KR101116570B1 - Photosensitizer - metal nanoparticle complex and composition containing the complex for photodynamic therapy or diagnosis - Google Patents

Abstract

A photosensitive agent-metal nanoparticle complex and a composition for photodynamic therapy or diagnosis containing the same are provided. The composite includes a photosensitive agent, metal nanoparticles, and a backbone connecting the photosensitive agent and the metal nanoparticles. The backbone has a polypeptide substrate that can be specifically degraded by proteases. The complex may inhibit the generation of fluorescence and reactive oxygen species in normal tissues and activate the generation of fluorescence and reactive oxygen species in tumor tissues to effectively destroy tumor tissues. In addition, selective fluorescence in tumor tissue can further improve the diagnostic accuracy of the tumor using the complex.

Description

Photosensitizer-metal nanoparticle complex and composition containing the complex for photodynamic therapy or diagnosis}

The present invention relates to a photosensitive agent, and more particularly, to a photosensitive agent-metal nanoparticle complex and a composition for photodynamic therapy or diagnosis containing the same.

Photodynamic therapy using photosensitizers is gaining attention as a treatment that can solve the side effects of surgery, radiation therapy, drug therapy, and aftereffects of cancer, which are conventional cancer treatments.

The photosensitizer is excited when irradiated with light of a specific wavelength, and the photosensitizer in the excited state reacts with the surrounding substrate or oxygen to produce reactive oxygen species, and the generated reactive oxygen species is generated in the surrounding tumor. Apoptosis or necrosis of cells.

However, in the case of the photosensitive agent currently used for cancer treatment, when the patient is exposed to light after administration of the photosensitive agent, the photosensing agent that is accumulated and remains in the skin or eye is excited, which causes the normality of the skin or the eye. Skin photosensitive side effects that kill cells are problematic.

It is an object of the present invention to provide a photosensitive agent-metal nanoparticle complex and a composition for photodynamic therapy or diagnosis containing the same, in which a photodynamic reaction may occur more selectively in a target lesion to reduce reaction side effects.

One aspect of the present invention to achieve the above object provides a photosensitizer-metal nanoparticle composite. The composite includes a photosensitive agent, metal nanoparticles, and a backbone connecting the photosensitive agent and the metal nanoparticles. The backbone has a polypeptide substrate that can be specifically degraded by proteases.

In order to achieve the above object, another aspect of the present invention provides a composition for photodynamic therapy or diagnosis. The composition comprises a photosensitive agent-metal nanoparticle complex and a pharmaceutically acceptable carrier.

According to the present invention, in the normal tissue, the photosensitizer-metal nanoparticle complex is present in a state where the photosensitizer and the metal nanoparticles are connected by the backbone, thereby suppressing the generation of fluorescence and reactive oxygen species. However, in the tumor where the protease is overexpressed, since the polypeptide in the backbone is specifically degraded, the photosensitizer and the metal nanoparticle may exist in a separated state, thereby activating fluorescence and reactive oxygen species generation. As a result, photosensitive reactions in normal tissues can be suppressed while tumor tissues can be effectively destroyed. In addition, selective fluorescence in tumor tissue can improve the diagnostic accuracy of the tumor using the complex.

The effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings in order to describe the present invention in more detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms.

A photosensor  - Metal nanoparticles  Complex

1 is a schematic view showing a photosensitive agent-metal nanoparticle composite according to an embodiment of the present invention.

Referring to FIG. 1, the photosensitive agent-metal nanoparticle composite 10 according to an embodiment of the present invention includes a metal nanoparticle 11, a photosensitive agent 15, and the metal nanoparticle 11 and the And a backbone 13 connecting the photosensitizers 15.

The backbone 13 includes a polypeptide substrate 13a that can be specifically degraded by protease that is overexpressed in diseased tissue such as tumors, for example tumors. As an example, the tumor may be composed of proliferative cells.

Examples of such proteases include cathepsins, matrix metalloproteinases (MMPs), membrane-type MMPs, collagenases, gelatinases, stromelysins, urokinase- Urokinase-type plasminogen activator (uPA), caspases, viral proteases, HIV proteases, HSV protease HSV proteases, gelatinases, eurokinases, secretases, endosomal hydrolase, prostate specific antigens, plasminogen activators , CMV (Cytomegalovirus) protease, and thrombin. Specifically, the kadipsin may be Kadipsin A, Kadipsin B, Kadipsin D, Kadipsin H, Kadipsin K, Kadipsin L, or Kadipsin S, wherein the MMP MMP1, MMP2, MMP3, MMP7, MMP8 , MMP9, MMP10, MMP11, MMP12, or MMP13.

