WO2017123052A1 - Photodegradable nanoparticles for imaging and multiple phototherapy, and uses thereof - Google Patents

Abstract

The present invention provides nanoparticles formed by self-assembly of an amphipathic compound in which at least one biocompatible hydrophilic polymer is linked to a cypate or a derivative thereof, and the uses thereof for photodynamic therapy, photothermal therapy and cancer imaging. The nanoparticles of the present invention can be collapsed by near-infrared irradiation and can exhibit fluorescence by absorption of light. In addition, the nanoparticles of the present invention can simultaneously generate active oxygen and heat by single near-infrared irradiation. The nanoparticles of the present invention exhibit more excellent fluorescence properties, active oxygen generation properties, and exothermic properties compared to the same amount of cypate.

Description

Photolytic Nanoparticles for Imaging and Multiple Phototherapy and Their Uses

The present invention was made by the task number HI03C2181 under the support of the Ministry of Health and Welfare of the Republic of Korea, the research and management professional organization of the task is Korea Health Industry Development Institute, the research project name "development of future fusion medical device", the research title is "specific gastric endoscopy cancer Development of Fluorescent Probe ", the lead institution is Korea Advanced Institute of Science and Technology.

This patent application claims priority to Korean Patent Application No. 10-2016-0004810 filed with the Korean Patent Office on January 14, 2016, the disclosure of which is hereby incorporated by reference.

The present invention relates to photodegradable nanoparticles and their use for imaging and multiple phototherapy.

Nanotechnology-based light therapy because nanoparticles can preferentially accumulate in tumors through enhanced permeability and retention (EPR) effects, and subsequent light therapy can accurately destroy tumors in a spatiotemporal manner. (nanotechnology-assisted phototherapy) has been regarded as a promising strategy for the treatment of cancer ^1-6. However, single phototherapy methods such as photodynamic therapy (PDT) or photothermal therapy (PTT) have shown limited in vivo treatment effects. PDT depletes oxygen in tumor tissues and destroys tumor-associated blood vessels, leading to decreased blood flow to the tumor and consequently severe local hypoxia, which contributes to subsequent poor response to radiation and chemotherapy. ⁷ . PTT, which utilizes photoreactive nanomaterials to convert near infrared into heat, typically results in incomplete tumor destruction due to uneven heat transfer and tumor destruction, resulting in tumor recurrence initiated by living cancer cells in the remaining tumor mass ^{6 , 11-14} . Recent attempts to combine PDT and PTT to overcome the limitations of single ^phototherapy have resulted in significantly improved therapeutic outcomes ^15-22 . Local hyperthermia caused by PTT increased tumor blood flow and oxygenation, which contributes to PDT. In contrast, reactive oxygen species (ROS) produced by PDT make cancer cells susceptible to high fever caused by PTT. However, previously reported systems for PDT / PTT combination therapy, prepared by loading or conjugating known photosensitizers (PS) to near infrared (NIR) -absorbing nanoparticles, still have some drawbacks. First, mismatches in the absorption wavelengths of PS and NIR-absorbing nanoparticles require two types of lasers to excite the two ^{, 15, 19-20, and 23} , which complicates the treatment process. Second, PS-loaded NIR nanoparticles showed significantly reduced production of singlet oxygen ( ¹ O ₂ ) due to fluorescence resonance energy transfer (FRET) between adjacently located PS and NPs, ^{21 , 24-25} , which is PDT It resulted in a decrease in effect. Third, most of the NIR nanoparticles used in the non-PTT - ^{16 -18, 25,} long-term toxicity's biodegradable material is made of (e. G., Carbon, gold and other precious metal-based nanomaterials) remained intact for a long time in the body, (long-term toxicity) may be a concern. In addition, the complexity of manufacturing previously developed PDT / PTT combination systems has hampered future clinical applications.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

In order to improve the above problems of the prior art, the present inventors can (1) effectively treat and image cancer using a single laser, (2) be disassembled and excreted, and (3) easily scale up. Research efforts have been made to develop new nanoparticles for PDT / PTT combination therapy. As a result, the phosphate nanoparticles were prepared by introducing a hydrophilic block into the hydrophobic cypate to form a phosphate self-assembly in an aqueous system, and the ability of the phosphate to penetrate into the cells, photolysis due to near-infrared irradiation, and active oxygen and heat By confirming the developmental capacity and the cancer imaging ability in vivo, the present invention has been completed.

Accordingly, it is an object of the present invention to provide nanoparticles formed by self-assembly of a phosphate derivative.

Another object of the present invention is to provide a pharmaceutical composition for photodynamic therapy, photothermal therapy or a combination thereof.

Another object of the present invention to provide a composition for cancer imaging.

Another object of the present invention to provide a composition for the simultaneous diagnosis and treatment of cancer.

Another object of the present invention to provide a method of treating cancer.

Another object of the present invention is to provide a method for imaging cancer.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides nanoparticles formed by self-assembly of the phosphate derivative of Formula 1 below:

Formula 1

Wherein n is an integer from 0-5, A ₁ and A ₂ are each independently a biocompatible hydrophilic polymer or OH, and at least one of A ₁ and A ₂ is a biocompatible hydrophilic polymer.

