130034079A - Nano ion-complex for photodynamic theraphy comprising hydrophile cationic polymer photosensitizer derivatives and anionic polysaccharide quencher derivatives - Google Patents

Abstract

PURPOSE: A nanoion complex containing a hydrophilic cationic polymer photosensitizer derivative and an anionic polysaccharide quencher derivative for photodynamic therapy is provided to enhance accumulation rate to a cancer tissue and to generate single oxygen or free radical. CONSTITUTION: A nanoion complex for photodynamic therapy contains a hydrophilic cationic polymer photosensitizer derivative and an anionic polysaccharide quencher derivative. The hydrophilic cationic polymer photosensitizer derivative is a copolymer containing a cationic polymer conjugated with a photosensitizer.

Description

Nano ion-complex for photodynamic theraphy comprising hydrophile cationic polymer photosensitizer derivatives and anionic polysaccharide quencher derivatives}

The present invention relates to a nano-ionic complex for photodynamic therapy, which can effectively internalize cancer cells by effectively internalizing cancer cells, thereby effectively treating cancer by irradiating light, and hydrophilic cationic polymer photosensitive agent derivatives and anionics. The present invention relates to a nanoionic complex for photodynamic therapy comprising a polysaccharide quencher derivative.

Photodynamic therapy (PDT) is a technique that can treat incurable diseases such as cancer without surgery by using photosensitizer, which is selective and sensitive to various tumors, and has side effects such as chemotherapeutic agents. This is a kind of curiosity without. For example, by administering the photosensitive material to a subject by intravenous injection and irradiating with appropriate light, the resulting photosensitive material activates an oxygen molecule to convert to a singlet oxygen or to convert new radicals into a single radical. It selectively attacks and destroys only cancer cells or various tumor tissues. Such photodynamic therapy can selectively remove only diseased cells while preserving normal cells, and in most cases, can eliminate the risk of general anesthesia and can be operated simply by local anesthesia. Therefore, there is no need to remove damaged organs, and there is an advantage in that the recovery is quick after the minimally invasive procedure, the hospitalization period is shortened, and the patient's welfare is improved.

 Such photosensitive materials are typical of porphyrin-type compounds. Porphyrin-based compounds extracted from jambun, mulberry leaves, and green algae have spectroscopic properties suitable for use as photosensitive materials, and the most important property is relatively cellular. The red light (700-900 nm) having a large transmittance can efficiently generate an electron transition and an excited state accordingly.

 Porphyrin derivatives as photosensitizers selectively penetrate and accumulate in cancer cells or tumor tissues, and are also used as early diagnosis of tumors because they exhibit fluorescence or phosphorescence due to the characteristics of the compounds. Porphyrin's cancer cell selectivity is explained by the fact that the receptor for low density lipoprotein (LDL), which is responsible for the transport of porphyrin in the body, is expressed in cancer cells more than normal cells. However, most photosensitizers are poorly soluble and have a long phototoxicity due to the long residence time of the human body. In addition, photo-sensitizers are expensive and metabolism is slow in the human body, so side effects of phototoxicity have been found, and treatment efficiency decreases due to aggregation of photo-sensitizers in the body.

 On the other hand, the current photodynamic therapy using the photosensitizers was to induce selective attack or death of cancer cells or tumors by administering the photosensitizers to the subjects by intravenous injection and then irradiating with appropriate light. In addition, current photodynamic therapy is not used in bulky tumors due to the restriction of light transmission, and in particular, side effects of phototoxicity have been found due to the slow metabolism of photosensitizers in the human body. In addition, the concentration of the photosensitizer in the tumor is low, it does not show an effective therapeutic effect.

Accordingly, the inventors of the present invention have invented a new photosensitizer having a high selectivity and accumulation rate for cancer tissues and significantly reducing side effects of phototoxicity of the existing photosensitizers.

An object of the present invention is to prepare a hydrophilic photosensitizer for photodynamic therapy that can significantly increase the selectivity and accumulation rate of cancer tissue and can generate monoantioxidant or free radical by laser irradiation. Is to provide a photodynamic therapeutic nano-ion complex comprising a hydrophilic cationic polymer photosensitive agent and an anionic polysaccharide quencher derivative.

In order to achieve the above object, the present invention provides a nano-ionic composite for photodynamic therapy comprising a hydrophilic cationic polymer photosensitive agent and an anionic polysaccharide quencher derivative.

In one embodiment of the present invention, the hydrophilic cationic polymer photosensitive agent may be a copolymer in which a hydrophilic cationic polymer and a photosensitizer are combined.