The polypeptide substrate 13a comprises RPLALWRS (SEQ ID NO: 1), GGLGQRGRSANAILE (SEQ ID NO: 2), GVSQNYPIVG (SEQ ID NO: 3), LVLASSSFGY (SEQ ID NO: 4), PLGMWSR (SEQ ID NO: 5), PGNWT (SEQ ID NO: 6), PAGLLGC (SEQ ID NO: 7), LGGSGRSANAILE (SEQ ID NO: 8) , RR, RRG, GPICFFRLG (SEQ ID NO: 9), HSSKLQG (SEQ ID NO: 10), PIC (Et) FF (SEQ ID NO: 11), HSSKLQ (SEQ ID NO: 12), P Amino acid sequences, including but not limited to (L / Q) G (I / L) AG (SEQ ID NO: 13), GVVQASCRLA (SEQ ID NO: 14), or KK. In one embodiment, examples of such polypeptide substrates 13a include PLGVRG (SEQ ID NO: 15, -Pro-Leu-Gly-Val-Arg-Gly-), or cathepsin S (which can be specifically degraded by MMP2). It may be LR (-Leu-Arg-) that can be specifically degraded by cathepsin S).

The backbone 13 is chemically bonded to both of the metal nanoparticles 11 and the photosensitive agent 15, and the polypeptide substrate 13a itself is the metal nanoparticles 11 and the light. And chemically bind to the sensory agent 15. Alternatively, the backbone 13 may chemically connect the first substrate 13b, the polypeptide substrate 13a, and the photosensitive agent 15, which chemically connect the polypeptide substrate 13a and the metal nanoparticle 11 to each other. It may further include at least one of the second connector (13c) for connecting. For example, the metal nanoparticles 11 and / or the photosensitive agent 15 may be coupled to side chains of the backbone 13.

Specifically, the backbone 13 may be chemisorbed to the metal nanoparticles 11 and may be covalently bonded to the photosensitive agent 15. As an example, the first connector 13b may be connected to the polypeptide substrate 13a by a peptide bond, and may be connected to the surface of the metal nanoparticle 11 by a thiol-metal bond. Examples of the first linker 13b for this purpose include cysteine having a thiol group in the side chain, or a polypeptide including cysteine, or an amino group or a carboxyl group in the main chain (or side chain) and a thiol group in the side chain (or main chain). It may be an alkylene. The second connector 13c may be connected to the polypeptide substrate 13a by a peptide bond, and the photosensitive agent 15 may be connected to an amide bond. Examples of the second linker 13c for this may be lysine containing an amino group in the side chain, or a polypeptide comprising lysine, or alkyl or alkylene having amino groups in the side chain and / or main chain. In addition, the photosensitive agent 15 may contain a carboxyl group as a substituent so that the carboxyl group may react with the amine group of the second linker 13c to form an amide bond. However, the present invention is not limited thereto, and when the one terminal amino acid of the polypeptide substrate 13a is cysteine, the first connector 13b may be omitted, and when the other terminal amino acid of the polypeptide substrate 13a is lysine, The second connector 13c may be omitted.

The backbone 13 may suitably include the constituent amino acid number or the carbon number of the alkylene group so that the photosensitive agent 15 is located within a distance of about 20 nm from the surface of the metal nanoparticle 11.

The photosensitive agent 15 may be a porphyrin-based compound or a nonporphyrin compound in the form of a free base or a metal complex. The porphyrins compound is, for example, porphyrin derivatives (porphyrin derivatives); At least one pyrrole constituting porphyrin is reduced porphyrins such as chlorin, bacteriochlorin, in which pyrrole is reduced to pyrroline; Or porphyrin analogues such as phthalocyanine, naphthalocyanine. The non-porphyrin compound may be hypericin, rhodamine, rose bengal, psoralen, phenothiazinium-based dye or merocyanine. .

The photosensitive agent 15 is not toxic when in the ground state, but is absorbed in a single state when absorbing light of a specific wavelength. Some of the photosensitizers in the singlet state return to the ground state while emitting energy in the form of fluorescence, and most of them are transferred to the triplet state through intersystem crossing. The photosensitive agent in one or three states reacts with the surrounding substrate or oxygen to react reactive oxygen species, for example, singlet oxygen, oxygen radicals, super oxides or peroxides. Create The resulting reactive oxygen species can kill or necrosis surrounding tumor cells.

The photosensitive agent 15 may be a material that is excited by light having a wavelength of 450 to 950 nm, preferably 600 to 900 nm, in consideration of the possibility of photopermeation in the tissue and the generation efficiency of reactive oxygen species.