In order to improve the above problems of the prior art, the inventors of the present invention have shown that a novel nano for PDT / PTT combination therapy that can effectively treat cancer and image, decompose and excrete and easily scale up using a single laser. Research efforts have been made to develop particles. As a result, the phosphate nanoparticles were prepared by introducing a hydrophilic block into the hydrophobic phosphate to form a phosphate self-assembly in the aqueous system, and the ability of the phosphate nanoparticles to penetrate into the cells, photolysis and reactive oxygen and heat generation ability according to near-infrared irradiation, And the cancer imaging ability in vivo was confirmed.

According to an embodiment of the present invention, n in Chemical Formula 1 is an integer of 1-5. If both A ₁ and A ₂ are OH and n is 2, the compound of formula 1 is a phosphate. In one specific example, n of Chemical Formula 1 is an integer of 2-5, an integer of 2-4, or an integer of 2-3.

According to an embodiment of the present invention, the nanoparticles of the present invention are particles formed by self-assembly of the phosphate derivative of Formula 1, to which at least one biocompatible hydrophilic polymer is bound, and nanoparticles formed by self-assembly in an aqueous system. May be particles.

According to one embodiment of the present invention, the nanoparticles of the present invention have a diameter of 1 nm to 100 nm. In one specific example, the nanoparticles have a diameter of 10 nm to 100 nm. This small size contributes to the preferential accumulation of nanoparticles of the present invention in cancer tissues through the EPR effect.

According to one embodiment of the present invention, the nanoparticles of the present invention are disintegrated (decomposed) by near-infrared (700 nm-1500 nm wavelength) irradiation. The nanoparticles of the present invention are disintegrated (decomposed) into compounds of the general formula (1), which are monomers that constituted the nanoparticles by near-infrared irradiation, and the monomer compounds thus produced do not exhibit toxicity, like nanoparticles, and are smaller in size than nanoparticles. Easily discharged to the outside of the body. Thus, this disintegration property of the present invention contributes to the safe use of the nanoparticles of the present invention in clinical practice.

According to one embodiment of the present invention, the nanoparticles of the present invention may generate active oxygen by near-infrared irradiation.

According to one embodiment of the present invention, the nanoparticles of the present invention may generate heat by near-infrared irradiation.

According to one embodiment of the present invention, the nanoparticles of the present invention may generate heat together with active oxygen by near-infrared irradiation in the same wavelength band. Therefore, using the nanoparticles of the present invention, a combined therapeutic effect of photodynamic therapy (PDT), which generates free radicals and therapeutic effects, and photothermal therapy (PTT), which generates therapeutic effects, is obtained. Unlike nanoparticles used in conventional PDT / PTT combination treatment, active oxygen and heat can be generated together by a single near-infrared irradiation, so that PDT / PTT combination treatment can be obtained in a more economical and convenient manner. That has the advantage.

In one specific example, the active oxygen generated by the nanoparticles of the present invention is singlet oxygen.

According to one embodiment of the present invention, the nanoparticles of the present invention absorb near infrared rays and exhibit fluorescence. The nanoparticles of the present invention can be preferentially accumulated in cancer tissues when administered in the body, and can absorb fluorescence by absorbing light, and thus the imaging of cancer in vivo by irradiating near infrared rays after administering the nanoparticles of the present invention to the body. And diagnostics are possible.

The compound of Formula 1 includes at least one biocompatible hydrophilic polymer. As the hydrophilic polymer usable in the present invention, for example, polyethylene glycol (PEG), poly (acrylic acid), poly (acrylate), poly (acrylamide) ( poly [acrylamide]), poly [vinyl ester], poly [vinyl alcohol], polystyrene, polyoxide, cellulose, starch ( starch, polysaccharide, polyelectrolyte, poly (1-nitro propylene), poly (N-vinylpyrrolidone) ), Poly [vinyl amine], poly (beta-hydroxyethyl methacrylate), poly ethyleneoxide, poly (ethylene oxide-b-propylene oxide) (Poly [ethylene oxide-b-propylene oxide]) and polylysine (Polylysine).

Still other examples of hydrophilic polymers usable in the present invention include chitosan, hyaluronic acid, collagen, gelatin, acacia gum, dextran, fibrin, pectin, agar, galactomannan, xanthan and alginate Etc.

Another example of a hydrophilic polymer that can be used in the present invention is a hydrophilic peptide consisting of 2-100 (eg 2-50) amino acids. The amino acids include natural amino acids as well as non-natural amino acids. Hydrophilic amino acids include glutamine, aspartic acid, glutamic acid, threonine, asparagine, arginine, serine, and the like, and hydrophobic amino acids include phenylalanine, tryptophan, isoleucine, leucine, proline, methionine, valine, and alanine. Uncoded hydrophilic amino acids include, for example, Cit and hCys. Those skilled in the art can easily synthesize hydrophilic peptides based on this information and peptide synthesis techniques, and use them to prepare nanoparticles of the present invention.

The range of the hydrophilic polymer includes not only the above-mentioned polymers, but also derivatives thereof.

In one particular embodiment, the hydrophilic polymer is polyethylene glycol or derivatives thereof. The polyethylene glycol derivative is, for example, methoxy polyethylene glycol (PEG), succinimide of PEG propionic acid, succinimide of PEG butanoic acid, eggplant Branched PEG-NHS, PEG succinimidyl succinate, succinimide of carboxymethylated PEG, benzotriazole carbonate of PEG, PEG-glycidyl ether, PEG-oxycarbonylimidazole, PEG nitrophenyl carbonates, PEG-aldehyde, PEG succinimidyl carboxymethyl Ester (PEG succinimidyl carboxymethyl ester), PEG succinimidyl ester, and the like.