In one embodiment of the present invention, the hydrophilic cationic polymer is glycol chitosan (GC), chitosan, poly-L-lysine (PLL), poly-beta-amino ester polymer, polyethyleneimine (PEI), polyethylene glycol, It may be at least one selected from the group consisting of poly (amidoamine) (PAMAM) dendrimers and derivatives thereof.

In one embodiment of the present invention, the photosensitizer is a porphyrin-based (phorphyrins) compounds, chlorins (chlorins) compounds, bacteriochlorins (bacteriochlorins) compounds, phthalocyanine (phtalocyanine) compounds, naphthalocyanines (naphthalocyanines) compounds And it can be at least one selected from the group consisting of 5-aminoevulinline ester compounds.

In one embodiment of the present invention, the hydrophilic cationic polymer photosensitizer derivative is composed of a copolymer of polyethylene glycol, polyethyleneimine and a photosensitizer, the photosensitizer may be hydrophilic by polyethylene glycol and polyethyleneimine have.

In one embodiment of the present invention, the polyethylene glycol may have a molecular weight of 300 ~ 50,000 (Da).

In one embodiment of the present invention, the polyethyleneimine is branched and may have a molecular weight of 100 ~ 25,000 (Da).

In one embodiment of the present invention, the anionic polysaccharide quencher derivative may be a combination of an anionic matrix polymer and a quencher.

In one embodiment of the invention, the anionic matrix polymer is specific for esterase enzymes and cancer cells, the anionic substrate polymer is chondroitin-6-sulfate (C6S), heparan sulfate (HS), heparan sulfate proteoglycan (HSPG), heparin, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronic acid (HA) It may be more than one species.

 In one embodiment of the present invention, the quencher may be a blackhole quencher (BHQ) or a blackberry quencher (BBQ).

In one embodiment of the present invention, the hydrophilic cationic polymer and the photosensitizer may be combined with the photosensitive agent in a molar ratio of 1 to 30 moles with respect to 1 mole of the hydrophilic cationic polymer.

In one embodiment of the present invention, the anionic matrix polymer and the quencher may be combined with the quencher in a molar ratio of 1 to 30 moles with respect to 1 mole of the anionic matrix polymer.

In one embodiment of the present invention, the nano-ion complex may be a mixture of a hydrophilic cationic polymer photosensitive agent to an anionic polysaccharide quencher derivative is mixed in a weight ratio of 1: 0.1 ~ 1:10.

In one embodiment of the present invention, the nano-ion complex is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, One or more cancers selected from the group consisting of rectal cancer, gastric cancer, bladder cancer, ovarian cancer, bile duct cancer and gallbladder cancer can be prevented or treated.

The hydrophilic cationic photosensitizer according to the present invention shows higher singlet oxygen generation when irradiated with fluorescence and laser than conventional hydrophobic photosensitizer in the water phase. This may prevent side effects such as aggregation and phototoxicity, which are problems of hydrophobic photosensitizers in vivo, and may contribute to improving targeting rates. In addition, when a nano ion complex with a cancer cell target anionic quencher is formed, it is quenched and specifically internalized only to cancer cells, and is degraded to reduce side effects in normal cells, which is a problem of conventional photodynamic drugs, and to enhance cancer cell targeting. The effect can be enhanced.