The metal nanoparticles 11 may be nanoparticles having a length of several tens to several tens of nanometers in at least one direction, and may include metals capable of resonance energy transfer with the photosensitive agent 15. The metal contained in the metal nanoparticles 11 may be, for example, gold, silver, copper, platinum, palladium, nickel, iron, or a mixed metal of two or more thereof. Specifically, when the photosensitive agent-metal nanoparticle composite 10, in which the metal nanoparticle 11 and the photosensitive agent 15 are connected by the backbone 13, is exposed to light of a specific wavelength, a single state Energy transfer may occur from the photosensitive agent 15 to the surface of the metal nanoparticles 11, and thus the photosensitive agent 15 may not fluoresce and generate reactive oxygen species. . As a result, the photosensitive agent-metal nanoparticle composite 10 in which the metal nanoparticles 11 and the photosensitive agent 15 are connected by the backbone 13 may not exhibit cytotoxicity.

In one embodiment, the metal contained in the metal nanoparticles 11 has a molar extinction coefficient of 10 ⁹ or more and can absorb light more than ten thousand times more efficiently than the photosensitive agent 15, and the introduction of a substance into the surface It can be easy gold. The metal nanoparticles 11 may have a sphere shape, a rod shape, a pyramid shape, a star shape, and a core-shell shape. The wavelength of the metal nanoparticles 11 may vary depending on its constituent material, shape, or size. Therefore, the constituent material, shape, or size of the metal nanoparticles 11 may be adjusted so that the fluorescence spectrum of the photosensitive agent 15 and the absorption spectrum of the metal nanoparticles 11 may overlap.

Specifically, the hydrophilic polymer 19 may be further connected to an end of the backbone 13 adjacent to the photosensitive agent 15. The hydrophilic polymer may be biocompatible polyalkylene glycol (poly (alkylene glycol)), such as low immunogenicity. The polyalkylene glycol is polyethylene glycol (PEG); Methoxy polyethylene glycol (MPEG); Methoxypolypropylene glycol; Copolymers of polyethylene glycol and methoxy polypropylene glycol; Dextran; Hyaluronic acid; Polylactic-polyglycolic acid copolymers; Polyethylene glycol-diacid; Polyethylene glycol monoamines; Methoxypolyethylene glycol monoamine; Methoxy polyethylene glycol hydrazide; Methoxy polyethylene glycol imidazole; Or a copolymer of two or more of polyethylene glycol, methoxy polypropylene glycol, polyethylene glycol-diacid, polyethylene glycol monoamine, methoxy polyethylene glycol monoamine, methoxy polyethylene glycol hydrazide, and methoxy polyethylene glycol imidazole. . In addition, the hydrophilic polymer may be a copolymer of polyalkylene glycol and another polymer such as polyethylene glycol and polypeptide, polyethylene glycol and polysaccharide, polyethylene glycol and polyamidoamine, polyethylene glycol and polyethyleneamine or polyethylene glycol Copolymers with polynucleotides.

An example of the photosensitive agent-metal nanoparticle composite 10 may be represented by the following Chemical Formula 1.

Figure 2a and Figure 2b is a schematic diagram showing the reaction by the light of the photosensitizer-metal nanoparticle complex according to an embodiment of the present invention accumulated in the affected tissue overexpressed normal tissue and protease, respectively.

Referring to FIG. 2A, the photosensitive agent-metal nanoparticle composite 10 accumulated in the normal tissue maintains the metal nanoparticle 11 and the photosensitive agent 15 connected by the backbone 13. Therefore, when the composite 10 is exposed to light having a wavelength capable of exciting the photosensitive agent 15, energy transfer from the photosensitive agent 15 in one state to the surface of the metal nanoparticle 11 is performed. That is, resonance energy transfer may occur. In this case, the metal nanoparticles 11 may act as a quencher to suppress fluorescence generation and reactive oxygen species generation of the photosensitive agent 15. As a result, the complex 10 may not exhibit cytotoxicity.

On the other hand, referring to Figure 2b, the photosensitizer-metal nanoparticle complex (10) accumulated in the affected part meets the protease (P) overexpressed in the affected part, the protease (P) is the complex (10) The polypeptide substrate 13a in the backbone 13 is specifically decomposed to separate the metal nanoparticle 11 and the photosensitive agent 15. As a result, the distance between the surface of the metal nanoparticle 11 and the photosensitive agent 15 is greater than 20 nm, for example, a distance where quenching is not possible. Thereafter, when the composite 10 is exposed to light having a wavelength capable of exciting the photosensitive agent 15, the photosensitive agent 15 is excited in a single state. Some of the photosensitizers in the singlet state return to the ground state while emitting energy in the form of fluorescence, and most of them are transferred to the triplet state through intersystem crossing. The photosensitive agent in one or three states reacts with the surrounding substrate or oxygen to react with reactive oxygen species such as singlet oxygen, oxygen radicals, super oxides or peroxides. Create The resulting reactive oxygen species can kill or necrosis surrounding tumor cells.

