Nanoparticles capable of charge conversion and medical use thereof

Abstract

The present invention relates to nanoparticles capable of charge conversion and medical use thereof. More specifically, provided are: nanoparticles formed by self-assembling a compound in which a photosensitizer is encapsulated in a cyclodextrin derivative conjugated with a zwitterionic near-infrared fluorophore; and a photodynamic or photodynamic / chemotherapeutic composition thereof for diagnosing or treating cancer, thereby enhancing anticancer efficacy through synergistic effects thereof.

Description

Charge-switchable nanoparticles and their medical uses

[0001] The present invention relates to charge-switchable nanoparticles and their medical uses.

[0002] Solid tumors, such as colorectal cancer, which includes colon and rectal cancer, possess a tumor microenvironment characterized by a dense extracellular matrix (ECM) and high interstitial fluid pressure. This tumor microenvironment hinders the penetration and homogeneous distribution of anticancer drug-delivering nanoparticles (NPs) deep into the tumor, which is known to be a major factor in inhibiting their anticancer efficacy. Therefore, enhancing the tumor penetration of anticancer nanoparticles is crucial for enhancing the anticancer efficacy of anticancer therapeutics, and various nanoparticle design strategies are continuously being studied to achieve this goal.

[0003] Among these, the tumor penetration strategy utilizing transcytosis of positively charged nanoparticles has recently attracted attention as an effective strategy for increasing tumor penetration of nanoparticles because it can bypass the dense extracellular matrix and high interstitial fluid pressure. While a positive surface charge has the advantage of enhancing the tumor penetration ability of nanoparticles, it has a fatal drawback in that it has a stronger nonspecific interaction with biomolecules than a negative or neutral surface charge, which causes particle accumulation in normal tissues. Therefore, it is desirable for the surface charge of nanoparticles to remain negative or neutral during blood circulation, while selectively converting to a positive charge inside the tumor.

[0004] To achieve selective charge conversion of nanoparticles, previous studies have developed nanoparticles whose structures change in response to tumor-specific conditions such as low pH, specific enzymes, and hypoxia. However, these strategies, which utilize the tumor microenvironment as a stimulus for charge conversion, suffer from problems such as low tumor selectivity, slow charge conversion rates, and non-uniform charge conversion within tumors.

[0005] The purpose of the present invention is to provide a new charge-convertible material that can overcome the problems of existing charge-converting strategies.

[0006] Another object of the present invention is to provide an anticancer therapeutic material and treatment method having improved tumor penetration ability and enhanced anticancer therapeutic efficacy by using the novel charge conversion material described above.

[0007] In order to achieve the above object, the present invention provides a cyclodextrin derivative represented by the following chemical formula 1, to which a heptamethine cyanine-based zwitterionic near-infrared fluorophore is bound:

[0008] <Chemical Formula 1>

[0009]

[0010] In the above formula, X and Y may be the same or different, and ASO3 - , ACO2 - , or a salt thereof, wherein A is aromatic or (CH2) a is selected from the alkyl corresponding to , a is selected from an integer from 1 to 8, m is selected from an integer from 6 to 8, and n can be selected from an integer from 1 to 40.

[0011] The present invention provides a cyclodextrin derivative represented by the following chemical formula 2, wherein a heptamethine cyanine-based zwitterionic near-infrared fluorophore and a tumor-targeting moiety are bound to cyclodextrin:

[0012] <Chemical Formula 2>

[0013]

[0014] In the above formula, X and Y may be the same or different, and ASO3 - , ACO2 - , or a salt thereof, wherein A is aromatic or (CH2) a is selected from alkyl corresponding to, a is selected from an integer from 1 to 8, R is a tumor targeting moiety, selected from compounds having a molecular weight of less than 10,000 Da used for active targeting, m is selected from an integer from 6 to 8, and n can be selected from an integer from 1 to 40.

[0015] The present invention provides a compound in which a photosensitizer is incorporated into the cyclodextrin derivative according to the above.

[0016] The present invention provides nanoparticles formed by self-assembly of the above-described encapsulated compound.

[0017] The present invention provides a composition for photodynamic therapy of cancer, comprising the above nanoparticles as an active ingredient.

[0018] The present invention provides a composition for combined photodynamic and chemotherapy treatment of cancer, comprising the above nanoparticles as an active ingredient.

[0019] In addition, the present invention provides a composition for near-infrared fluorescence imaging for cancer diagnosis, which comprises the above nanoparticles as an active ingredient.

[0020] The novel cyclodextrin derivative according to the present invention has excellent tumor targeting ability by introducing a tumor targeting moiety, and can improve tumor penetration ability by introducing a zwitterionic fluorescent dye as a new charge conversion strategy and converting it to a positive charge through near-infrared irradiation.

[0021] Nanoparticles formed by self-assembling a compound in which a photosensitizer is incorporated into a cyclodextrin derivative according to the present invention under aqueous conditions have improved tumor targeting ability and tumor penetration ability, and in addition, can enhance anticancer efficacy through the synergistic effect of photodynamic and chemodynamic activities by the photosensitizer.

[0022] Figure 1 is a schematic representation of the design and mechanism of novel nanoparticles (P-ZWNIR NPs) for tumor targeting and penetration. (A) shows the chemical structures of cyclodextrin derivatives (P-ZWNIR CD) conjugated with phenylboronic acid (PBA) and zwitterionic near-infrared fluorophores and pheophorbide (Pheo) modified with ferrocene (Fc) (Pheo-Fc), and the photobleaching-mediated charge transfer mechanism of P-ZWNIR NPs, and (B) schematically shows the delivery cascade and phototherapy regimen of P-ZWNIR NPs.

[0023] Figure 2 is an analysis of compound 6 synthesized according to reaction scheme 1, (A) is compound 6 1 H-NMR spectrum, (B) is the process for preparing a complex of compound 6 and 2,5-dihydroxybenzoic acid (DHB) for MS analysis, and (C) is the MALDI-TOF spectrum of the complex prepared according to (B).

[0024] Figure 3 shows the analysis of compound 7 (P-ZWNIR NP), (A) is the P-ZWNIR CD in DMSO-d6 / D2O (90:10, v / v). 1H NMR spectrum, (B) and (C) are standard calibration curves for quantifying the PBA moiety and the near-infrared fluorophore moiety, respectively, using 3-acetamidoPBA and IR-78 as standards for quantification. (D) compares the conjugated number of the near-infrared fluorophore moiety in P-ZWNIR CD with the 7-conjugated PBA moiety, and the ratio of PBA to the near-infrared fluorophore is 1.01, confirming that the near-infrared fluorophore moiety is also 7-conjugated.

[0025] Figure 4 shows the analysis of compound 10 (Pheo-Fc), (A) 1 (B) is the H NMR spectrum and (B) is the electrospray ionization-mass spectrometry (ESI-MS) spectrum.

[0026] Figure 5 shows the analysis of the characteristics of P-ZWNIR NPs. (A) shows the photo-oxidative degradation mechanism of heptamethine cyanine using Gly-S-IR783 (a heptamethine cyanine fluorophore model), (B) is a schematic diagram of the photobleaching-mediated charge conversion of P-ZWNIR NPs, and (C) shows the near-infrared laser irradiation (808 nm, 0.8 W cm) of the P-ZWNIR NP suspension. -2 , 5 min), digital images before and after laser irradiation (D), UV-vis spectra of P-ZWNIR NPs in phosphate buffer solution (PBS, pH 7.4) before and after laser irradiation, (E) shows the zeta potential change of P-ZWNIR NPs under laser irradiation, and the data are expressed as the mean ± standard deviation (SD) (n = 3). (F) shows the size distribution profile before and after near-infrared laser irradiation, and (G) shows the transmission electron microscope (TEM) image of P-ZWNIR NPs. (H) schematically shows the photodynamic and chemodynamic effects of Pheo-Fc, and (I) shows the 660 nm laser irradiation (350 mW, 2 mW cm -2 ) singlet oxygen of Pheo-Fc ( 1O2) generating ability, (J) shows the hydroxyl radical (·OH) generating ability of Pheo-Fc (500 μM) in PBS / DMSO co-solvent (9:1, v / v), and shows UV-Vis spectra of various mixtures, and (K) shows the total reactive oxygen species (ROS) generating ability of Pheo-Fc (4 μM Pheo-Fc in PBS) and P-ZWNIR NPs under 660 nm laser irradiation.