Figure 1a is a photograph showing the solubility results of hydrophobic free sensitizer (free drug) and hydrophilic cationic photosensitizer (WPS). Where (i) was treated with Ce6 (0.2 mg / ml), (ii) WPS (Ce6, 0.2 mg / ml), and (iii) WPS (Ce6, 0.3 mg / ml).
FIG. 1B is a graph showing fluorescence intensity in tertiary distilled water of hydrophobic and hydrophilic cationic photosensitizers and near-infrared fluorescence image at Ce6, 1 ug / ml.
FIG. 1C is a near infrared fluorescence image graph of distilled water (DDW), a hydrophobic photosensitizer, and a hydrophilic cationic photosensitizer.
1D is a graph showing the UV-Vis absorption spectra of hydrophobic and hydrophilic cationic photosensitizers in an aqueous phase.
FIG. 1E is a graph showing UV-Vis absorption spectra of hydrophobic and hydrophilic cationic photosensitizers in an organic solvent (dimethyl sulfoxide).
FIG. 2A is a graph showing near infrared fluorescence quenching characteristics (λex = 650 nm, λex = 670 nm) according to mass ratio of anionic polymer quencher (BHQ-CS) / hydrophilic cationic photosensitizer (WPS). Where F0 represents the fluorescence intensity of the hydrophilic cationic photosensitizer and Fi represents the fluorescence intensity of the nano ion composite (NPS).
2b is a graph showing particle size and zeta-potential (n = 3) in tertiary distilled water.
Figure 2c is a graph showing the particle distribution of the nano ion composite.
Figure 2d is a field emission scanning electron microscope image of the nano-ion composite.
Figure 3a is a graph showing the decomposition characteristics (n = 3) according to the ionic strength of the nano-ion composite (NPS5).
Figure 3b is a near infrared fluorescence image according to the ionic strength of the nano-ion composite (NPS5).
Figure 4a is a graph and near-infrared fluorescence image showing the change in fluorescence intensity of the nano-ion complex according to the esterase treatment.
Figure 4b is a graph and near-infrared fluorescence image showing the change in fluorescence intensity of the nano-ion complex according to the hyaluronidase type 2 treatment.
Figure 4c is a graph showing the monooxygen generation (Esterase or Hyaluronidase type 2, 10 U / ml, n = 3) of the nano-ion complex according to the enzyme treatment.
Figure 5a is a graph showing the influx of cancer of the nano ion complex (NPS5) using flow cytometry (FACS). Where (i) represents the cell influx over time of the hydrophobic photosensitizer. (Ii) shows cell influx over time of nano ion complex (NPS5).
Figure 5b is a photograph showing the intracellular distribution of the nano ion complex using a confocal microscope.
5C is a graph measuring in vitro cytotoxicity of nano ion complexes.
Figure 6a is a picture of fluorescence image observed over time after the injection of NPS5 (0.1mg / kg of Ce6) in the lower left part (tumor) and the upper right part (normal) part of the cancer-induced nude mouse, respectively. Here (iii) was taken 10 minutes after injection and (ii) was observed 10 hours after injection and (iii) 20 hours after injection.
Figure 6b is a graph showing the fluorescence recovery rate compared to normal tissue at the tumor site after injecting nano-ion complex in cancer-induced nude mouse.

The present invention covalently binds a hydrophilic cationic polymer with a photosensitizer to impart hydrophilicity to the hydrophobic photosensitizer, in order to overcome the fact that most of the photosensitizers used in photodynamic therapy are hydrophobic. It is characterized by providing a nano-ionic complex for photodynamic therapy comprising a combination of sexy quencher.

More specifically, the present invention may provide a nano-ionic complex for photodynamic therapy, including a hydrophilic cationic polymer, a photosensitive agent, and an anionic polysaccharide quencher derivative.

In the present invention, the hydrophilic cationic polymer is glycol chitosan, chitosan, poly-L-lysine (PLL), poly-beta-amino ester polymer, polyethylenimine (PEI), poly (amidoamine) (PAMAM) dendrimer And the derivatives thereof may be selected from the group consisting of, the hydrophilic cationic polymer can form the ionic composite nanoparticles by the anionic substrate polymer and electrostatic attraction.

In an embodiment of the present invention, polyethylene glycol and polyethyleneimine were used as the hydrophilic cationic polymer. The polyethylene glycol (PEG) has a structural formula represented by HO- (CH ₂ CH ₂ O) nH, and is repeatedly connected to ethylene oxide. Structural properties with ((CH ₂ CH ₂ O)-) show strong hydrophilicity and also have the property of imparting biocompatibility when combined with proteins or compounds.

In addition, polyethylene glycol is also present in the form of methoxy polyethylene glycol (mPEG) in which one end is substituted with a methoxy group (CH ₃ O-), and the structural formula is represented by CH ₃ O- (CH ₂ CH ₂ O) nH. . In particular, in the development of pharmaceuticals, in the case of a preparation having a form of polyethylene glycol-protein, methoxy polyethylene glycol derivatives are mostly used as the polyethylene glycol. This is because the terminal of the polyethylene glycol is protected with a methoxy group to maintain the stability of the structure.

In the present invention, the polyethylene glycol usable may be polyethylene glycol having a carboxyl group at the terminal having a molecular weight of 300 to 50,000. In addition, the polyethylene glycol may be methoxy polyethylene glycol in which one end is substituted with a methoxy group.

In addition, polyethyleneimine, which is a hydrophilic cationic polymer, is a cationic polymer electrolyte that has been utilized in the papermaking field for a long time. Generally, polyethyleneimine is divided into linear and branched forms according to its structure, and the synthesis method of the two is different. Polyethyleneimines generally used are branched, where the number of primary amines, secondary amines and tertiary amines is present in a ratio of 1: 2: 1. Branched polyethyleneimine is known to exist in about one to three to 3.5 of the main chain nitrogen atom, the polyethyleneimine is dissolved in water, alcohol, glycol, dimethylformamide, tetrahydrofuran, esters, etc. It is known to be insoluble in high molecular weight hydrocarbons, oleic acid and diethyl ether.