On the other hand, when the photosensitive agent-metal nanoparticle complex 10 is administered in vivo with a tumor, the nano-sized complex 10 is easy to the tumor due to the loose neovascular tissue present around the tumor. It can infiltrate and accumulate in the tumor and stay longer due to lack of drainage through the lymphatic vessels. However, the complex 10 may also accumulate nonspecifically in normal tissues such as eyes or skin.

However, since the protease is overexpressed in the tumor than in the normal tissue, as described with reference to FIG. 2A, in the normal tissue, the complex 10 includes the photosensitive agent 15 and the metal nanoparticle 11 as the backbone ( Fluorescence and reactive oxygen species generation can be suppressed even when exposed to light having a wavelength capable of exciting the photosensitive agent 15 by being present in a connected state. However, as described with reference to FIG. 2B, in the tumor, the photosensitive agent 15 and the metal nanoparticles 11 may exist in a separated state, and thus the wavelength at which the photosensitive agent 15 may be excited Fluorescence and reactive oxygen species generation can be activated when exposed to light having. As a result, photosensitive reactions in normal tissues can be suppressed while tumor tissues can be effectively destroyed. In addition, selective fluorescence in tumor tissue may further improve the accuracy of tumor diagnosis using the complex.

A photosensor  - Metal nanoparticles  Pharmaceutical Compositions Containing Complexes

Yet another embodiment of the present invention provides a pharmaceutical composition containing the photosensitive agent-metal nanoparticle complex described with reference to FIG. 1.

The pharmaceutical composition according to the present embodiment includes a pharmaceutically effective amount of a photosensitive agent-metal nanoparticle complex and a pharmaceutically acceptable carrier. The carrier may be a diluent. The composition may further comprise adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants.

Such pharmaceutical compositions may be formulated to match the intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, such as intravenous, intradermal, subcutaneous, intranasal, transdermal (topical), mucosal, and rectal administration.

Suitable carriers that can be used for parenteral administration are well known to those skilled in the art. Aqueous vehicles, including but not limited to, for example, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactate containing Ringer's injection; Water-miscible carriers, including but not limited to ethyl alcohol, polyethylene glycol, and polypropylene glycol; And non-aqueous carriers, including but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Such pharmaceutical compositions can be used for tumor treatment or tumor diagnosis. When used for tumor treatment or tumor diagnosis, the effective amount can be determined by the physician on a case-by-case basis. At this time, the age, sex, weight, or severity of the condition to be treated or diagnosed may be considered. As an example, the photosensitive agent-metal nanoparticle complex may be administered at 0.001 μg to 10 mg / kg based on the equivalent weight of the photosensitive agent and the patient's weight. In certain instances the complex may be administered at 0.1 mg-1 mg / kg.

Experimental examples ( Examples )

Next, preferred examples are provided to help understanding of the present invention. However, the following examples are only for illustrating the present invention, and do not limit the content of the present invention.

Manufacturing example  One: Of metal nanoparticles  Produce

Metal nanoparticles were prepared using a seed mediated method. Specifically, 7.5 ml of CTAB aqueous solution dissolved in 100 mM mol of CTAB (cetyltrimethylammonium bromide) in tertiary distilled water and 250 μl of 10 mM HAuCl ₄ aqueous solution (HAuCl ₄ .3H ₂ O) were mixed therein, followed by 10 mM NaBH _4. 600 µl of aqueous solution was added thereto. After stirring for 2 minutes, the mixture was left at 25 ° C. for 2 hours to prepare a seed solution.

Meanwhile, 1.7 ml of 10 mM HAuCl ₄ aqueous solution (HAuCl ₄ .3H ₂ O) was added to 40 ml of 100 mM CTAB aqueous solution, and then 250 µl of 10 mM AgNO ₃ aqueous solution and 270 µl of 100 mM ascorbic acid were added thereto. Put them sequentially. 420 μl of the seed solution was added thereto and reacted for 12 hours, followed by centrifugation at 15000 g, 25 ° C., and 15 minutes to obtain gold nanorods.

3 is a transmission electron microscope photograph of the gold nanorods prepared according to Preparation Example 1. FIG.

Referring to FIG. 3, it can be seen that gold nanorods having an average length of 34 nm and a width of 9 nm are formed.