[0027] Figure 6 shows the results of an in vitro cytotoxicity test, where (A) and (B) show the cell viability of Pheo-Fc and P-ZWNIR NPs in HT-29 cells under dark and 660 nm laser irradiation conditions, respectively, and (C) shows the cell viability of blank P-ZWNIR NPs according to the incubation time.

[0028] Figure 7 shows (A) flow cytometry results and (B) laser scanning confocal microscopy (LSCM) images of HT-29 cells treated with P-ZWNIR NPs and 2′,7′-dichlorofluorescein diacetate (DCFH-DA), respectively.

[0029] Figure 8 shows the results of (A) H2O2 production in HT-29 and NIH3T3 cells and (B) cell viability for dark toxicity of P-ZWNIR NPs.

[0030] Figure 9 is about ZWNIR CD and ZWNIR NP as negative control, (A) is a schematic diagram showing the synthesis process of ZWNIR CD, (B) is a schematic diagram showing the synthesis process of ZWNIR CD 1 H-NMR spectrum (DMSO-d6 / D2O (90:10, v / v)), (C) was obtained using an 808 nm laser (0.8 W cm -2 , 5 min) before and after irradiation, the hydrodynamic diameter and (D) zeta potential of ZWNIR NPs are shown, and the data are expressed as mean ± SD (n = 3).

[0031] Figure 10 shows the in vitro cellular uptake, ROS generation, transcytosis and penetration properties of P-ZWNIR NPs, Pheo-Fc, ZWNIR NPs, ZWNIR NPs + 808 nm (laser pre-treated ZWNIR NPs, 0.8 W cm -2 , 5 min), P-ZWNIR NP, and P-ZWNIR NP + 808 nm (laser-pretreated P-ZWNIR NP, 0.8 W cm -2 , 5 min) and cultured for 4 h. (A) Flow cytometry analysis and (B) LSCM images of HT-29 cells. The data of flow cytometry analysis are presented as mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001), and the scale bar of the LSCM image is 250 μm. (C) is a schematic diagram of the transcytosis experiment using the Transwell® system (Scale bar = 250 μm), and (D) is the result of the tumor penetration experiment of P-ZWNIR NPs in the HT-29 multicellular tumor spheroid (MTS) model (Scale bar = 100 μm). (E) shows the fluorescence intensity at a depth of 150 μm plotted as a function of distance from the left edge of the spheroid, (F) is a representative MTS image from the antitumor efficacy study in the HT-29 MTS model, and (G) is the growth profile of the corresponding MTS after laser treatment on day 0, and data are presented as mean ± SD (n = 8, **p < 0.01, and ****p < 0.0001).

[0032] Figure 11 is about the photothermal effect of P-ZWNIR NP, (A) is a schematic diagram of near-infrared (808 nm) light-mediated photobleaching and photothermal effect, (B) is an 808 nm laser (0.8 W cm -2 ) As a result of the temperature increase of P-ZWNIR NPs at various concentrations under investigation, P-ZWNIR NPs showed dose-dependent hyperthermia due to the photothermal effect.

[0033] Figure 12 shows the analysis of the tumor targeting and penetration potential of P-ZWNIR NPs. (A) In vivo real-time near-infrared fluorescence (NIRF) imaging after intravenous injection of ZWNIR NPs or P-ZWNIR NPs into orthotopic (rectal) HT-29 tumor-bearing mice (white dotted circle indicates rectal tumor area), (B) temporal changes in the average radiant efficiency in the tumor area, (C) ex vivo NIRF radiant efficiency of the resected tumors and major organs from HT-29 rectal tumor-bearing mice at 8 hours post-injection, data are presented as mean ± SD (n = 3) [He: heart, Lu: lung, Li: liver, Sp: spleen, Ki: kidney, Tu: tumor]. (D) Ex vivo NIRF images of HT-29 tumors located in the rectum (Rec), muscle (Mu), small intestine (SI), and large intestine (LI), (E) tumor-to-background ratio (TBR) of ZWNIR NPs and P-ZWNIR NPs, data are presented as mean ± SD (n = 3). (F) In vivo NIRF images of rectal tumors before and after 808 nm laser irradiation, (G) matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) of dissected rectal tumor tissues before and after 808 nm laser irradiation, scale bar length 2 mm. (H) Schematic diagram of in vivo tumor penetration study using two-photon microscopy, (I) two-photon microscopy images of P-ZWNIR NPs in tumor tissue before and after 808 nm laser irradiation, and (J) plots the fluorescence intensity as a function of distance from blood vessels, and the fluorescence intensity values ​​of each image were normalized by the fluorescence intensity of blood vessels at 5 μm before and after laser irradiation, respectively.

[0034] Figure 13 shows P-ZWNIR NP-assisted combined phototherapy in a rectal cancer (RC) mouse model. (A) is a schematic diagram of P-ZWNIR NP-assisted phototherapy, (B) is the tumor growth profile of subcutaneous HT-29 tumor-bearing mice after various interventions, and the data are presented as the mean ± SD (n = 6). (C) is the excised tumor weight and (D) the tumor image on day 20, (E) is the tumor growth profile of HT-29 subcutaneous tumor-bearing mice after various laser treatment regimens, and (F) is the excised tumor weight and (G) the tumor image on the last day of the experiment. (H) is the bioluminescence image during the experimental period after various interventions, (I) is the bioluminescence intensity and (J) is the weight profile of mice after each intervention, and the data are presented as the mean ± SD (n = 6). (K) Ex vivo bioluminescence images at day 20 and (L) corresponding bioluminescence intensities for each group, data are presented as mean ± SD (n = 6) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

[0035] Hereinafter, the present invention will be described in detail.

[0036]

[0037] The inventor of the present invention, as a method to overcome the problems of the existing charge conversion strategy, took advantage of the fact that heptamethine cyanine, a near-infrared fluorescent dye, undergoes asymmetric structural decomposition when photobleaching occurs by a near-infrared laser, and modified a cyclodextrin derivative with heptamethine cyanine in a zwitterionic state and confirmed that the charge of the material was converted to a positive charge when photobleaching occurred by irradiating the cyclodextrin derivative with a near-infrared laser, thereby completing the present invention.

[0038]

[0039] The present invention provides a novel cyclodextrin derivative.

[0040] The cyclodextrin derivative according to the present invention may have a zwitterionic near-infrared fluorescent moiety bound thereto.

[0041] Preferably, a cyclodextrin derivative represented by the following chemical formula 1, to which a heptamethine cyanine-based zwitterionic near-infrared fluorophore using heptamethine cyanine as a target, is bound, can be provided:

[0042] <Chemical Formula 1>

[0043]

[0044] In the above formula, X and Y may be the same or different, and ASO3 - , ACO2 - , or a salt thereof, wherein A is aromatic or (CH2) a is selected from the alkyl corresponding to , a is selected from an integer from 1 to 8, m is selected from an integer from 6 to 8, and n can be selected from an integer from 1 to 40.

[0045] Specifically, the heptamethine cyanine-based zwitterionic near-infrared fluorophore may include, but is not limited to, a heptamethine cyanine-based dye selected from IR-783, IR-780, MHI-148, IR-820, or IR-825.

[0046] The above cyclodextrin is a circularly bonded cyclic substance, and may be selected from cyclodextrins in which m is 6 to 8, and preferably may be β-cyclodextrin in which m is 7, but is not limited thereto.

[0047] More preferably, it may be a cyclodextrin derivative represented by the following chemical formula 1-1:

[0048] <Chemical Formula 1-1>

[0049]

[0050] The cyclodextrin derivative containing the above zwitterionic near-infrared fluorophore can be converted to a positive charge by photobleaching upon near-infrared laser irradiation.