According to an embodiment of the present invention, in Chemical Formula 1, the biocompatible hydrophilic polymer is bonded through -CO- and an amide bond. To this end, the biocompatible hydrophilic polymer has an amine group at the side chain or terminal.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for photodynamic therapy, photothermal therapy or a combination thereof comprising the nanoparticles of the present invention as an active ingredient.

Since the pharmaceutical composition of the present invention uses the above-described nanoparticles of the present invention as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

The nanoparticles of the present invention generate free radicals and heat by near-infrared irradiation, and thus may be used for photodynamic therapy and / or photothermal therapy.

According to one embodiment of the invention, the composition of the invention is a pharmaceutical composition for the combined treatment of photodynamic therapy and photothermal therapy.

According to one embodiment of the invention, the composition of the invention is a pharmaceutical composition for the treatment of cancer, psoriasis, acne or warts. Cancers to which the present invention is applicable include, for example, melanoma, skin cancer, colon cancer, pancreatic cancer, biliary tract cancer, neuroendocrine tumor, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, bladder cancer, Colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, thyroid cancer, parathyroid cancer and ureter cancer.

The composition of the present invention includes a pharmaceutically effective amount of nanoparticles. The term "pharmaceutically effective amount" means an amount sufficient for the nanoparticles of the present invention to be applied in the treatment of the disease to achieve a therapeutic effect.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical compositions of the present invention are capable of parenteral administration, such as intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration. In addition, oral administration, rectal administration, inhalation administration, nasal administration and the like are possible.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment.

The pharmaceutical compositions of the present invention are prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulations may then be in the form of solutions, suspensions or emulsions in oils or aqueous media, and may further comprise dispersants or stabilizers.

According to another aspect of the present invention, the present invention provides a composition for cancer imaging comprising the nanoparticles of the present invention.

Since the composition for cancer imaging of the present invention uses the above-described nanoparticles of the present invention as an active ingredient, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

Since the nanoparticles of the present invention can be preferentially accumulated in cancer tissues when administered in vivo, they can be used to image cancer tissues and cells.

According to another aspect of the present invention, the present invention provides a composition for the simultaneous diagnosis and treatment of cancer comprising the nanoparticles of the present invention.

According to another aspect of the present invention, the present invention provides a method for treating cancer, comprising administering to a subject a composition comprising the nanoparticles of the present invention as an active ingredient.

More specifically, the method comprises the steps of i) administering a pharmaceutical composition comprising the nanoparticles of the invention as an active ingredient to a subject; ii) allowing a predetermined time until the administered nanoparticles accumulate in cells in the cancer tissue of the subject; And iii) irradiating near-infrared rays to the site of the cancer tissue of the subject.

In addition, according to another aspect of the present invention, the present invention provides a method for imaging cancer tissue comprising the step of administering to the subject a composition comprising the nanoparticles of the present invention as an active ingredient.

More specifically, the method comprises the steps of i) administering to the subject a composition comprising the nanoparticles of the invention as an active ingredient; ii) allowing a predetermined time until the administered nanoparticles accumulate in cells in the cancer tissue of the subject; iii) imaging the cancer tissue by irradiating near-infrared to identify the site of cancer tissue of the subject; And iv) analyzing the image data to determine the exact location of the cancerous tissue.

Imaging of the cancer tissue may preferentially accumulate in cancer tissues when the nanoparticles of the present invention are administered in the body, and may absorb and fluoresce when irradiated with near infrared rays. When irradiated, it means the visualization of the location of cancer tissue by detecting the fluorescence of the nanoparticles absorbing near infrared rays. The imaging may use a variety of diagnostic devices that can absorb the fluorescence emitted by the nanoparticles of the present invention, regardless of the type of device, such as a fluorescence detection device,

As used herein, the term “administration” or “administer” refers to administering a therapeutically effective amount of a composition of the invention directly to a subject (an individual) in need thereof so that the same amount is formed in the subject's body. Say that.

A "therapeutically effective amount" of a composition means a content of the composition that is sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is to be administered, and includes "prophylactically effective amount". As used herein, the term "subject" includes, without limitation, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

Since the present invention is a method for treating cancer, it is a method comprising administering a pharmaceutical composition comprising the nanoparticles of the present invention as an active ingredient, which is an aspect of the present invention. To avoid this.

As described above, the nanoparticles of the present invention may preferentially accumulate in cancer tissues due to the EPR effect when administered in the body, and absorb fluorescence by absorbing near-infrared rays, as well as active oxygen and heat that induce the death of cancer cells. It can be applied to the simultaneous diagnosis and treatment of cancer.

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides nanoparticles formed by self-assembly of amphiphilic compounds in which at least one biocompatible hydrophilic polymer is linked to a phosphate or derivative thereof, and uses thereof for photodynamic therapy, photothermal therapy and cancer imaging.

(ii) The nanoparticles of the present invention may be disintegrated by near-infrared irradiation, and may absorb light to fluoresce. In addition, the nanoparticles of the present invention can generate heat together with active oxygen by a single near infrared irradiation.

(iii) The nanoparticles of the present invention exhibit better fluorescence, reactive oxygen production and exothermic properties than the same amount of phosphate.