Manufacturing example  2: A photosensor -Polypeptide conjugates ( MMP2 Probe ) Produce

Rink amide MBHA resin as a solid support phase, Fmoc-mini-PEG ^™ (PEPNET, 9-Fluorenylmethoxycarbonyl-8-Amino-3,6-Dioxaoctanoic Acid), and amino groups protected with Fmoc (Fluorenylmethoxycarbonyl) groups The amino acids were used, and the compound represented by (2) was obtained through conventional solid-phase synthesis. Specifically, (a) Fmoc deprotection using 20% piperidine DMF (dimethylformamide) solution, (b) Fmoc-mini-PEG ^TM addition, (c) DIPEA (N, N-Diisopropylethylamine), HOBT (Hydroxybenzotriazole) Carboxyl activation and coupling were repeated four times using DMF solution containing HBTU (O- (Benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate), and (d) Fmoc deprotection using 20% piperidine DMF solution, (e) Fmoc-amino acid addition, (f) carboxylic acid activation and coupling with DMF solution comprising DIPEA, HOBT, and HBTU (2) To give a compound. The Fmoc-amino acid, in turn, has a residue of Fmoc-Lys (Alloc), a lysine protected with an Alloc (allyloxycarbonyl) group, Fmoc-Gly, and a residue of Fmoc-Arg (an arginine protected with a Pbf (pentamethyldihydro-benzofuran-5-sulfonyl) group. Pbf), Fmoc-Val, Fmoc-Gly, Fmoc-Leu, Fmoc-Pro, Fmoc-Gly, Fmoc-Gly, residues were Fmoc-Cys (Trt) protected with Trt (trityl) group.

To the compound (2), a DMF solution containing (Boc) ₂ O and DIPEA was added to replace the terminal amino group of the compound (2) with a Boc (t-butoxycarbonyl) group to obtain the compound of (3). Then, MC (Methylene Chloride) solution containing Pd (PPh ₃ ) ₄ (Tetrakis (triphenylphosphine) palladium (0)) and 1,3-dimethylbarbituric acid was added to compound (3). Alloc which protects the residue of the lysine of the compound (3) was deprotected, and the compound of (4) was obtained. Thereafter, DMF solution containing pyropheophorbide a (ppa), DIPEA, HOBT, and HBTU was added to compound (4) to couple compound (4) and ppa to form compound (5). Got it. Subsequently, compound (5) of trifluoroacetic acid (TFA) / TIS (triisopropylsilane) / EDT (1,2-ethanedithiol) / thioanisole / water (volume ratio = 90: 2.5: 2.5: 2.5: 2.5) The mixture was added to obtain a mixed solution obtained by cutting the compound of (6) from the link amide MBHA resin.

 The resultant mixed solution was treated with an excess of diethyl ether to form a precipitate. The precipitate was centrifuged to completely precipitate the precipitate, and the procedure of removing excess TFA, TIS, EDT, thionazole, etc. was repeated twice. Solidified precipitate was obtained. The obtained precipitate was purified using high performance liquid chromatography (HPLC). The purified material was lyophilized to obtain the photosensitive agent-polypeptide conjugate, Compound 6, MMP2 probe (MMP2P).

Figure 4a is the HPLC results of the MMP2 probe prepared in Preparation Example 2, Figure 4b is a mass spectrometry of the MMP2 probe prepared in Preparation Example 2.

4A and 4B, the MMP2 probe prepared through Preparation Example 2 exhibited a purity of about 98%, and the molecular weight of the MMP2 probe was confirmed to be 2039 g / mol, thereby predicting that Compound (6) was produced. .

Manufacturing example  3: A photosensor - Metal nanoparticles  Composite ( MMP2P - GNR ) Produce

The gold nanorod prepared in Preparation Example 1 was added to 1 ml of distilled water to prepare a gold nanorod dispersion having a concentration of 100 nM, and 200 μl and 2 mM K in which the MMP2 probe prepared in Preparation Example 2 was dissolved at a concentration of 1 mM. 100 μl of a ₂ CO ₃ aqueous solution was added, followed by reaction at room temperature for 5 days. Thereafter, dialysis was carried out using distilled water using a filtration membrane having a molecular weight of 50,000 (MWCO) to remove MMP2 probe that did not participate in the reaction with the gold nanorods, thereby providing a photosensitizer-polypeptide conjugate connected to the gold nanorods. A metal nanoparticle composite solution, that is, MMP2P-GNR was obtained.

5A is a UV / Vis absorption spectrum of gold nanorods prepared according to Preparation Example 1. FIG.

Referring to FIG. 5A, it was found that the maximum absorption wavelength was about 750 nm and a strong absorption spectrum in the 650 to 780 nm region, which is the fluorescence wavelength of pyrophorovide a.

5B is a UV / Vis absorption spectrum of the photosensitive agent-metal nanoparticle complex (MMP2P-GNR) according to Preparation Example 3. FIG.