The polyethyleneimine that can be used in the present invention may use a non-toxic branched polyethyleneimine, and when the molecular weight of the polyethyleneimine is less than 100, the copolymer produced according to the present invention cannot bind well with useful bioactive substances. If the molecular weight is more than 25,000, there is a problem that is difficult to be discharged out of the body through the kidney in the body. Therefore, the polyethyleneimine may have a molecular weight of 100 to 25,000, preferably 100 to 2000.

In the nano-ion complex according to the present invention, the photosensitizer is a porphyrin compound, a chlorins compound, a bacteriochlorins compound, a phthalocyanine compound, a phthalocyanine compound, a naphthalocyanine compound And 5-aminolevulin ester-based (5-aminoevuline esters) compound may be selected from the group consisting of.

In the present invention, the anionic polysaccharide quencher derivative is specifically degraded by enzymes, especially esterases, and has specific characteristics for cancer cells, and as anionic matrix polymer, chondroitin-6-sulfate (C6S), heparan sulfate ( HS), heparan sulfate proteoglycan (HSPG), heparin, chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronic acid (HA) One selected from the group consisting of can be used.

In addition, the quencher may be selected from the group consisting of black hole quencher (BHQ), blackberry quencher (BBQ), and derivatives thereof.

As described above, the present invention provides hydrophilicity to a photosensitive agent having hydrophobicity by using a cationic polymer, that is, a hydrophilic cationic polymer, which has not been used as a nano-ionic complex for photodynamic therapy. There is a characteristic that does not dissolve to form a precipitate.

In addition, the nano-ionic complex for photodynamic therapy according to the present invention contains an anionic polymer (polysaccharide) quencher, an anionic polymer (polysaccharide) quencher is specific for cancer cells because it can bind to a tumor cell-specific receptor It can be targeted internally and specifically degraded by enzymes, especially esterases.

The anionic polymer quencher contained in the nano-ion composite of the present invention is configured in the form of a quencher coupled to an anionic substrate polymer, and the materials that can be used as the anionic substrate polymer and quencher are as described above.

Furthermore, the present invention can provide a method of preparing a nano-ionic complex for photodynamic therapy, including a hydrophilic cationic polymer, a photosensitive agent, and an anionic polysaccharide quencher derivative.

The method of preparing a nano-ionic complex for treating photodynamics may include: synthesizing a copolymer of a hydrophilic cationic polymer and a photosensitizer; Forming a complex of the anionic polymer and the quencher; And mixing a copolymer of the hydrophilic cationic polymer and the photosensitizer and the anionic polymer and the quencher.

In the preparation of the nano-ion composite, the hydrophilic cationic polymer and the photosensitizer may use materials as described above. According to one embodiment of the present invention, the activated polyethylene glycol is first activated, and then the activated polyethylene The co-conjugate of polyethylene glycol and polyethyleneimine was obtained by reacting a solution in which glycol was dissolved with a solution in which polyethyleneimine was dissolved.

The copolymer in which the polyethylene glycol and polyethyleneimine are covalently bonded is then mixed with a solution in which a hydrophobic photosensitizer is dissolved to prepare a copolymer in which polyethylene glycol-polyethyleneimine-photosensitizer is combined.

The hydrophobic photosensitizer in this bonded copolymer is endowed with hydrophilicity by the cationic polyethyleneglycol-polyethyleneimine.

When the copolymer of polyethylene glycol-polyethyleneimine-photosensitive agent is completed, a complex of anionic polymer and quencher is prepared.

In an embodiment of the present invention, chondroitin sulfate was used as the anionic polymer, BHQ3 was used as a quencher, and chondroitin sulfate dissolved in distilled water and BHQ3 dissolved in DMSO were mixed and stirred to prepare a complex of anionic polymer and quencher. .

In addition, a light sensitizer or quencher that can be bound to 1 mole of the cationic polymer or anionic polymer for the preparation of the nano-ion composite according to the present invention may be used in a ratio of 1 to 30 moles. When used in an amount outside this molar ratio, the properties of the polymer may change due to the change of the structure or the charge due to the photosensitizer or quencher used, and there is a problem that the photoactive or quenching effect may not occur. It is preferable to use within.

The copolymer of the hydrophilic cationic polymer and the photosensitizer prepared as described above and the anionic polymer and the quencher may be mixed in a weight ratio of 1:01 to 1:10 to form a nano ion composite according to the present invention.

Since the cationic polymer and the anionic polymer used to prepare the nano-ion composite of the present invention are bound by electrostatic attraction, it is preferable to mix them within the range of the weight ratio described above. There is also a possibility that no nano ion composite is formed because this does not occur.

Therefore, it is preferable to react in the range of the weight ratio described above.