1 shows the characteristics of SP ³ NP for cancer diagnosis and treatment. (A) Schematic explanation of NIR fluorescence imaging of SP ³ NPs and the degradation of laser-induced nanoparticles and synergistic photodynamic and photothermal anticancer effects. (B) DLS data showing the hydrodynamic size of SP ³ NP in PBS (pH 7.4). (C) TEM image over time after NIR irradiation (808 nm, 0.8 W cm ⁻² ). Scale bar: 100 nm. (D) Fluorescence images of SP ³ NP series diluents in phosphate and PBS (Ex / Em: 710-760 nm / 810-875 nm). (E) 525 nm (Ex: 488) in the presence or absence of azide sodium (100 μM) of PBS, free phosphate and SP ³ NP after 808-nm laser irradiation (0.8 W cm ⁻² , 5 min) single oxygen sensor green (SOSG) fluorescence intensity (FI) at nm. ^* P <0.05 compared to other groups. (F) Photothermal heating curves of SP ³ NPs at various concentrations for 5 minutes of 808-nm laser irradiation.

2 shows the in vitro endocytosis, NIR-induced ROS and heat generation, and cytotoxicity of SP ³ NP. CLSM image (A) and flow cytometry (B) of B16F10 melanoma cells treated with free lysate or SP ³ NP for 1 hour. Sulfate- or SP ³ NP-treated cells were irradiated with an 808-nm laser (8 W cm ⁻² , 3 min). Intracellular ROS production (C) was detected by H ₂ DCFDA (10 μM). After light irradiation, the thermal image (D) of the cells was recorded using an IR thermal camera. (E) Relative viability of B16F10 cells treated with different concentrations of SP ³ NP for 1 hour and irradiated with 808-nm laser (0.2, 0.4 or 0.8 W cm ⁻² , 3 minutes) and then further incubated for 24 hours.

Figure 3 shows the in vivo bio-distribution in the tumor of mouse SP ³ NP. (A) Systemic NIR fluorescence images 0.5, 1, 2, 4, 8, 12 and 24 hours after administration of B16F10 melanoma mice receiving free phosphate or SP ³ NP at 1 mg / kg phosphate dose. (B) Semi-quantitative analysis of fluorescence intensity in tumor sites using Living Image Software Version 2.6. (C) CLSM image of tumor tissue excised and cryo sectioned 4 hours after intravenous administration of free phosphate or SP ³ NP. Scale bar: 50 μm.

4 shows the NIR laser induced antitumor effect of SP ³ NP. (A) Real-time thermography of B16F10 melanoma mice exposed to 808-nm laser (0.4 W cm ⁻² ) for 10 minutes at 4 hours of PBS, free phosphate or SP ³ NP (5 mg / kg phosphate) administration . The circle indicates the tumor site. (B) Photothermal heating curve of tumor after 10 minutes of irradiation with 808-nm laser. B16F10 intravenously administered with PBS, free phosphate or SP ³ NP (5 mg / kg phosphate, single dose, n = 6-7) with or without 808-nm laser irradiation (0.4 W cm ⁻² , 10 minutes) Tumor growth curve (C), weight change (D) and survival rate (E) of melanoma mice. ^* P <0.05 compared to other groups at each time point. H & E (E) and TUNEL (F) staining of tumor tissues from each group of mice 12 hours after light irradiation. Scale bar: 25 μιη.

Figure 5 shows the results of the characterization of pegylated phosphate by ¹ H NMR (A) and MALDI-TOF / MS (B).

6 shows the UV-vis spectral change according to colloidal stability (A) and NIR laser irradiation of SP ³ NP.

7 shows the effect of various ROS scavengers on intracellular ROS generated by SP ³ NP after NIR laser irradiation. (A) Scavenging of ROS generated in cells by ROS scavengers. (B) Cytoprotective effect of ROS scavenger.

8 relates to the cytotoxicity assessment of NIR-cut fragments of SP ³ NP. (A) Schematic description of SP ³ NP digested by NIR laser used to test cytotoxicity of digested fragments. (B) Cytotoxicity of NIR-digested fragments of SP ³ NPs. The results are expressed as mean ± sem of 4 independent experiments.

9 shows digital images of tumors treated with NIR laser treatment with each treatment. The circle indicates the tumor site.

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

Example

1. Experimental Materials and Methods

Peg Sipate ( PEGylated cypate ) synthesis

Pegylated phosphate was prepared by conjugating the phosphate with methoxypolyethylene glycol (mPEG ₂₀₀₀ -NH ₂ ) through an amide bond. Briefly, N, N′-dicyclohexylcarbodiimide (DCC; 90 μmol) and N-hydroxysuccinimide (NHS; 90 μmol) were added to phosphate (60 μmol) in dichloromethane (10 ml). After stirring for 30 minutes, insoluble byproducts were removed by filtration. MPEG ₂₀₀₀ -NH ₂ (20 μmol) was added to the activated phosphate and the mixture was allowed to react at room temperature for 24 hours. The crude product was purified by silica gel column chromatography using chloroform / methanol (75:25, v / v). The resulting pegylated phosphate was analyzed for ¹ H NMR characterization using a Bruker AVANCE-500 MHz FT-NMR spectrometer (Bruker, Billerica, Mass., USA). The molecular weight of PEGylated phosphate was measured by matrix-assisted laser desorption / ionization-time of flight spectrometry (MALDI-TOF) using an Autoflex III MALD-TOF system (Bruker).