5A and 5B, the UV / Vis absorption spectrum of the photosensitive agent-metal nanoparticle composite according to Preparation Example 3 is compared with the UV / Vis absorption spectrum of the gold nanorod according to Preparation Example 1 (FIG. 5A). FIG. 5B is 387 nm (F ₁ , which is an absorption peak by the photosensitive agent). Soret band) and additional absorption peaks at 670 nm (F ₂ , Q band). It was confirmed from the UV / Vis absorption spectral analysis of FIG. 5B that the photosensitive agent concentration in the aqueous solution of the photosensitive agent-metal nanoparticle composite obtained after dialysis was 75uM equivalent.

Analysis example  One: A photosensor - Metal nanoparticles  Composite ( MMP2P - GNR Fluorescence activation assay

HT1080 fibrosarcoma, a cancer cell line that overexpresses MMP2, and BT20 mammary adenocarcinoma, a cell line that underexpresses MMP2 (see Bremer C et al., Nature Medicine 7 (6) 743-748, 2001). LebTek II Chambered coverglass) was seeded 90,000 per well, and then incubated for 24 hours. Thereafter, the photosensitive agent-metal nanoparticle complex solution according to Preparation Example 2 was diluted using the cell culture solution to contain 5 μM equivalent of the photosensitive agent, and then added to 600 μl per well, and then placed in an incubator at 37 ° C. for 15 hours. Thereafter, the cell culture medium containing the complex was removed, the cells were washed three times with fresh cell culture, and fluorescence was observed under a confocal microscope (Ex. 405 nm, Em. 646-753 nm).

6a and 6b are photographs taken of fluorescence of the HT1080 cell line and the BT20 cell line treated with the photosensitive agent-metal nanoparticle complex, respectively.

6A and 6B, it can be seen that much stronger fluorescence occurs in HT1080, a cancer cell line that overexpresses MMP2, compared to BT20, a cell line that expresses MMP2 low. From these results, it can be seen that the photosensitive agent is separated from the metal nanoparticles as MMP2 decomposes the polypeptide of the photosensitive agent-metal nanoparticle complex, and the separated photosensitive agent exhibits strong fluorescence. Therefore, when the photosensitive agent-metal nanoparticle complex is administered to a patient and the fluorescence according to light irradiation is measured, it can be seen that MMP2 can be more accurately diagnosed with the location of tissues overexpressed.

Analysis example  2: Photodynamics  Cell survival analysis after treatment

Analysis Example 2-1 Analysis of Cell Line HT1080>

HT1080 (MMP2 +) was seeded in 96 well plates at a concentration of 9,000 cells / well. The following day, the photosensitive agent-polypeptide conjugate used in Preparation Example 2 and the photosensitive agent-metal nanoparticle complex prepared in Preparation Example 2 were diluted using a cell culture solution, and the final concentration of the MMP2 probe (MMP2P) solution having a final concentration of 5uM equivalent of the photosensitive agent. And a photosensitizer-metal nanoparticle complex (MMP2P-GNR) solution were prepared, 200 μl per well was added to some wells, and then placed at 37 ° C. for 15 hours. On the other hand, the control group was added the same volume of a new cell culture containing no photosensitizer. Subsequently, after washing twice with the cell culture solution, the PDT treatment group was subjected to photodynamic therapy using a 670 nm laser under conditions of an irradiation dose density of 40 mW / cm ² and a dose of 10 J / cm ² . After 24 hours, MTT assay was performed to determine cell viability.

Analysis Example 2-2 Analysis of Cell Line BT20

BT20 (MMP2-) was seeded in 96 well plates at a concentration of 9,000 cells / well. The following day, the photosensitive agent-polypeptide conjugate used in Preparation Example 2 and the photosensitive agent-metal nanoparticle complex prepared in Preparation Example 2 were diluted using a cell culture solution, and the final concentration of the MMP2 probe (MMP2P) solution having a final concentration of 5uM equivalent of the photosensitive agent. And a photosensitizer-metal nanoparticle complex (MMP2P-GNR) solution were prepared, 200 μl per well was added to some wells, and then placed at 37 ° C. for 15 hours. On the other hand, the control group was added the same volume of a new cell culture containing no photosensitizer. Subsequently, after washing twice with the cell culture solution, the PDT treatment group was subjected to photodynamic therapy using a 670 nm laser under conditions of an irradiation dose density of 40 mW / cm ² and a dose of 10 J / cm ² . After 24 hours, MTT assay was performed to determine cell viability.

The conditions of the groups analyzed in Analysis Examples 2-1 and 2-2 are shown.