[0051]

[0052] The cyclodextrin derivative according to the present invention may be in the form of a zwitterionic near-infrared fluorescent moiety and a tumor-targeting moiety bound to cyclodextrin.

[0053] Preferably, the present invention provides a cyclodextrin derivative represented by the following chemical formula 2, wherein a heptamethine cyanine-based zwitterionic near-infrared fluorophore and a tumor-targeting moiety are bound to cyclodextrin:

[0054] <Chemical Formula 2>

[0055]

[0056] In the above formula, X and Y may be the same or different, and ASO3 - , ACO2 - , or a salt thereof, wherein A is aromatic or (CH2) a is selected from alkyl corresponding to, a is selected from an integer from 1 to 8, R is a tumor targeting moiety, selected from compounds having a molecular weight of less than 10,000 Da used for active targeting, m is selected from an integer from 6 to 8, and n can be selected from an integer from 1 to 40.

[0057] The above-mentioned zwitterionic near-infrared fluorescent moiety can be converted to a positive charge by photobleaching upon near-infrared laser irradiation, and the above-mentioned tumor-targeting moiety can have tumor-targeting ability.

[0058] Specifically, the heptamethine cyanine-based zwitterionic near-infrared fluorophore may include, but is not limited to, a heptamethine cyanine-based dye selected from IR-783, IR-780, MHI-148, IR-820, or IR-825.

[0059] According to one embodiment of the present invention, the heptamethine cyanine-based zwitterionic near-infrared fluorophore may be a heptamethine cyanine dye converted into a zwitterionic state having both positive and negative charges through a ring-opening reaction with 2-iminothiolane.

[0060] Specifically, the tumor targeting moiety may be at least one compound selected from the group consisting of phenylboronic acid (PBA), peptides containing Arginine-Glycine-Aspartate (RGD), IL-4 receptor targeting peptide (IL4RPep-1), biotin, folate, glucose, galactose, mannose, fructose, sorbitol, N-acetylglucosamine, N-acetylmannosamine, adenosine, thiamine, riboflavin, methotrexate, anisamide, and anacardic acid, but is not limited thereto. It is not.

[0061] The above phenylboronic acid has binding affinity to sialic acid overexpressed in tumor cells, and can target tumor cells.

[0062] The above cyclodextrin may be selected from cyclodextrins in which m is 6 to 8, and preferably may be β-cyclodextrin in which m is 7, but is not limited thereto.

[0063] More preferably, it may be a cyclodextrin derivative represented by the following chemical formula 2-1:

[0064] <Chemical Formula 2-1>

[0065]

[0066]

[0067] The above near-infrared fluorophore and tumor-targeting moiety can be coupled to cyclodextrin via a linker.

[0068] The above linker may include, but is not limited to, glutamic acid.

[0069] The above cyclodextrin derivative may be one in which a tumor-targeting moiety and a zwitterionic near-infrared fluorophore are conjugated to each terminal of the pegylated cyclodextrin derivative at a molar ratio of 1:1, and preferably, may be one in which each terminal of the β-cyclodextrin is heptconjugated, but is not limited thereto.

[0070] Preferably, the cyclodextrin derivative can be provided with charge conversion function and tumor targeting ability by simultaneously introducing zwitterionic heptamethine cyanine and phenylboronic acid.

[0071]

[0072] The present invention provides a compound in which a photosensitizer is incorporated into the novel cyclodextrin derivative.

[0073] The above cyclodextrin derivative is a cyclic substance bonded in a circular shape, and the photosensitizer can be incorporated into the circular shape.

[0074] The photosensitizer may be selected from pheophorbide or a derivative thereof, and preferably, may be pheophorbide a to which ferrocene is bound, but is not limited thereto.

[0075] More preferably, the ferrocene-bound pheophorbide a can be represented by the following chemical formula 3:

[0076] <Chemical Formula 3>

[0077]

[0078]

[0079] The above ferrocene has a high affinity for the cyclodextrin cavity, so it can enhance the inclusion effect for cyclodextrin, and at the same time, it can have a chemodynamic effect, so it can increase cytotoxicity by converting H2O2 generated by photodynamic therapy using a photosensitizer into hydroxyl radicals.

[0080] The above compound may be one in which ferrocene is bound in a host-guest form within the cyclodextrin.

[0081] The above compound may be a cyclodextrin derivative in which the photosensitizer is incorporated at a molar ratio of 1:1.

[0082] The above compound can be utilized in cancer treatment using the synergistic effect of photodynamics or photodynamics and chemodynamics or the combined therapy of photodynamics and chemotherapy by introducing the above photosensitizer.

[0083]

[0084] The present invention provides nanoparticles formed by self-assembly of a compound in which a photosensitizer is incorporated into the novel cyclodextrin derivative.

[0085] The above compound has amphiphilic properties, in which a hydrophobic photosensitizer is entrapped in a hydrophilic cyclodextrin derivative, and can spontaneously self-assemble under aqueous conditions to form nanoparticles.

[0086] The above nanoparticles may have a zwitterionic charge, but may be converted to a positive charge by photobleaching upon near-infrared irradiation.

[0087] The above near-infrared rays have a wavelength of 600 to 900 nm and a power of 0.5 to 4 mW cm -2 It can be investigated in a range of strengths, but is not limited thereto.

[0088] The positively charged nanoparticles converted by near-infrared irradiation can promote cellular uptake and enhance tumor penetration through the transcytosis pathway. The photothermal effect of near-infrared irradiation can also contribute to enhanced tumor penetration.

[0089]

[0090] The present invention provides a composition for photodynamic therapy of cancer, comprising the above nanoparticles as an active ingredient.

[0091] The above nanoparticles can be converted to positive charges through near-infrared irradiation (first near-infrared irradiation), thereby enhancing tumor targeting ability and tumor penetration ability, and can be utilized for photodynamic therapy of cancer by activating a photosensitizer through additional near-infrared irradiation (second near-infrared irradiation). In particular, the efficiency of photodynamic therapy can be further enhanced as the tumor penetration ability is enhanced.

[0092] The above first near-infrared ray has a wavelength of 800 to 900 nm and 0.5 to 1 mW cm -2 The intensity range can be, the second near-infrared has a wavelength of 600 to 700 nm and an intensity of 1 to 3 mW cm -2 It can be selected from a range of intensities, but is not limited thereto.

[0093] The cancer may be selected from solid cancers such as colon cancer, liver cancer, and lung cancer, and may preferably be colon cancer including rectal cancer and squamous cell carcinoma, but is not limited thereto.

[0094]

[0095] The present invention provides a composition for combined treatment of cancer with photodynamic and chemotherapy, comprising the above nanoparticles as an active ingredient.

[0096] The photosensitizer bound to ferrocene constituting the above nanoparticles has excellent photodynamic and chemodynamic activities, and the tumor penetration ability is improved by the nanoparticles converted to positive charges through near-infrared irradiation, and then the photosensitizer bound to ferrocene is activated through additional near-infrared irradiation, thereby enhancing the cancer treatment efficiency through the synergistic effect of photodynamic and chemodynamics.

[0097]

[0098] The present invention provides a composition for near-infrared fluorescence imaging for cancer diagnosis or treatment, comprising the above nanoparticles as an active ingredient.

[0099] The above composition can be used as a composition for optical imaging to determine the extent of cancer diagnosis and cancer treatment, as it can confirm the extent of tumor cell distribution in a living body and the tumor cell targeting of drugs using near-infrared fluorescence imaging.

[0100]

[0101] In addition, the present invention provides a method for treating cancer, comprising the steps of: administering the nanoparticles or a cancer treatment composition containing the nanoparticles to a subject; irradiating a first near-infrared ray; and irradiating a second near-infrared ray.

[0102] In the above treatment method, the step of administering the nanoparticles or the composition to a subject may be administered to all subjects requiring cancer treatment, or to all subjects excluding humans, and the nanoparticles may accumulate at a tumor site in the body. The administration may be performed in the form of intravenous injection or intratumoral injection.