SP ³ NP  Manufacturing and Characterization

PEGylated phosphate (1 mg) in methanol was dried and the resulting thin membrane was hydrated and vortexed with 1 ml PBS (pH 7.4). After sonication for 10 minutes, large aggregates were removed by filtration through a 0.2-μm polycarbonate membrane filter (Millipore Corp., Billerica, Mass., USA) to obtain SP ³ NP and stored at 4 ° C. until use. The size and shape of SP ³ NP was confirmed by TEM using the JEM1010 system (Jeol Ltd, Tokyo, Japan). The hydrodynamic diameter and zeta potential of SP ³ NPs were measured using a Nanosizer ZS90 instrument (Malvern Instruments Ltd, Malvern, UK). The colloidal stability of SP ³ NP was tested by monitoring the size change for at least 4 weeks. UV-Vis spectra of SP ³ NPs were recorded using a UV-Vis spectrophotometer (NEOSYS2000; Scinco, Twin Lakes, WI, USA).

NIR  Probe-induced SP ³ NP  Disruption

SP ³ NP (100 μg ml ⁻¹ ) in PBS was irradiated with light for 20 minutes using a BWF2 continuous-wave NIR laser (808 nm, 0.8 W cm ⁻² ; B & W Tek Inc., Newark, DE, USA). . Absorption spectra and shape changes of SP ³ NP during light irradiation were recorded using a UV-Vis spectrophotometer and transmission electron microscope (TEM), respectively. The appearance of SP ³ NP during NIR irradiation was recorded with a digital camera.

NIR  Fluorescence, and SP ³ NP Metropolitan  Characteristics and Heat  characteristic

Fluorescence images of the same concentration of free phosphate in PBS and SP ³ NPs were recorded using the Xenogen IVIS Lumina Imaging System (Perkin Elmer Inc., Waltham, Mass., USA). The photodynamic effect was tested by irradiating (808 nm, 0.8 W cm ^{- 2} ) light for 5 minutes to the glass silicate and SP ³ NP (25 μg ml ^-1 ) in the presence and absence of azide sodium (100 μM), The generated singlet oxygen was detected using a singlet oxygen sensor green (SOSG) Invitrogen, Grand Island, NY, USA). PBS was used as a negative control. Photothermal effects were evaluated by irradiating different concentrations of SP ³ NP with an 808-nm NIR laser (0.8 W cm ^-2 , 5 minutes). Temperature was quantified using an IR thermal imaging system (FLIR T420; FLIR Systems Inc., Danderyd, Sweden). As a negative control, the photothermal heating curve of PBS was measured.

In vitro intracellular uptake test

Intracellular uptake of SP ³ NP was confirmed by confocal microscopy and flow cytometry using the intrinsic fluorescence of the phosphate. B16F10 rat melanoma cells (American Type Culture Collection, Rockville, MD, USA) were added to DMEM (Gibco-BRL Life Technologies, Carlsbad, CA) with 10% fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin. , USA). B16F10 cells were dispensed into cover glasses of 24-well plates at a density of 1 × 10 ⁵ cells / well. After reaching 70% confluency, cells were treated with free lysate or SP ³ NP equally with the throughput of lysate. After 1 hour of incubation the cells were washed with cold PBS, fixed for 15 minutes with 4% paraformaldehyde and stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Cell fluorescence was observed using confocal laser microscopy (LSM 5 Exciter; Carl Zeiss, Inc., Jena, Germany). Flow cytometry was performed by washing the first harvested cells three times with cold PBS containing 2% fetal bovine serum. Cells were then analyzed with a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA) using Cell Quest Pro software.

Heat generation due to light irradiation and intracellular ROS

B16F10 cells were aliquoted into 24-well plates at a density of 1 × 10 ⁵ cells / well. The next day, the cells were treated with free sulphate or SP ³ NP (same amount of phosphate) at a concentration of 5 μg ml ⁻¹ for 1 hour. After washing with cold PBS, cells were irradiated with NIR laser (808 nm, 0.8 W cm ^{- 2} ) for 3 minutes and fresh with ROS detection dye H ₂ DCFDA (10 μM; Life Technologies, Carlsbad, CA, USA) Incubate for 1 hour in media. DCF (green) was observed using a fluorescence microscope (Leica, DM IL, Germany). Several ROS scavengers, including glutathione, azide sodium (single singlet oxygen scavenger), D-mannitol (hydroxyl radical scavenger) or sodium pyruvate (hydrogen peroxide scavenger), were tested during NIR irradiation. The type of ROS generated by addition to the medium was confirmed. The subsequent procedure is as described above. Intracellular heat generation was assessed by treating B16F10 cells with free lysate or SP ³ NP (20 μg ml ⁻¹ , equal amount of phosphate) for 1 hour. After washing with cold PBS, cells were irradiated with NIR for 3 minutes (808 nm, 0.8 W cm ⁻² ) and thermal images were recorded as described above using an IR thermal camera.

Laser induced SP ³ NP  For tumor cells Phototoxicity

In vitro anti-tumor effects of SP ³ NP following NIR irradiation were tested by MTT assay. Briefly, B16F10 cells were dispensed in 96 well plates at a density of 1 × 10 ⁴ cells / well. The next day, cells were treated with SP ³ NP series diluent for 1 hour. After washing with cold PBS, the cells were irradiated with 808-nm NIR laser for 3 minutes. Cells were incubated for 24 hours and cell viability was quantified by MTT assay. SP ³ in NP ^cells the (100 μg ml ¹⁾ for 1 h, before addition to the MTT assay ROS scavenger is a medium containing (sodium pyruvate, glutathione, sodium azide or D- mannitol, 100 μM) Incubation was performed for 30 minutes to test the cytoprotective effects of various ROS scavengers. Cytotoxicity of the NIR-cut fragments of SP ³ NP was tested by completely digesting SP ³ NP with NIR lasers and then treating the colorless product obtained on B16F10 cells for 24 hours. Cell viability was quantified by MTT assay and expressed in proportion to the results of the untreated control group.