Cell line Condition name Condition Cell line treatment conditions Photodynamic therapy HT1080 Media control Cell culture × (MMP2P-GNR)-PDT Photosensitive Agent-Metal Nanoparticle Complex × (MMP2P)-PDT Photosensitizer-polypeptide conjugates × (MMP2P-GNR) + PDT Photosensitive Agent-Metal Nanoparticle Complex ○ (MMP2P) + PDT Photosensitizer-polypeptide conjugates ○ BT20 Media control Cell culture × (MMP2P-GNR)-PDT Photosensitive Agent-Metal Nanoparticle Complex × (MMP2P)-PDT Photosensitizer-polypeptide conjugates × (MMP2P-GNR) + PDT Photosensitive Agent-Metal Nanoparticle Complex ○ (MMP2P) + PDT Photosensitizer-polypeptide conjugates ○

7A and 7B are graphs showing cell viability according to conditions according to Assays 1-1 and 1-2, respectively.

Referring to FIGS. 7A and 7B, when photodynamic therapy was performed after treatment with the photosensitizer-polypeptide conjugate (MMP2P + PDT), it was found that high phototoxicity was shown by showing low cell viability in both the HT1080 cell line and the BT20 cell line. have. However, photodynamic therapy after photosensitive agent-metal nanoparticle complexes in which the photosensitive agent-polypeptide conjugate was bound to the metal nanoparticles (MMP2P-GNR + PDT) resulted in 16% cell survival. BT20 cell lines showed 63% cell viability. From this, it can be seen that the phototoxic agent-induced phototoxicity of the metal nanoparticle complex is selectively activated by MMP2. In other words, it can be seen that the photosensitizer-metal nanoparticle complex causes phototoxicity only in tissues overexpressing MMP2, and phototoxicity is not induced in tissues with low MMP2 expression, for example, normal tissues. In conclusion, it can be seen that when the photosensitive agent-metal nanoparticle complex is administered to a patient and subjected to photodynamic therapy, only tissues overexpressing MMP2 can be selectively treated.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may implement the present invention in other specific forms without changing the technical spirit or essential features thereof. I can understand that. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention is shown by the following claims rather than the above description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

1 is a schematic view showing a photosensitive agent-metal nanoparticle composite according to an embodiment of the present invention.

Figure 2a and Figure 2b is a schematic diagram showing the reaction by the light of the photosensitizer-metal nanoparticle complex according to an embodiment of the present invention accumulated in the affected tissue overexpressed normal tissue and protease, respectively.

3 is a transmission electron microscope photograph of the gold nanorods prepared according to Preparation Example 1. FIG.

Figure 4a is the HPLC results of the MMP2 probe prepared in Preparation Example 2, Figure 4b is a mass spectrometry of the MMP2 probe prepared in Preparation Example 2.

5A is a UV / Vis absorption spectrum of a gold nanorod according to Preparation Example 1, and FIG. 5B is a UV / Vis absorption spectrum of a photosensitive agent-metal nanoparticle composite according to Preparation Example 2. FIG.

6a and 6b are photographs taken of fluorescence of the HT1080 cell line and the BT20 cell line treated with the photosensitive agent-metal nanoparticle complex, respectively.

7A and 7B are graphs showing cell viability according to conditions according to Assays 2-1 and 2-2, respectively.

<110> National Cancer Center <120> Photosensitizer-metal nanoparticle complex and composition          containing the complex for photodynamic therapy or diagnosis <130> 9017 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 1 Arg Pro Leu Ala Leu Trp Arg Ser   1 5 <210> 2 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 2 Gly Gly Leu Gly Gln Arg Gly Arg Ser Ala Asn Ala Ile Leu Glu   1 5 10 15 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 3 Gly Val Ser Gln Asn Tyr Pro Ile Val Gly   1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 4 Leu Val Leu Ala Ser Ser Ser Phe Gly Tyr   1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 5 Pro Leu Gly Met Trp Ser Arg   1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 6 Pro Gly Asn Trp Thr   1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 7 Pro Ala Gly Leu Leu Gly Cys   1 5 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 8 Leu Gly Gly Ser Gly Arg Ser Ala Asn Ala Ile Leu Glu   1 5 10 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 9 Gly Pro Ile Cys Phe Phe Arg Leu Gly   1 5 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 10 His Ser Ser Lys Leu Gln Gly   1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <220> <221> VARIANT <222> (3) <223> Xaa = Cys (Et) which is ethylcysteine <400> 11 Pro Ile Xaa Phe Phe   1 5 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 12 His Ser Ser Lys Leu Gln   1 5 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <220> <221> VARIANT <222> (2) <223> Xaa = Leu or Gln <220> <221> VARIANT <222> (4) <223> Xaa = Ile or Leu <400> 13 Pro Xaa Gly Xaa Ala Gly   1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 14 Gly Val Val Gln Ala Ser Cys Arg Leu Ala   1 5 10 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> polypeptide substrate <400> 15 Pro Leu Gly Val Arg Gly   1 5  