[0103] The step of irradiating the first near-infrared ray is performed at a wavelength of 800 to 900 nm and 0.5 to 1 mW cm -2 By irradiating near-infrared light in the intensity range, the nanoparticles accumulated in the tumor area can be converted to positive charges by photobleaching and penetrate into the tumor.

[0104] The step of irradiating the second near-infrared ray has a wavelength of 600 to 700 nm and a power of 1 to 3 mW cm -2 By irradiating near-infrared light in the intensity range, cancer can be treated by exhibiting anticancer efficacy through photodynamic or photodynamic / chemodynamic activity by photosensitizers of nanoparticles that have penetrated inside the tumor.

[0105] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0106]

[0107] <Experimental substance>

[0108] O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate [O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HBTU], 1-Hydroxybenzotriazole hydrate [HOBt], 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC], trifluoroacetic acid [TFA], triethylamine [TEA], N,N-Diisopropylethylamine [DIEPA], IR-783, 2-iminothiolane hydrochloride (Traut's reagent), ferrocenecarboxylic acid, anhydrous N,N-dimethylformamide (DMF), anhydrous dimethylsulfoxide (DMSO), 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA), and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).3-Aminophenylboronic acid (3-APBA), N-Boc-ethylenediamine, 3,3',5,5'-tetramethylbenzidine (TMB), and 2,5-dihydroxybenzoic acid (DHB) were purchased from Tokyo Chemical (Tokyo, Japan). Boc-L-glutamic acid α-methyl ester was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and T-boc-N-amido-PEG8-amine was purchased from Broadpharm (San Diego, CA, USA). Heptakis-(6-amino-6-deoxy)-β-cyclodextrin heptahydrochloride was purchased from Cyclodextrin-shop (Tilburg, Netherlands), and pheophorbide a was purchased from Frontier Scientific (Newark, DE, USA).

[0109] NMR solvents including deuterium oxide (D2O), dimethyl sulfoxide-d6 (DMSO-d6), chloroform-d (CDCl3) and methanol-d4 (CD3OD) were purchased from Zeochem AG (R ti, Switzerland). Dialysis membrane (Spectra / Por® 7 Dialysis Tubing, MWCO 1000 Da) was purchased from Repligen Corporation (Rancho Dominguez, CA, USA). RPMI 1640 and phosphate-buffered saline (PBS) were purchased from Corning (Manassas, VA, USA). Penicillin-streptomycin solution (100X), fetal bovine serum (FBS), and trypsin solution (10X) were purchased from Welgene (Gyeongsangbuk-do, Republic of Korea). D-luciferin was purchased from Perkin Elmer (Boston, MA, USA). All other reagents were of analytical grade.

[0110] HT-29 (human colorectal adenocarcinoma) and NIH3T3 cell lines (fibroblasts) were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). A 660 nm laser (CYL20ADJ) was purchased from Laser Solution Korea (Gyeonggi-do, Republic of Korea), and a continuous wave near-infrared (808 nm) diode laser beam was purchased from CNILaser (Changchun, China).

[0111]

[0112] <Example 1> Design of phenylboronic acid and zwitterionic near-infrared fluorophore-modified nanoparticles (P-ZWNIR NPs)

[0113] Phenylboronic acid (PBA) and zwitterionic near-infrared (NIR) fluorophore-modified nanoparticles (P-ZWNIR NPs) are composed of an inclusion compound formed by a hydrophilic β-cyclodextrin (CD) derivative (host) moiety and a hydrophobic photosensitizer (guest) moiety (Fig. 1A). Given that only organic photosensitizers are currently used clinically, we designed a nanomaterial capable of organic charge conversion using cyclodextrin as the core material.

[0114] Proton and carbon NMR analyses were performed using a JNM-ECZ400s (JEOL, Tokyo, Japan), and the molecular weights of the synthesized small-molecule compounds were measured by an electrospray ionization-mass spectrometer (Agilent Technologies 6430 Triple Quad LC / ESI-MS system; Agilent Technologies, Santa Clara, CA, USA). The molecular weights of the synthesized cyclodextrin derivatives were determined by a Voyager-DE STR Biospectrometry Workstation (matrix-assisted laser desorption / ionization time-of-flight MS; Applied Biosystems Inc., Waltham, MA, USA). The compounds were matrixed with 2,5-dihydroxybenzoic acid (DHB).

[0115]

[0116] 1. Synthesis of cyclodextrin derivatives

[0117] Cyclodextrin derivatives conjugated with phenylboronic acid and a zwitterionic near-infrared fluorophore (P-ZWNIR CD) were synthesized by tethering phenylboronic acid and a heptamethine cyanine fluorophore to the cyclodextrin core using glutamic acid as a tri-arm linker. Phenylboronic acid was used to target sialic acid-overexpressing cancer cells, and the heptamethine cyanine fluorophore served as a charge-switching moiety.

[0118]

[0119] The following reaction scheme 1 shows the specific synthesis process of P-ZWNIR CD.

[0120] <Reaction Scheme 1>

[0121]

[0122] In the above reaction scheme 1, the synthesis conditions are as follows:

[0123] a) HATU, DMF, DIPEA, RT, overnight; b) LiOH in MeOH / DDW (50:50, v / v), RT, 4 h; c) HBTU, DMF / DMSO, DIPEA, RT, 48 h; d) TFA / ACN (50:50, v / v), RT, 2 h; e) DMSO, TEA, 24 h.

[0124]

[0125] Referring to the above reaction scheme 1, the PBA moiety (compound 2) was first introduced into the PEGylated CD (compound 4) to obtain compound 5, which was then deprotected under acidic conditions to obtain compound 6. The heptaconjugation of the PBA moiety of compound 6 is 1This can be confirmed through H-NMR and MALDI-TOF MS analysis (Fig. 2). Next, a heptamethine cyanine (IR-783) moiety was introduced into compound 6. In order for the nanomaterial to be converted from a zwitterionic state to a positively charged state through photobleaching, it is important that the heptamethine cyanine moiety is linked to the cyclodextrin structure as a zwitterion. Therefore, compound 6 was synthesized by conjugating IR-783 with 2-iminothiolane, a Traut reagent. 2-iminothiolane was used as a linker to convert the negatively charged IR-783 into a zwitterionic heptamethine fluorophore (ZWNIR) by generating a positive charge at the amidine bond through a ring-opening reaction.

[0126] Briefly, compound 6 (0.0034 mmol) and IR-783 (0.0714 mmol) were dissolved in DMSO (3 mL), and then TEA (0.1666 mmol) was added with stirring. DMSO (0.0238 mmol mL -1 ) was slowly added dropwise to the reaction mixture. After stirring for 24 h, the reaction mixture was transferred to a dialysis bag (MWCO: 1000 Da; Cellu Sep, Membrane Filtration Products, Seguin, TX, USA) and dialyzed against MeOH / DDW (1:1, v / v) for 48 h. The resulting compound 7 was lyophilized and stored at -20°C for further experiments. Meanwhile, for ZWNIR CD, compound 4 was conjugated to IR-783 using 2-iminothiolane using the same procedure described above.

[0127] Sequential conjugation of PBA and ZWNIR moieties onto the PEGylated CD core yielded P-ZWNIR CD (compound 7). Referring to Figure 3, the degree of ZWNIR moiety conjugation in compound 6 was calculated by comparing the integrated values ​​of the anomeric protons of CD (~ 4.7 ppm) and the distinct signal of IR-783 (~ 6.2 ppm) in the P-ZWNIR CD, and NMR analysis showed that the degree of ZWNIR moiety conjugation in the P-ZWNIR CD was about 7. The molar ratio of ZWNIR moiety to PBA moiety in the P-ZWNIR CD was determined to be about 1:1 by UV-Vis spectroscopy using alizarin red S, which also indicates heptaconjugation of ZWNIR.