SP ³ NP  In vivo tumor detection ability

In vivo experiments were performed using 6-week-old male C57BL / 6 mice (Orient Bio. Lab. Animal Inc., Seungnam, Kyonggi-do, South Korea). 1 x 10 ⁶ B16F10 cells were subcutaneously inoculated into the right back region of the hairless C57BL / 6 to allow tumors to grow. When the tumor volume reached about 100 mm ³ , the amount of sulphate was injected equally (1 mg / kg) with free sulphate or SP ³ NP into the tail vein. In vivo distribution of fluorescent free phosphate and SP ³ NPs in mice at predetermined times after dosing was confirmed using the Xenogen IVIS Lumina Imaging System (Perkin Elmer Inc.). In other experiments, tumors were excised and cryosectioned and then confocal microscopy confirmed the intratumoral distribution of free phosphate or SP ³ NPs as described above.

NIR  According to the investigation SP ³ NP  In vivo tumor ablation

Mice with B16F10 tumors were randomly divided into 6 groups based on body weight and tumor volume (n = 6-7 mice / group): (1) PBS, (2) PBS + NIR irradiation, (3) between glass Pate, (4) free phosphate + NIR irradiation, (5) SP ³ NP, (6) SP ³ NP + NIR irradiation. Mice were injected intravenously with free sulphate or SP ³ NP such that the dose of phosphate (5 mg / kg) was the same. Mice were anesthetized 4 hours after administration. Tumor sites were irradiated with 808-nm continuous wave NIR lasers (0.4 W cm ⁻² , 10 minutes). Temperature changes induced by light at the tumor site were recorded using a real-time IR thermal imaging system (FLIR T420). Tumor volume, body weight, tumor appearance and survival were monitored after treatment. Tumor volume was measured in two dimensions using an electron ruler and calculated according to the formula "axb ² x 0.5 (a is the largest dimension, b is the smallest dimension)". In another experiment, tumor tissue was extracted 12 hours after irradiation, fixed with 4% paraformaldehyde, and then embedded in paraffin to prepare 10 μm sections. Tumor tissue slides were stained with H & E and observed with light microscopy. Killed cells in tissue sections were identified by TUNEL assay (Bio Vision).

Statistical analysis

Student-Newman-Keuls tests for analysis of variance (ANOVA) and post hoc pairwise comparisons were used to analyze experimental data. All statistical analyzes were performed using SigmaStat software (version 3.5; Systat Software, Richmond, Calif., USA), where p <0.05 was considered significant.

2. Experimental Results

The present inventors have attempted to develop a phosphate-based nanoparticle that can be used in combination therapy of photodynamic therapy (PDT) / photothermal therapy (PTT). In order to form the phosphate-based nanostructures, the inventors have introduced a hydrophilic, biocompatible polyethyleneglycol (PEG) into the phosphate to prepare pegylated phosphate. The inventors expected that the amphiphilic PEGylated phosphate spontaneously self-assembled into small nanoparticles (named SP ³ NP) and exhibit photodegradability, photodynamic properties and photothermal properties (FIG. 1A).

PEGylated phosphate in a one-step process in which the carbodiimide-activated form of one of the two carboxylic acids of the phosphate is reacted with methoxypolyethyleneglycolamine (mPEG ₂₀₀₀ -NH ₂ ) Were synthesized and characterized by ¹ H NMR and mass spectrometry (FIG. 5). When placed in an aqueous solution, as shown in Fig. 1A, PEGylated phosphate hydrophobic phosphate forms the core of the nanoparticles, hydrophilic PEG is a micelle-type nanoparticles (SP ³ NP) exposed to water The furnace was assembled. Dynamic light scattering (DLS) measurements confirmed that SP ³ NP had a narrow hydrodynamic diameter with an average hydrodynamic diameter of 60 ± 8 nm (FIG. 1B). High-resolution in situ transmission electron microscopy (HRTEM) imaging showed that SP ³ NP was a circular nanostructure with a clear outline of ˜40 nm diameter in dry state (FIG. 1C). In addition, SP ³ NP did not have a significant change in size even in more than 4 weeks in PBS showed good colloidal stability (A of Figure 6). The zeta potential of SP ³ NP in PBS at pH 7.4 was 0.06 ± 0.22 mV, and these results show that the nanoparticles have a nearly neutral charge. In addition, SP ³ NP contained in PBS showed strong absorption in the NIR region (B of FIG. 6). FIG. 1D shows that NIR fluorescence images of free phosphate and SP ³ NPs were excited at 710-760 nm. SP ³ NP showed much stronger fluorescence intensity than free sulphate. On the other hand, SP ³ NP retained and maintained the strong fluorescence of pristine phosphate under physiological conditions, indicating that it is suitable for using SP ³ NP as an NIR imaging probe.