Claims (14)

Photosensitizers; Metal nanoparticles capable of resonant energy transfer with the photosensitive agent and having a size of 10 nm or more; And A length of the polypeptide substrate which is connected to the photosensitive agent and the metal nanoparticles so that the photosensitive agent is located within a distance of 20 nm from the surface of the metal nanoparticle, and is specifically decomposed by a protease Photosensitive agent-metal nanoparticle composite comprising a backbone having a. The method of claim 1, The polypeptide substrate is RPLALWRS (SEQ ID NO: 1), GGLGQRGRSANAILE (SEQ ID NO: 2), GVSQNYPIVG (SEQ ID NO: 3), LVLASSSFGY (SEQ ID NO: 4), PLGMWSR (SEQ ID NO: 5), PGNWT (SEQ ID NO: 6), PAGLLGC (SEQ ID NO: 6) 7), LGGSGRSANAILE (SEQ ID NO: 8), RR, RRG, GPICFFRLG (SEQ ID NO: 9), HSSKLQG (SEQ ID NO: 10), PIC (Et) FF (SEQ ID NO: 11), HSSKLQ (SEQ ID NO: 12), P (L / Q) A photosensitive agent-metal nanoparticle complex having an amino acid sequence comprising G (I / L) AG (SEQ ID NO: 13), GVVQASCRLA (SEQ ID NO: 14), KK, PLGVRG (SEQ ID NO: 15), or LR. The method of claim 1, The polypeptide substrate is cathepsins, matrix metallopeptidases, membrane type MMPs, collagenases, stromelysins, urokinase-type plasminogen activators (urokinase-type). plasminogen activator (uPA), caspases, viral proteases, HIV proteases, HSV proteases, gelatinase , Urokinases, secretases, endosomal hydrolase, PSA (Prostate Specific Antigen), plasminogen activator, and thrombin Photosensitive agent-metal nanoparticle complex which is specifically cleaved by any one selected protease. The method of claim 1, The backbone further comprises at least one of a first linker connecting the polypeptide substrate and the metal nanoparticles and a second linker connecting the polypeptide substrate and the photosensitive agent. 5. The method of claim 4, Said first linker is a cysteine having a thiol group, or a polypeptide comprising a cysteine, or an alkylene having a thiol group in the side chain or the main chain. 5. The method of claim 4, The second linker is a lysine containing an amine group in the side chain, or a polypeptide comprising lysine, or an alkyl or alkylene having an amine group in the side chain or main chain, The photosensitizer has a carboxyl group as a substituent photosensitive agent-metal nanoparticles composite. The method of claim 1, The photosensitizer is a porphyrin-based compound in the form of a free base or a metal complex, or a photosensitive agent-metal nanoparticle complex, which is a nonporphyrin compound. The method of claim 7, wherein The photosensitive agent is a pyropheophorbide a photosensitive agent-metal nanoparticle complex. The method of claim 1, The metal nanoparticles are gold, silver, copper, platinum, palladium, nickel, iron, or a photosensitive agent-metal nanoparticle composite containing two or more of these. 10. The method of claim 9, The metal nanoparticles may be formed in the form of spheres, rods, pyramids, star-shaped core-shells, and photosensitive agent-metal nanoparticles. Complex. The method of claim 1, And a hydrophilic polymer coupled to an end adjacent to said photosensitive agent of said backbone. The method of claim 11, The hydrophilic polymer is polyethylene glycol (PEG); Methoxy polyethylene glycol (MPEG); Methoxypolypropylene glycol; Dextran; Hyaluronic acid; Polylactic-polyglycolic acid copolymers; Polyethylene glycol-diacid; Polyethylene glycol monoamines; Methoxypolyethylene glycol monoamine; Methoxy polyethylene glycol hydrazide; Methoxy polyethylene glycol imidazole; Or copolymers of two or more of polyethylene glycol, methoxy polypropylene glycol, polyethylene glycol-diacid, polyethylene glycol monoamine, methoxy polyethylene glycol monoamine, methoxy polyethylene glycol hydrazide, and methoxy polyethylene glycol imidazole; Or a photosensitive agent-metal nanoparticle complex which is a copolymer of polyethylene glycol and polypeptide, polyethylene glycol and polysaccharide, polyethylene glycol and polyamidoamine, polyethylene glycol and polyethyleneamine, or polyethylene glycol and polynucleotide. The method of claim 1, The photosensitizer-metal nanoparticle composite is a photosensitizer-metal nanoparticle composite represented by the following formula (1). [Formula 1]

14. A composition for photodynamic therapy or diagnosis comprising the photosensitive agent-metal nanoparticle complex of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

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