[0128]

[0129] 2. Synthesis of photosensitizers

[0130] Pheophorbide a (Pheo) was selected as a phototherapy agent and modified with ferrocene (Fc) to take advantage of the synergistic effect of photodynamic / chemodynamic therapy and the binding affinity of Fc for CD cavities.

[0131] The following reaction scheme 2 shows the specific synthetic process of ferrocene-bonded pheophorbide a (Pheo-Fc).

[0132] <Reaction Formula 2>

[0133]

[0134] In the above reaction scheme 2, the synthesis conditions are as follows:

[0135] a) HOBt, EDC, DIPEA in anhydrous DMF, RT, N2, 24 h; b) TFA / DCM (40:60), RT, 2 h. c) EDC, HOBt, DIPEA in anhydrous DMF, RT, N2, 48 h.

[0136]

[0137] Specifically, compound 8 was synthesized according to a previously reported method. Ferrocene carboxylic acid (250 mg, 1.08 mmol), HOBt (175 mg, 1.30 mmol), and EDC (225 mg, 1.30 mmol) were dissolved in anhydrous DMF (10 mL), followed by the addition of DIPEA (0.84 mL, 5.40 mmol). After stirring at room temperature for 30 min under a nitrogen atmosphere, N-Boc-ethylenediamine (0.34 g, 2.17 mmol) was added and stirred at room temperature for 12 h. The reaction mixture was diluted with double-distilled water (DDW) and extracted with ethyl acetate (EtOAc) (3 × 50 mL). The EtOAc layer was dried over anhydrous sodium sulfate (Na2SO4) and concentrated in vacuo to obtain the crude product. Compound 8 (yield 66%) was obtained by purification by flash column chromatography using hexane: EtOAc (1:3).

[0138] 1 H NMR (400 MHz, CDCl3)δ6.62 (s, 1H), 5.09 (s, 1H), 4.72 - 4.67 (m, 2H), 4.34 - 4.32 (m, 2H), 4.21 (dd,J= 3.9, 2.1 Hz, 2H), 4.20 (s, 3H), 3.51 - 3.46 (m, 2H), 3.40 - 3.33 (m, 2H), 1.45 (s, 9H). 13 C NMR (100 MHz, CDCl3)δ171.18, 79.94, 75.96, 70.52, 69.86, 68.23, 41.20, 41.11, 40.72, 28.51.

[0139] Subsequently, Boc-deprotection of compound 8 was carried out at room temperature for 2 h using 50% TFA in DCM. After the reaction, the reaction solvent was evaporated in vacuo, and the crude product was further purified by flash column chromatography using EtOAc:MeOH to obtain compound 9 (yield 83%).

[0140] 1 H NMR (400 MHz, CD3OD)δ4.83 - 4.76 (m, 2H), 4.44 (t,J= 1.9 Hz, 2H), 4.21 (s, 5H), 4.10 (q,J= 7.1 Hz, 1H), 3.58 (t,J= 6.1 Hz, 2H), 3.11 (t,J= 6.1 Hz, 2H), 2.00 (d,J= 10.6 Hz, 2H), 1.24 (t,J= 7.1 Hz, 2H).

[0141] Finally, to synthesize Pheo-Fc of compound 10, pheophorbide a (Pheo) (50 mg, 0.084 mg), HOBt (13.5 mg, 0.10 mmol), EDC (20 mg, 0.10 mmol), and DIPEA (74 μL, 0.425 mmol) were dissolved in anhydrous DMF (5 mL). The reaction mixture was stirred at room temperature for 30 min under a nitrogen atmosphere. Compound 9 (38.6 mg, 0.10 mmol) was dissolved in 2 mL of anhydrous DMF to which DIPEA (74 μL, 0.425 mmol) was added, and then added to the reaction mixture. The reaction was carried out at room temperature for 48 h. The crude product was obtained by concentration in vacuo, and purified by flash column chromatography using hexane: EtOAc (1:1, v / v) to obtain compound 10 (yield 80%).

[0142] 1H NMR (400 MHz, CDCl3)δ9.46 - 9.24 (m, 2H), 8.48 (d,J= 21.3 Hz, 1H), 8.07 - 7.85 (m, 1H), 6.55 (d,J= 6.9 Hz, 1H), 6.39 - 6.11 (m, 4H), 4.59 - 4.41 (m, 3H), 4.24 - 4.08 (m, 3H), 3.97 (s, 4H), 3.89 (s, 1H), 3.83 (s, 2H), 3.69 - 3.55 (m, 3H), 3.48 (s, 3H), 3.35 (d,J= 5.7 Hz, 4H), 3.31 - 3.13 (m, 7H), 2.96 - 2.85 (m, 1H), 2.72 - 2.55 (m, 1H), 2.51 - 2.27 (m, 2H), 2.14 - 1.98 (m, 1H), 1.77 (d,J= 7.2 Hz, 3H), 1.72 - 1.57 (m, 5H), 1.27 (h,J= 5.1 Hz, 1H). ESI-MS: m / z [M+H]+ found 847.6. (Figure 4).

[0143]

[0144] 3. Preparation of nanoparticles (P-ZWNIR NP)

[0145] The preparation of P-ZWNIR NPs consists of (1) formation of inclusion complexes and (2) self-assembly of the inclusion complexes in an aqueous medium.

[0146] ZWNIR NPs, which are synthesized by synthesizing ZWNIR CDs lacking PBA in the terminal structure for use as a negative control, are also manufactured by the same process.

[0147] Briefly, 15 mg of P-ZWNIR CD (0.013 mmol) or 13 mg of ZWNIR CD (0.013 mmol) was mixed with 1.12 mg of Pheo-Fc (0.013 mmol) in 0.5 mL of DMSO. The mixture of CD derivative and Pheo-Fc was vortexed overnight to form an inclusion complex. The formed inclusion complex was slowly dropped into 4 mL of DDW with constant stirring for 1 h. The resulting P-ZWNIR NP or ZWNIR NP solution was placed in a dialysis bag (MWCO 12-16 kDa, Membra-Cel, Viskase Corporation, Chicago, IL, USA) and dialyzed against DDW for 6 h. The DDW was replaced every hour to completely remove residual DMSO.

[0148] The concentration of Pheo-Fc in the NPs was measured by HPLC-Fluorescence (Ex / Em wavelength = 400 / 685 nm). The concentration of NIR dye in the NPs was analyzed by UV-Vis spectrometry (Orion AquaMate 8100, Thermo Fisher Scientific, Waltham, MA, USA) at 780 nm absorbance.

[0149] As shown in Fig. 1(A), Pheo-Fc was loaded onto P-ZWNIR CDs to form inclusion complexes by utilizing the host-guest interaction between the Fc and CD cavities. The P-ZWNIR CD / Pheo-Fc inclusion complex was prepared in dimethyl sulfoxide (DMSO) with a molar ratio of P-ZWNIR CD to Pheo-Fc of 1:1, and the prepared P-ZWNIR CD / Pheo-Fc solution was dropped into DDW and stirred to produce P-ZWNIR NPs by self-assembly. Since the outer ZWNIR structure of P-ZWNIR NPs can be asymmetrically decomposed by oxidative decomposition under NIR (~808 nm) laser irradiation, zwitterionic P-ZWNIR NPs can be converted to positively charged NPs after NIR laser irradiation.

[0150]

[0151] P-ZWNIR NPs manufactured in this way can be utilized as nanoparticles for intravenous injection.

[0152] Referring to Fig. 1(B), once P-ZWNIR NPs are administered intravenously, (1) they circulate in the bloodstream in a zwitterionic state and (2) progressively accumulate in tumor tissues through extravasation and active targeting. However, they penetrate only a short distance and remain primarily in the perivascular region. Upon irradiation of the tumor site with an 808 nm laser, (3) the NPs in the tumor are charge-converted to cationic NPs, which (4) enhances tumor penetration through transcytosis. Transcytosis-mediated deep tumor penetration of the NPs enables intracellular delivery and uniform distribution of the photosensitizer (PS), which, upon subsequent 660 nm laser irradiation, (5) will enhance the anticancer efficacy by combined photodynamic and chemodynamic therapy of pheophorbide (Pheo) and ferrocene (Fc), respectively.