It was investigated whether SP ³ NP could be decomposed by light. As also shown in C of Figure 1, continuous wave ^laser was ^{(808 nm, 0.8 W cm 2} ) a according to the NIR irradiation SP ³ NP using markedly, and experienced a non-reversible shape change which appeared after a minute investigation, After 10 minutes of irradiation, no detectable nanoparticles remained. This NIR-induced photolysis was accompanied by rapid bleaching (C in FIG. 1, illustration), leaving only 5.5% of the unique absorption peak of SP ³ NP at 790 nm 10 minutes after irradiation (FIG. 7). This NIR-induced degradation and decay character shows that SP ³ NP can be easily released from the body after treatment with NIR lasers, thus alleviating the toxicity problem. In addition, SP ³ NP can be used as a photoreactive drug carrier by mounting a drug.

Next, it was investigated whether SP ³ NP generates singlet oxygen ( ¹ O ₂ ) for photodynamic effects or heat for photothermal treatment in response to NIR irradiation. As shown in E of Figure 1, by measuring the fluorescence intensity of SOSG (singlet oxygen sensor green), it was confirmed that ¹ O ₂ was generated by the laser irradiation of 808-nm. SP ³ NP showed much stronger (~ 3.8-fold) SOSG fluorescence intensity compared to the free phosphate. This fluorescence intensity was markedly reduced in the presence of NaN ₃ , a representative ¹ O ₂ scavenger, which supports that SP ³ NP can produce ¹ O ₂ in response to NIR laser irradiation. Next, it was examined whether SP ³ NP can generate heat in response to NIR irradiation. The temperature of the sample solution was recorded during 808 nm laser irradiation (F in FIG. 1). The inventors found that the temperature rose rapidly with increasing concentration of SP ³ NP and reached a plateau within 2 minutes of irradiation, and that the SP3NP of 100 and 200 μg ml ⁻¹ increased the temperature by 17.4 ° C. and 25.3 ° C., respectively. . PBS control did not show this photothermal effect. These results show that SP ³ NP also has good photothermal properties. Interestingly, the temperature of SP ³ NP gradually decreased after reaching their peak, indicating that SP ³ NP is undergoing NIR-induced photobleaching and photolysis (FIG. 7). Overall, the simultaneous and efficient generation of these NIR-induced heat and ¹ O ₂ suggests a promising use in combined PDT and PTT for effective cancer treatment of SP ³ NPs.

Next, intracellular ROS production levels following NIR laser (0.8 W cm ⁻² , 3 min) exposure in SP ³ NP-treated cancer cells were measured using H ₂ DCFDA, a ROS-detected fluorescent probe. As expected, SP ³ NP-treated cancer cells showed strong fluorescence as a result of activation of H ₂ DCFDA, indicating high levels of intracellular ROS generation (FIG. 2C). Without NIR irradiation, no ROS were produced in SP ³ NP-treated cancer cells (FIG. 8). In addition, when the case of treatment with glutathione or sodium azide ⁽¹ O ₂ scavenger) cells whereas the fluorescence intensity decreases to the background level, the process D- mannitol (hydroxyl scavengers) include the fluorescent intensity There was no effect, through which it was confirmed that ¹ O ₂ is a major intracellular ROS produced by light irradiation of SP ³ NP. This increase in ROS production raised the temperature of SP ³ NP-treated cells by ˜4 ° C.

Since SP ³ NP can produce ¹ O ₂ and heat in the cells upon exposure to NIR, we have confirmed their cancer cell killing effect in B16F10 mouse melanoma cells. As it is shown in Fig. 2 E, cytotoxicity of SP NP ³ was characterized by the density and the relationship between the laser intensity and the amount used in the SP ³ NP. As expected, higher concentrations of nanoparticles irradiated with higher light power exhibited the best anticancer effects. However, SP ³ NP did not show cytotoxicity in the absence of NIR irradiation. Addition of hydroxyl radical scavenger (pyruvate sodium or D-mannitol) did not affect cell viability, while co-treatment of ¹ ₂ scavenger (glutathione or azide sodium) was SP ³ NP-induced cells Toxicity was significantly reduced (FIG. 9). In addition, the product of the cut is broken SP ³ NP generated according to the NIR irradiation are did not show cytotoxicity, which NIR light-after showing the induced anti-cancer effect suggests decomposed into pieces SP ³ NP represents a lower toxicity . These in vitro cell experiments show that SP ³ NP can be efficiently introduced into cancer cells after NIR irradiation and can kill cancer cells through the combined effect of intracellular production and hyperthermia of ¹ O ₂ .

SP ³ NP was expected to be preferentially located at the tumor site due to its relatively small size (~ 60 nm) and EPR effects due to its high PEGylation surface. To test this, free lysate or SP ³ NP (0.25 mg / kg, equivalent amount of phosphate) was administered via tail vein to mice bearing B16F10 melanoma allograft. 3A shows real time biodistribution and tumor accumulation at various times. Only weak fluorescence was observed at the tumor site of mice administered free lysate. On the other hand, SP ³ NP showed a much stronger fluorescence signal at the tumor site as well as prolonged circulation / retention of more than 24 hours compared to free phosphate. After 4 hours, the difference in tumor-to-background intensity was evident, resulting in a clear boundary at the tumor site. Measurement of the amount of light in the tumor site over time showed that the fluorescence intensity of tumors in SP ³ NP-treated mice was ˜18-fold stronger than mice treated with free phosphate after 1 hour of injection (FIG. 3). B). Tumor tissue was excised and frozen sectioned 4 hours after injection. CLSM imaging showed that SP ³ NP was uniformly distributed in tumor tissue at much higher intensity than free lysate (FIG. 3C). The high levels of tumor accumulation and distribution observed in NIR-irradiated SP ³ NPs are very advantageous for effective phototherapy using imaging of cancer.