In addition, the particle size of the nano-ion composite of the present invention prepared by the above method may have a size of 50 ~ 800nm, the distance between the photosensitizer and the quencher in the nano-ion composite is to maintain within 20nm.

Thus prepared nano-ion complex of the present invention can be used for cancer treatment through photodynamic therapy, the cancer is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, It may be selected from the group consisting of skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, ovarian cancer, bile duct cancer and gallbladder cancer.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited to these examples.

< Example  1>

Preparation of cancer cell specific nano ion complex

<1-1> hydrophilicity Cationic Photosensitive agent Polyethylene glycol Synthesis of Polyethylenimine Copolymer

<1-1-1> Polyethylene glycol  Activation

After the reflux condenser was installed, 10 g of methoxy polyethylene glycol (mPEG-COOH) (Sigma, 5000 Da) was dissolved in 50 ml of methylene chloride (CHCl2) using a 250 ml flask. Thereafter, 0.52 g of N-hydroxysuccinylimide and 0.74 g of dicyclocarbodiimide were added, followed by reaction at room temperature for 20 hours. Dicyclohexyl urea was removed through a filter process and then precipitated in diethylether to obtain activated polyethylene glycol (mPEG-NHS).

<1-1-2> Polyethylene glycol and  Covalent Formation of Polyethylenimine

2 g of the polyethylene glycol of the activated form obtained in Example <1-1-1> was dissolved in 200 ml of chloroform. Thereafter, 0.5 g of polyethyleneimine (Alfa Aesar, 1800da) was dissolved in 50 ml of chloroform, and then a covalent reaction of polyethylene glycol and polyethyleneimine was performed by dropping a solution of the polyethylene glycol dissolved therein drop by drop. At this time, the reaction was carried out for 24 hours, after completion of the reaction was concentrated to a total volume of 30ml using a vacuum concentrator, and then precipitated in diethyl ether to obtain a covalent conjugate of polyethylene glycol and polyethyleneimine It was.

<1-1-3> hydrophilicity Cationic Photosensitive agent Polyethylene glycol Polyethyleneimine Photosensitive  synthesis

1 g of the polyethyleneglycol-polyethylenimine (mPEG-PEI) covalently obtained in Example <1-1-2> was added to Ce6 (eq, 0.07 mmol), DCC (1.2 * Ce6 in moles) and N-hydroxystone Sinylimide (HOSu; 1.2 * Ce6 in moles) was dissolved in 20 ml of DMSO and stirred for 3 hours. Thereafter, the two solutions were mixed and reacted at room temperature for 24 hours. After filtering to remove water-insoluble dicyclohexylurea, dialysis was performed using dialysis membrane (Spectra / Por; mol. Wt. Cutoff size, 1,000) for 2 days using primary distilled water, and then the final reaction was frozen. Dried through drying. Through this process, a hydrophilic cationic photosensitive agent of Chemical Formula 2 was obtained.

[Formula 2]

<1-2> cancer cell specific Anionic  Polysaccharide Quenching agent BHQ3 join Chondroitin Sulphate  synthesis

0.1 g of chondroitin sulfate was dissolved in 20 ml of distilled water. After dissolving BHQ3 in dried DMSO, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimine hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) were each added 1.5-fold to the molar ratio of BHQ. . Then, the two solutions were each stirred at room temperature for 3 hours, then mixed and reacted for 24 hours. Removal of unreacted quencher (BHQ3) was removed by dialysis using primary distilled water for 2 days using a dialysis membrane (Spectra / Por; mol. Wt. Cutoff size, 1,000), and the final reaction product was freeze-dried after dialysis. Dried through. Through this process, a cancer cell specific anionic polysaccharide quencher such as the following Chemical Formula 3 was obtained.

(3)

<1-3> Preparation of Nano Ion Composite

The hydrophilic cationic photosensitive agent and the cancer cell specific anionic polymer quencher prepared in Examples <1-1> and <1-2> were dissolved in tertiary distilled water, respectively, and then based on the mass ratio of 1.0: It mixed at the ratio of 0.0, 1.0: 0.3, 1.0: 0.6, 1.0: 1.2, 1.0: 2.5, and 1.0: 5.0, respectively. After 2 hours, the cancer cell specific nano ion complexes formed by the mixing were obtained by filtering using a 0.8 μm syringe filter.

< Experimental Example  1> hydrophilic cation Photosensitive  Physical / chemical characterization in the water phase

We used only hydrophobic photosensitizers as controls to analyze the properties in the water phase of the hydrophilic cationic photosensitisers used in the present invention. That is, after dissolving 0.2 mg / ml of hydrophobic photosensitizer and hydrophilic cationic photosensitizer (Ce6 0.2mg / ml, 0.3mg / ml) in distilled water and checking the solubility, the hydrophobic photosensitizer had low solubility in the aqueous phase. On the other hand, the hydrophilic cationic photosensitizer was found to have almost all of the high water solubility characteristics without precipitation even at a concentration of 0.3mg / ml (see Figure 1a).