[0153]

[0154] <Analysis Example 1> Characteristic Analysis of P-ZWNIR NPs

[0155] 1-1. Confirmation of the charge-conversion mechanism

[0156] To investigate the charge transfer mechanism, a near-infrared fluorophore model of Gly-S-IR783 was synthesized.

[0157] Glycine (5 mg, 0.067 mmol), 2-iminothiolane hydrochloride (9.2 mg, 0.067 mmol), and IR-783 (50 mg, 0.067 mmol) were dissolved in 1 mL MeOH and 20 mM aqueous sodium bicarbonate solution (pH 8.5) (1:1, v / v) and vortexed for 4 h at room temperature. The reaction mixture was loaded onto a Sep-Pak C18 20 cc Vac Cartridge (Waters Co., Milford, MA, USA) and purified by gradient elution. The mobile phase consisted of MeOH and DDW, and the gradient elution procedure was as follows: 70 mL MeOH:DDW (50:50, v / v) was used to remove hydrophilic impurities, and then the MeOH percentage was increased to 60% to obtain Gly-S-IR-783. The product was dried under reduced pressure (yield 56%). ESI-MS: m / z [M]+ found 866.5.

[0158] Gly-S-IR783 prepared by the above method was dissolved in PBS and exposed to a NIR (808 nm) laser (0.8 W cm -2) was irradiated for 5 min to induce photobleaching. The resulting solution was analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). As a result, laser irradiation induced the asymmetric cleavage of Gly-S-IR783, generating a combination of two ketone and aldehyde fragments (a + b or c + d) (Fig. 5A). It should be noted that both pathways left a cation at the amidine bond conjugation moiety. Since the amidine bond is connected to the cyclodextrin (CD) core in P-ZWNIR CD, only the cation moiety may remain after NIR laser irradiation of the NP (Fig. 1A).

[0159] Next, the photobleaching properties of P-ZWNIR NPs were investigated under the same irradiation conditions (Fig. 5B). The NIR laser bleached the ZWNIR portion of the P-ZWNIR NPs, transforming the green P-ZWNIR NPs into a dark yellow suspension with reduced light absorption and disappearance of light absorption at long wavelengths (Fig. 5C and D). This indicates cleavage of the heptamethine conjugation system due to photobleaching.

[0160] The average hydrodynamic diameter, size distribution, polydispersity index (PI), and zeta potential of the NPs were determined using a Zetasizer (Malvern Panalytical, Worcestershire, UK), and the morphology of the P-ZWNIR NPs before and after irradiation with an 808 nm laser was observed using a transmission electron microscope (TEM, JEM1010, JEOL, Tokyo, Japan) operating at an 80 kV accelerating voltage. Referring to Fig. 5(E), the zeta potential of the P-ZWNIR NPs gradually increased upon NIR laser irradiation and reached a plateau at 5 min, indicating that the photobleaching-mediated charge conversion of the NPs was completed within 5 min. P-ZWNIR NPs and cationic NPs (photobleached P-ZWNIR NPs) showed similar size distribution profiles in dynamic light scattering (DLS) analysis (Fig. 5F), and transmission electron microscopy (TEM) images also showed corresponding particle sizes (Fig. 5G).

[0161]

[0162] 1-2. Confirmation of the photodynamic and chemodynamic activities of Pheo-Fc

[0163] Next, the photodynamic and chemodynamic activities of Pheo-Fc were evaluated (Fig. 5H).

[0164] Singlet oxygen (singlet oxygen, 1 In the experiments on O2) and hydroxyl radical (·OH) generation, only Pheo-Fc was used without P-ZWNIR CD because P-ZWNIR CD exhibits strong absorbance in the wavelength range used for reactive oxygen species analysis, which interferes with the analysis. In the measurement of reactive oxygen species (ROS), DMSO / PBS co-solvent was used as a solvent because Pheo-Fc has low solubility in water.

[0165] 1O2 generating ability was analyzed by absorbance using 9,10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA) by dissolving Pheo-Fc (50 μM) and ABDA (100 μM) in PBS / DMSO co-solvent (50:50, v / v). Specifically, Pheo-Fc solution (100 μmol L in DMSO -1 , 1 mL) in ABDA solution (200 μmol L in PBS -1 , 1 mL) were mixed and irradiated with a 660 nm laser at an intensity of 350 mW from a distance of 2 cm. The absorption spectrum of the mixture was measured every 2 minutes in the range of 300 to 500 nm for 10 minutes using a UV-Vis spectrometer. Controls were performed with the same procedure using only Pheo-Fc solution or ABDA solution in a darkroom or under irradiation conditions. Referring to Fig. 5(I), the absorbance of ABDA rapidly decreased under 660 nm laser irradiation, suggesting a reaction with singlet oxygen generated from Pheo-Fc.

[0166] ·OH production from the ferrocene moiety of Pheo-Fc was evaluated using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. 500 μL Pheo-Fc solution (4 mmol L in DMSO -1) was mixed with 4 mL of test solution. The compositions of the test solutions are as follows: 1) TMB, H2O2, PBS, pH 5.0; 2) TMB, H2O2, PBS, pH 7.4; 3) TMB, PBS, pH 5.0; 4) H2O2, PBS, pH 5.0. The reaction mixture was incubated in a shaking bath at 37°C for 15 min, and the absorption spectrum of the mixture was measured in the range of 500 - 800 nm using a UV-Vis spectrometer. Colorless TMB is oxidized by ·OH generation from the reaction between Fc and H2O2 to form 3,3',5,5'-tetramethylbenzidine diamine as a blue product. Referring to Figure 5J, the ·OH production of Pheo-Fc (500 μM) in PBS / DMSO co-solvent (9:1, v / v) was prominent only in the presence of H2O2 at pH 5.0. This selective production of ·OH may translate into higher ·OH production within the acidic intracellular compartment of cancer cells.

[0167] The total ROS generating capacity of Pheo-Fc and P-ZWNIR NPs was compared using dichlorodihydrofluorescein (DCFH) as an indicator.

[0168] 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was hydrolyzed at room temperature under basic conditions (NaOH 10 mM) to generate DCFH. 8 μL DCFH solution (500 μM) in PBS was mixed with 8 μL Pheo-Fc solution or P-ZWNIR NPs (500 μM as Pheo-Fc), and then 984 μL PBS was added to the mixture. The final concentration of Pheo-Fc in the mixture was 4 μM. The mixture was irradiated with a 660 nm laser. The fluorescence intensity of the samples after laser treatment was measured with a FP-8350 Spectrofluorometer (λex / λem = 480 / 525 nm; JASCO Corp.), and the ROS production profiles of Pheo-Fc and P-ZWNIR NPs were expressed as a function of time and fluorescence intensity (I / I0-1). I0 and I represent the fluorescence intensities of the mixture at the initial time point and every 5 seconds for 1 minute, respectively. Referring to Fig. 5(K), upon laser irradiation, the fluorescence intensity of dichlorofluorescein (DCF) in P-ZWNIR NPs increased over time, whereas the fluorescence intensity of Pheo-Fc remained almost the same as in PBS. This difference may be mainly due to the low water solubility of Pheo-Fc and its solubilization by P-ZWNIR NPs.

[0169]

[0170] 1-3. Confirmation of photodynamic and chemodynamic activity in cells

[0171] Based on the high ROS-generating ability of P-ZWNIR NPs, the photodynamic / chemodynamic efficacy of P-ZWNIR NPs was further evaluated in HT-29 cells.