Next, the therapeutic effect of SP ³ NP according to NIR laser irradiation was investigated in tumor mice. Temperature at the tumor site following NIR exposure was continuously monitored (FIG. 4A). Mice treated with PBS did not show an increase in temperature during light irradiation, but mice in the SP ³ NP injection group showed a significant temperature increase up to 45 ° C., which was much higher than in mice injected with free lysate (˜38 ° C.). Temperature (FIG. 4B). Treatment efficacy after a single intravenous injection followed by NIR irradiation was assessed by measuring tumor volume, weight and survival rate (C in FIG. 4). Inhibition of tumor growth was not found in PBS alone or PBS + NIR group, and tumor volume of both groups was similar. Tumor growth in the SP ³ NP and free lysate alone groups was similar to the PBS control group, indicating that treatment in the absence of NIR irradiation showed no cytotoxicity. In contrast, tumors in the SP ³ NP + NIR group were completely ablated (FIG. 4A). Furthermore, continuous monitoring showed that tumors did not recur until after 42 days in the SP ³ NP + NIR group resulting in 100% survival (FIG. 4E). In addition, mice in the SP ³ NP + NIR group showed no sign of significant weight loss, indicating no nanoparticles themselves or acute side effects of light treatment (FIG. 4D). Although free phosphate + NIR treated mice inhibited tumor growth, all mice in this group died of tumors within 36 days.

Antitumor effects were again evaluated by H & E staining and TUNEL analysis. As shown in F and G of FIG. 4, PBS + NIR, free lysate and SP ³ NP alone group showed infiltration of cancer cells with high polymorphic nuclei and showed no evidence for apoptosis similar to PBS control. Did. Tumor tissue sections from SP ³ NP + NIR treated mice showed large disruption of the cell nucleus and significant apoptosis, demonstrating the potency of the combination treatment of PDT and PTT. The optical power intensity (0.4 W cm ^-2 ) used in this study is the lowest among the reported optical powers (~ 1-2 W cm ^-2 ) for in vivo PTT. These results indicate that SP ³ NPs may preferentially migrate to the tumor site due to the high PEGylation properties and proper size of the surface, and the combination of PTT and PDT after optical imaging-guided NIR laser irradiation. As a result of synergistic anti-tumor efficacy, it is shown that tumors can be effectively removed and prolonged survival of mice without tumor recurrence.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (16)

A nanoparticle formed by self-assembling a cypate derivative of Formula 1 below: Formula 1

Wherein n is an integer of 1-10, A ₁ and A ₂ are each independently a biocompatible hydrophilic polymer or OH, and at least one of A ₁ and A ₂ is a biocompatible hydrophilic polymer. The nanoparticle of claim 1, wherein the nanoparticle is formed by self-assembly in an aqueous system. According to claim 1, wherein the nanoparticles are nanoparticles, characterized in that having a diameter of 1 nm to 500 nm. According to claim 1, wherein the nanoparticles are nanoparticles, characterized in that collapsed by near-infrared irradiation. The nanoparticles of claim 1, wherein the nanoparticles generate active oxygen by near-infrared radiation. The nanoparticle of claim 1, wherein the nanoparticles generate heat by near-infrared radiation. The nanoparticles of claim 1, wherein the nanoparticles generate heat together with active oxygen by near-infrared radiation of the same wavelength band. The nanoparticle of claim 1, wherein the nanoparticles absorb near infrared rays and exhibit fluorescence. According to claim 1, wherein the biocompatible hydrophilic polymer is polyethylene glycol, poly (acrylic acid), poly (acrylate) (poly [acrylate]), poly (acrylamide) (poly [acrylamide]) , Poly [vinyl alcohol], polyoxide, cellulose, starch, polysaccharide, polyelectrolyte, poly (N-vinylpyrroli) (Poly [N-vinyl pyrrolidone]), poly [vinyl amine], poly (beta-hydroxyethyl methacrylate) (Poly [beta-hydroxyethylmethacrylate]), polyethylene oxide, Nanoparticles, characterized in that selected from the group consisting of poly (ethylene oxide-b-propylene oxide) and polylysine (Polylysine). A pharmaceutical composition for photodynamic therapy, photothermal therapy or a combination thereof comprising the nanoparticles of any one of claims 1 to 9 as an active ingredient. The composition of claim 10, wherein the composition is for treating cancer, psoriasis, acne or warts. The composition of claim 10, wherein the composition is for a combined treatment of photodynamic therapy and photothermal therapy. A composition for cancer imaging comprising the nanoparticles of any one of claims 1 to 9. The composition for the simultaneous diagnosis and treatment of cancer comprising the nanoparticles of any one of claims 1 to 9 as an active ingredient. i) administering to the subject a pharmaceutical composition comprising the nanoparticles of any one of claims 1 to 9 as an active ingredient; ii) allowing a predetermined time until the administered nanoparticles accumulate in cells in the cancer tissue of the subject; And iii) irradiating near-infrared rays to the site of cancer tissue of a subject. i) administering to the subject a pharmaceutical composition comprising the nanoparticles of any one of claims 1 to 9 as an active ingredient; ii) allowing a predetermined time until the administered nanoparticles accumulate in cells in the cancer tissue of the subject; And iii) imaging the cancer tissue by irradiating near-infrared to identify the site of cancer tissue of the subject; And iv) analyzing the image data to determine the exact location of the cancer tissue.

Images