In addition, the fluorescence intensity was measured by using a fluorescence imaging device (12-bit CCD camera, Kodak) and a fluorescence fluorescence analyzer (RF-5301PC; Shimadzu, Japan) to analyze the fluorescence characteristics of the hydrophilic cationic photosensitizer in the water phase. It was confirmed that the hydrophilic cationic photosensitizer in the aqueous phase was stronger in fluorescence intensity than the hydrophobic photosensitizer (see FIG. 1B).

In addition, the hydrophilicity in the water phase was measured after irradiating the laser using a reagent (Singlet oxygen sensor green, Molecular Probe Inc. Eugene, OR, USA) to determine the singlet oxygen generation characteristics of the hydrophilic cationic photosensitizer. Cationic photosensitizer was confirmed to be higher than the hydrophobic photosensitizer monohydrogen generation (see Figure 1c).

Furthermore, the UVVis absorption of the hydrophilic cationic photosensitizer in the aqueous phase was analyzed using UVVis spectrophotometer (Shimadzu, Japan) to analyze the spectroscopic characteristics of the cationic photosensitizer in the aqueous phase and the organic solvent, respectively. ) Is about 3 times better than the hydrophobic photosensitizer (see FIG. 1D), and the absorption form of the hydrophilic cationic photosensitizer in the organic solvent (DMSO) is almost similar to that of the hydrophobic photosensitizer. (See FIG. 1E).

Through this, the inventors have found that the hydrophilic cationic photosensitizer used in the present invention has better physicochemical properties in the water phase than the hydrophobic photosensitizer.

< Experimental Example  2> Characterization and shape analysis of nano ion composite particles

In order to analyze the particle characteristics and morphology of the nano ion composite of the present invention prepared by Example 1, the optical properties and particle characteristics according to the mixing ratio of the hydrophilic cationic photosensitizer and the cancer cell specific anionic polymer quencher were analyzed. . To this end, the quenching characteristics were analyzed using a fluorescence imaging device and a fluorescence spectrometer. The mass ratio of the hydrophilic cationic photosensitizer and the cancer cell specific anionic polymer quencher was 1: 0 to 1: 5. As the mass ratio increased, it was confirmed that the quenching characteristic became stronger (see FIG. 2A).

In addition, in order to measure the particle size and zeta potential of the nano-ion composite in the mass ratio range, the analysis was performed using Zetasizer Nano ZS (Malvern Instruments Ltd., UK). In addition, zeta potential was found to have a strong negative charge of about -20 as the mass ratio of the anionic polymer quencher increases (see Figure 2b).

On the basis of these experimental results, the particle size distribution and particle shape analysis were observed with a mixture of hydrophilic cationic photosensitizer and anionic polymer quencher prepared in a mass ratio of 1: 5. As shown in FIG. 2C, a uniform particle size distribution of about 150 nm could be confirmed. As shown in FIG. 2C, the Zetasizer Nano ZS (Malvern Instruments Ltd., UK) was analyzed. The particle morphology was observed using a scanning electron microscope (FE-SEM; S-4700; Hitachi, Japan). As a result, as shown in FIG. 2D, the spherical uniform nano ion complex was confirmed.

< Experimental Example  3> Ionic strength  Stability measurement

In order to confirm the stability according to the ionic strength of the nano-ion composite prepared in the present invention, the stability was measured by using a probe-probe quenching-dequenching method. To this end, different concentrations of NaCl were first mixed with the nano-ion complex for 2 hours, and then fluorescence intensities (ex650, em670) were analyzed. When the total fluorescence intensity (I) is assumed to be the sum of the fluorescence intensity (IC) of the hydrophilic cationic photosensitizer when forming a complex with the fluorescence intensity (If) of the hydrophilic cationic photosensitive agent, the following formula 1 is used. The percentage (x) of the decomposed complex was obtained.

 Equation 1 I = If + Ic = If 0 * x / 100 + Ic0 * (100 x) / 100

(If0: fluorescence intensity of the hydrophilic cation photosensitizer when no complex is formed with any polymer; Im0: initial fluorescence intensity when an ionic complex is formed; x: percentage of decomposed complex)

As a result, it was confirmed that the deion complex of the nano-ion complex was less than 10% at the NaCl concentration of 300mM, about 40% decomplex occurs at a concentration of 600mM (see Fig. 3a).