[0172] HT-29 cells were cultured in RPMI 1640 supplemented with 10% FBS (v / v) and 1% penicillin-streptomycin (v / v) at 37°C, 5% CO2, and 95% relative humidity. For cytotoxicity experiments, 5,000 cells were seeded in each well of a 96-well plate (SPL Life Sciences, Gyeonggi-do, Republic of Korea). After 24 h of incubation, the cells were treated with Pheo-Fc or P-ZWNIR NPs and incubated for 4 h. The complete medium was removed and replaced with fresh serum-free RPMI 1640 medium, and then the cells were exposed to a 660 nm laser (350 mW; 2 mW / cm 2 ) at a distance of 2 cm for 1 min per well, and then the cells were cultured for an additional 24 h. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS] reagent (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Milan, Italy) according to the manufacturer's instructions. The absorbance at 490 nm was measured with a microplate reader (Multiskan Microplate Spectrophotometer; Thermo Fisher Scientific, Massachusetts, USA). The in vitro cytotoxicity of blank P-ZWNIR CD was also evaluated similarly in HT-29 cells. HT-29 cells were treated with P-ZWNIR CD solution (0.2–100 μg mL -1 ) for 24, 48, and 72 hours, and cell viability was evaluated using the method described above.

[0173] Intracellular ROS detection was performed using DCFH-DA as a dye reagent. Intracellular esterase cleaves the diacetate group of DCFH-DA to generate DCFH, which can be converted to highly fluorescent fluorescein (DCF) upon exposure to ROS. HT-29 cells were stained with P-ZWNIR NPs (2 μmol mL -1 After culturing for 4 h in cell culture medium without phenol red with Pheo-Fc), the cells were washed three times with PBS, and then DCFH-DA solution was added (10 μmol mL in PBS). -1 ). After 15 minutes of incubation, the culture medium was replaced with cell culture medium without phenol red. In the laser treatment group, a 660 nm laser (350 mW) was irradiated for 5 minutes. The treated cells were analyzed using a flow cytometer (FACSCalibur, BD Biosciences). The flow cytometer measured the average fluorescence intensity of DCF converted within the cells. HT-29 cells treated in the same manner as above were observed using a confocal microscope (Leica TCS SP8), and fluorescence was measured at an excitation wavelength of 488 nm with a GFP filter.

[0174] H2O2 released from HT-29 or NIH3T3 cells was measured using the Amplex Red® Hydrogen Peroxide / Peroxidase assay kit (Thermo Fisher Scientific, Waltham, MA, US) according to the manufacturer's instructions. HT-29 and NIH3T3 cells were cultured in RPMI 1640 and DMEM medium, respectively, supplemented with 10% FBS (v / v) and 1% penicillin-streptomycin (v / v). For the H2O2 production assay, a reaction mixture containing 50 μM Amplex Red reagent and 0.1 U / mL horseradish peroxidase (HRP) in Krebs-Ringer phosphate (KRPG) buffer (pH 7.35) was prepared. Then, 100 μL of the reaction mixture was filled into each well of a 96-well microplate and incubated at 37°C for 10 min. 2 × 10 6 HT-29 or NIH3T3 cells (20 μL) were added to each well, and the amount of H2O2 produced was quantified by measuring the absorbance of the reaction mixture at 560 nm.

[0175] At the experimental concentrations, Pheo-Fc inhibited cancer cell growth under dark conditions and 660 nm laser irradiation conditions, with limited efficacy and similar between the two conditions (Fig. 6A). In contrast, P-ZWNIR NPs inhibited cancer cell growth under dark conditions and 660 nm laser irradiation conditions compared to those without Pheo-Fc (IC 50, 5.3 ± 0.1 and 3.5 ± 0.2 μM, respectively, under dark conditions and 660 nm illumination, compared to Pheo-Fc (Fig. 6B). The dark toxicity of P-ZWNIR NPs could be attributed mainly to ·OH generated from Pheo-Fc, as P-ZWNIR CDs showed negligible cytotoxicity against HT-29 cells at higher concentrations (Fig. 6C). The significant difference in efficacy between Pheo-Fc and P-ZWNIR NPs could be explained by the lower total ROS production of Pheo-Fc in PBS (Fig. 5K). The higher cytotoxicity observed for P-ZWNIR NPs (+ 660 nm) compared to P-ZWNIR NPs (dark) could be attributed to the activation of photodynamic agents by 660 nm laser irradiation (Fig. 7).

[0176] To confirm the H2O2-dependent dark toxicity and cancer cell-specific cytotoxicity of P-ZWNIR NPs, dark toxicity was compared between HT-29 cells (a cancer cell line) and NIH3T3 cells (a normal cell line). First, HT-29 cells showed significantly higher H2O2 production than NIH-3T3 cells (Fig. 8A). NIH3T3 cells, which have relatively low H2O2 production, showed lower dark toxicity of P-ZWNIR NPs compared to HT-29 cells, which may be due to their higher IC 50 The value (Pheo-Fc of 15.4 ± 0.5 μM) was demonstrated (Fig. 8B). These results suggest that the dark toxicity of P-ZWNIR NPs may be significantly higher in cancer cells than in normal cells, thereby minimizing the potential toxicity of P-ZWNIR NPs in normal tissues.

[0177]

[0178] <Analysis Example 2> Analysis of cellular uptake, transcytosis, penetration, and efficacy of P-ZWNIR NPs

[0179] Prior to cellular uptake studies, ZWNIR NPs were prepared by synthesizing ZWNIR CDs lacking PBA in the terminal structure and used as a negative control (Fig. 9). ZWNIR CDs were synthesized by conjugating NIR dyes to the PEGylated CD core using the same reaction used for the synthesis of P-ZWNIR CDs. The degree of NIR dye conjugation on ZWNIR CDs was determined by UV-vis spectroscopy using Alizarin Red S and 1 The value calculated from the H NMR spectrum was approximately 7, indicating heptaconjugation of the ZWNIR moiety within the CD core. ZWNIR NPs were prepared from the ZWNIR CD / Pheo-Fc inclusion complex, and the NPs also exhibited NIR photoresponsive charge-switching properties without significant changes in particle size distribution.

[0180]

[0181] 2-1. Analysis of cell uptake efficiency

[0182] The cellular uptake efficiency of P-ZWNIR NPs was evaluated in HT-29 cells using flow cytometry and laser scanning confocal microscopy (LSCM).

[0183] For flow cytometry analysis, 1×10 5 HT-29 cells were seeded in each well of a 6-well cell culture plate (SPL Life Sciences, Gyeonggi-do, Republic of Korea) and cultured at 37°C for 24 h. Then, Pheo-Fc, ZWNIR NPs, ZWNIR NPs + 808 nm (laser-pretreated ZWNIR NPs), P-ZWNIR NPs, and P-ZWNIR NPs + 808 nm (laser-pretreated P-ZWNIR NPs) (2 μM of Pheo-Fc) were treated to HT-29 cells and cultured for 4 h. Positively charged NPs were prepared by irradiating P-ZWNIR NPs with an 808 nm laser and were prepared before treatment to HT-29. Free sialic acid (500 μmol L) acting as a competitor for PBA-sialic acid interaction-1 ) or 3-aminophenylboronic acid (3-APBA, 500 μmol L -1 ) were incubated with the formulation for 15 min prior to cell treatment. Cells treated with nanoparticles were analyzed after 4 h of incubation. The mean fluorescence intensity of Pheo-Fc was analyzed and processed using FACSCalibur and CellQuest Pro Software (BD Biosciences, San Jose, CA, USA).

[0184] Referring to Fig. 10(A), the average fluorescence intensities of cells treated with P-ZWNIR NPs and P-ZWNIR NPs + 808 nm were significantly higher than those of cells treated with ZWNIR NPs and ZWNIR NPs + 808 nm, respectively. The cellular uptake of photobleached P-ZWNIR NPs (P-ZWNIR NPs + 808 nm) was inhibited in the presence of competitive inhibitors (free sialic acid or free 3-aminophenylboronic acid (3-APBA)). These results indicate that the PBA-sialic acid interaction promotes the cellular uptake of P-ZWNIR NPs and photobleached P-ZWNIR NPs. Photobleached ZWNIR NPs and P-ZWNIR NPs show higher cellular uptake than ZWNIR NPs and P-ZWNIR NPs themselves, indicating that cation transfer promotes the cellular uptake of the NPs.