In addition, as a result of confirming the stability of the nano-ion complex according to the ionic strength by using a fluorescence imager, it was found that until the 600mM to form a stable complex almost without decomplexing to maintain the quenching state, the strong fluorescence at the concentration of 600mM It can be seen that the decomplex form occurs (see FIG. 3B).

Through these results, the present inventors were able to confirm that the nano-ion composite of the present invention showed stability even in strong ionic strength, and in particular, the present invention maintains a stable form in vivo given that the ionic strength in vivo is 150 mM. It can be seen that the nanocomposite can act while.

< Experimental Example  4> Enzyme specific activity analysis of nano ion complex

In order to analyze the enzyme specific activation of the nano-ion complex of the present invention, 2 ml of a solution containing the enzyme esterase and hyaluronidase type 2, respectively, was added to 2 ml of phosphate buffer (pH7.4) in which the nano-ion complex was dispersed. It was. At this time, the concentration of enzyme was set to 1 or 10 Units / ml. The solution was reacted for 12 hours in a constant temperature stirred water bath (100rpm, 37 ° C) and analyzed for each sample by the fluorescence analyzer, fluorescence imager, oxygen monoxide measurement method.

As a result, it was confirmed that the esterase activity of the nano-ion complex showed strong fluorescence intensity and image depending on the enzyme concentration used (see FIG. 4A).

In addition, when the activity of the hyaluronidase type 2 of the nano-ion complex was observed, it was confirmed that the fluorescence intensity and the image were weaker than the esterase in FIG. 4A (see FIG. 4B).

As a result of confirming the enzyme's monoantigen-oxygen generating activity, it was confirmed that the mono-oxygen generation rate was increased when the laser was irradiated after being degraded by the esterase similarly to FIGS. 4a and 4b (see FIG. 4c).

As a result, it can be seen that the nano ion complex of the present invention is specifically degraded by esterase and exhibits activity when introduced into cancer cells.

< Experimental Example  5> of nano ion composite Within cancer cells  Influx and Cytotoxicity Testing

In order to investigate whether the nano-ion complex of the present invention is specifically introduced into cancer cells and irradiated with a laser, the following experiments were performed on cancer cells cultured in vitro in order to investigate whether cancer cells are toxic and thereby treat cancer. Was performed. That is, after culturing human colon cancer cells (HCT-116), the nano-ion complex according to the present invention treated with cells at a concentration of 1 μg / ml and washed with buffer solution after 30 minutes and 2 hours, and then flow cytometry ( Beckman, San Jose, CA, USA) was used to analyze the inclusion influx over time of the nano-ion composite of the present invention. In addition, the distribution of intracellular nano ion complexes was visually confirmed after fixation of 4% paraformaldehyde cells under confocal microscopy (LSM 510 Meta; Zeiss, Germany). In addition, in order to confirm cancer cell toxicity during photodynamic therapy of nano ion complexes, human colon cancer cells (HCT-116) were cultured in 96 well plates for one day and then 0.16 10 μg / ml in 100 μl SF-media. After 2 hours of incubation, the laser (HeNe laser, 633 nm, 5 mW / cm²) was irradiated to the cells for 4 minutes, and after culturing the cells for 12 hours, the cell viability was measured using the MTT test method. It was calculated by comparison and a group using only hydrophobic photosensitizer was used as a control.

 As a result of confirming the intracellular inflow through the flow cytometer, as shown in FIG. 5A, the nano ion complex (ii of FIG. 5A) of the present invention is also time-dependent as compared with the photosensitizer introduced by simple diffusion. It can be seen that similarly introduced into the cell (see Fig. 5a). Therefore, it can be seen that the nano-ion complex of the present invention is introduced into cancer cells.

In addition, as a result of confirming the intracellular distribution of the nano-ion complex using confocal microscopy, as shown in FIG. The distribution in the cytoplasmic part of the cell was confirmed (see FIG. 5B). Therefore, it can be seen from these results that the quenching effect of the nano ion complex of the present invention disappears due to degradation by enzymes in cells.

In addition, the hydrophobic photosensitizer (Ce6), the hydrophilic cationic photosensitizer (WS-Ce6), and the nano ion complex (NPS5) of the present invention, which were used as a control group without laser irradiation, showed little toxicity. , When irradiated with a laser it was confirmed that all similarly showing cytotoxicity (see Figure 5c).

Therefore, through the above experimental results, the nano-ion complex of the present invention is effectively introduced into cancer cells, decomposed and activated by enzymes in cancer cells, and the fact that it can induce toxicity to cancer cells by inducing toxicity to cancer cells by inducing laser to generate cancer, thereby treating cancer. And it was found.

< Experimental Example  6> Confirmation of Cancer Cell Specificity of Nano-Ion Complex in Cancer-induced Nude Mice

6-week-old male nude mice transplanted with human colon cancer cells (HTC-116) to confirm the in vivo cancer cell specific activity of the nano-ion complex of the present invention (Balb / C-nu mice, Institute of Medical Science, Tokyo). When human colon cancer cells (HCT-116) were implanted at 1.0 × 10 into the dorsal side of the mouse and the tumor size reached 150200 mm³, the nano ion complex of the present invention was administered to tumors and normal tissues in an amount of 0.1 mg / kg, respectively. It was. In order to observe the cancer cell specificity of the nano-ion complex in real time, the fluorescence pattern was observed using the fluorescence imaging apparatus.

As a result, the nano ion complex of the present invention showed strong activity in cancer cells compared to normal tissues in proportion to time (see FIG. 6A), and when the fluorescence image was quantified, the activity of the nano ion complexes was stronger in cancer cells than normal tissues. The activity was confirmed, and after 20 hours it was confirmed that the fluorescence intensity difference of about three times (see Fig. 6b).

Therefore, it was confirmed through the above experiments that the nano ion complex of the present invention is particularly active in cancer cells in vivo, and ultimately, the nano ion complex of the present invention presents great potential as a new cancer cell target therapeutic agent.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (14)

A nanoionic composite for photodynamic therapy comprising a hydrophilic cationic polymer photosensitive agent and an anionic polysaccharide quencher derivative. The method of claim 1,
The hydrophilic cationic polymer photosensitizer derivative is a nano-ionic composite for photodynamic therapy, characterized in that the copolymer is a combination of a hydrophilic cationic polymer and a photosensitizer. The method of claim 2,
The hydrophilic cationic polymer is glycol chitosan (GC), chitosan, poly-L-lysine (PLL), poly-beta-amino ester polymer, polyethyleneimine (PEI), polyethylene glycol, poly (amidoamine) (PAMAM) At least one selected from the group consisting of dendrimers and derivatives thereof. The method of claim 1,
The photosensitizer is a porphyrin compound, a chlorins compound, a bacteriochlorins compound, a phthalocyanine compound, a naphthalocyanine compound, and a 5-aminolevulin ester compound ( 5-aminoevuline esters) compound is a photodynamic therapeutic nano-ion complex, characterized in that at least one member selected from the group consisting of. The method of claim 1,
The hydrophilic cationic polymer photosensitizer derivative is composed of a copolymer of polyethylene glycol, polyethyleneimine and a photosensitizer, and the photosensitizer has hydrophilicity by polyethylene glycol and polyethyleneimine. . The method of claim 5,
The polyethylene glycol is a photodynamic therapy nano-ion composite, characterized in that the molecular weight is 300 ~ 50,000 (Da). The method of claim 5,
The polyethyleneimine is branched and has a molecular weight of 100 ~ 250,000 (Da) photodynamic therapeutic nano-ion composite, characterized in that. The method of claim 1,
The anionic polysaccharide quencher derivative is a nano-ionic composite for photodynamic therapy, characterized in that the combination of the anionic matrix polymer and the quencher. 9. The method of claim 8,
The anionic matrix polymer is specific for esterase enzymes and cancer cells, and the anionic matrix polymer is chondroitin-6-sulfate (C6S), heparan sulfate (HS), heparan sulfate proteoglycan (HSPG), heparin, chondroitin-4 Photodynamic therapy comprising at least one member selected from the group consisting of sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronic acid (HA) Nano ion complex for 9. The method of claim 8,
The quencher is a black hole quencher (BHQ) or a blackberry quencher (BBQ), characterized in that the nano-ionic composite for photodynamic therapy. The method of claim 2,
The hydrophilic cationic polymer and the photosensitizer is a photodynamic therapy nano-ion composite, characterized in that combined with the molar ratio of the photosensitive agent 1 to 30 moles per 1 mole of the hydrophilic cationic polymer. 9. The method of claim 8,
The anionic substrate polymer and the quencher is a photodynamic therapy nano ion composite, characterized in that the quencher is combined in a molar ratio of 1 to 30 moles with respect to 1 mole of the anionic substrate polymer. The method of claim 1,
The nano ion composite is a nano ion composite for photodynamic therapy, characterized in that a mixture ratio of a hydrophilic cationic polymer photosensitive agent to an anionic polysaccharide quencher derivative is mixed in a weight ratio of 1: 0.1 to 1:10. The method of claim 1,
The nano-ion complex is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, ovarian cancer, Photodynamic therapy nano-ion complex, characterized in that the prevention or treatment of at least one cancer selected from the group consisting of bile duct cancer and gallbladder cancer.

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