[0185] For confocal microscopy analysis, 1×10 5HT-29 cells were seeded into each well of a 4-well cell culture plate (SPL Life Sciences, Gyeonggi-do, Republic of Korea), cultured at 37°C for 24 h, and then processed in the same manner as the flow cytometry described above. After washing three times with PBS (pH 7.4), the cells were fixed with 4% (w / v) formaldehyde solution, stained with 4′,6-diamidino-2-phenylindole (DAPI; ThemoFisher Scientific, Eugene, OR, USA), and mounted in Fluoroshield (Sigma-Alrich, Saint Louis, MO, USA). Fluorescence images were acquired using a Leica TCS SP8 confocal microscope and analyzed using the Leica Application Suite X (Leica Microsystems, Wetzlar, Germany). As a result, similar to the flow cytometry results, LSCM analysis showed a strong fluorescence signal in the P-ZWNIR NP + 808 nm group (Fig. 10B).

[0186]

[0187] 2-2. Confirmation of transcytosis

[0188] Next, the transcytosis ability of P-ZWNIR NPs was investigated in a nested Transwell® culture system (1 μm-diameter microporous polyester membrane; Corning, Manassas, VA, USA) by observing the fluorescence of Pheo-Fc.

[0189] 1×10 5HT-29 cells were seeded in 12-well cell culture plates without round coverslips (SPL Life Sciences, Gyeonggi-do, Republic of Korea) (Plate 1) or plates with round coverslips (Plate 2) and cultured for 24 h to allow cell attachment. After 24 h, cells in Plate 1 were treated with P-ZWNIR NPs or 808 nm-pretreated P-ZWNIR NPs (2 μmol L -1 As Pheo-Fc) was treated and cultured for 4 hours to induce cellular uptake. Cells that had absorbed NPs were washed three times with PBS, detached with trypsin-EDTA solution, centrifuged at 250 × g for 5 minutes, and resuspended in RPMI 1640. Cells were transferred to transwell inserts and placed in the apical compartment of Plate 2, and the transwell system was further cultured at 37°C for 4 hours. Cells on the round coverslips in the basolateral compartment were washed and fixed. The fixed cells were stained with DAPI and observed with a Leica TCS SP8 confocal microscope.

[0190] Referring to Fig. 10(C), the fluorescence of Pheo-Fc taken up by the attached cells in the basolateral compartment was observed in this experiment. The fluorescence signal of the cells in the basolateral compartment indicates that the NPs were excreted from the HT-29 cells in the apical compartment, translocated across the membrane, and then taken up by the basolateral compartment cells. The group treated with P-ZWNIR NPs showed weak fluorescence, and the fluorescence intensity decreased in the presence of Exo1, an exocytic inhibitor. The fluorescence intensity of the group treated with P-ZWNIR NPs + 808 nm (laser-pretreated P-ZWNIR NPs) was much stronger than that of the group treated with P-ZWNIR NPs, and also decreased in the presence of Exo1. These results indicate that the transcytosis efficiency of P-ZWNIR NPs was enhanced by photobleaching-mediated charge transfer.

[0191]

[0192] 2-3. Confirmation of tumor penetration ability

[0193] Based on the higher transcytosis efficiency of photobleached NPs, the tumor penetration ability of NPs was investigated using 3D HT-29 multicellular tumor spheroids (MTS). Considering that the photothermal effect of P-ZWNIR NPs under 808 nm may affect the tumor penetration of NPs, the tumor penetration ability of the following groups was tested and compared:

[0194] 1) P-ZWNIR NP

[0195] 2) P-ZWNIR NPs + 808 nm (pretreated)

[0196] 3) P-ZWNIR NPs (in situ).

[0197] HT-29 3D multicellular spheroids were seeded at 500 cells per well in a 96-well SPL3D Cell Floater (SPL Life Sciences, Gyeonggi-do, Republic of Korea) containing complete RPMI 1640 medium at 37°C, 5% CO2, and 95% relative humidity. Spheroids were grown with fresh cell culture medium every 2 days until the average diameter of the spheroids reached 300 μm, and then treated with P-ZWNIR NPs or 808 nm laser-pretreated P-ZWNIR NPs (2 μmol L -1 as Pheo-Fc). For the P-ZWNIR NPs + 808 nm (pretreated) group, P-ZWNIR NPs were treated with an 808 nm laser (0.8 W cm) for 5 min before being treated on HT-29 spheroids. -2 ) were investigated, and for the P-ZWNIR NP + 808 nm (in situ) group, immediately after P-ZWNIR NP was treated on the spheroids, 808 nm laser (0.8 W cm -2, 5 min) were irradiated on HT-29 spheroids. After 6 h, the spheroids were carefully washed with PBS, fixed with 4% formaldehyde solution (w / v), and mounted on Fluoroshield in a Ø13 mm confocal dish. The fluorescence of Pheo-Fc was observed with a Leica TCS SP8 confocal scope over a z-stack range of 150 μm from the surface of the spheroids.

[0198] Referring to Fig. 10(D), zwitterionic P-ZWNIR NPs were mainly located at the periphery of the spheroids. In contrast, cationic NPs (P-ZWNIR NPs + 808 nm (pretreated)) penetrated deeper into the spheroids. Notably, the penetration ability of P-ZWNIR NPs + 808 nm (in situ) was higher than that of P-ZWNIR NPs + 808 nm (pretreated). The fluorescence intensity-distance plot corresponding to a 150 μm z-stack showed that the average fluorescence intensity was highest for P-ZWNIR NPs + 808 nm (in situ) (Fig. 10E). This result implies that in addition to the cationic charge, the photothermal effect of P-ZWNIR NPs may also contribute to the tumor penetration of the NPs (Fig. 11). In summary, in vitro experiments on cellular uptake, transcytosis, and tumor penetration demonstrated that photobleaching-mediated charge transfer could enhance the transcytosis efficiency of P-ZWNIR NPs, thereby promoting tumor penetration of P-ZWNIR NPs.

[0199]

[0200] 2-4. Confirmation of antitumor efficacy

[0201] The antitumor efficacy of phototherapy using P-ZWNIR NPs was investigated in the HT-29 MTS model.

[0202] MTS were cultured according to the protocol described above. After growing until the average diameter of the spheroids reached 300 μm, they were randomly divided into six groups: untreated, P-ZWNIR NP (darkroom condition), P-ZWNIR NP + 808 nm (in situ), P-ZWNIR NP + 660 nm, P-ZWNIR NP + 808 nm (pretreatment) + 660 nm, and P-ZWNIR NP + 808 nm (in situ) + 660 nm. The treatment concentration was 5 μM with Pheo-Fc. The laser irradiation schedule was the same as that described in the penetration efficiency evaluation experiment in 2-3. After 6 h of incubation of P-ZWNIR NP-treated spheroids, 660 nm irradiation was applied. The 660 nm or 808 nm laser was irradiated for 5 min. After laser treatment, the cell culture medium was replaced with fresh medium, and then every 3 days. Spheroids were observed using the Thunder Imaging System (Leica Microsystems, Wetzlar, Germany), and the spheroid volume was calculated according to the following equation:

[0203] <Formula 1>

[0204] V (mm 3 ) = 0.5 × longest diameter × (shortest diameter) 2

[0205]

[0206] Referring to Figures 10(F) and 10(G), P-ZWNIR NPs (dark) without laser treatment slightly inhibited the growth of MTS. Irradiation at 660 nm on P-ZWNIR NPs significantly inhibited spheroid growth compared to P-ZWNIR NPs (dark), and in particular, the P-ZWNIR NP + 808 nm (pretreatment) + 660 nm group more effectively inhibited the growth of MTS compared to the P-ZWNIR NP + 660 nm group (three times lower spheroid volume). This indicates that deeper penetration of the NPs enhanced the antitumor efficacy. In the spheroid penetration experiment, the P-ZWNIR NP + 808 nm (in situ) + 660 nm group, which showed the highest tumor penetration efficiency, exhibited the best anticancer efficacy. MTS treated with this therapy did not grow during the experimental period, and this excellent therapeutic efficacy may be attributed to the combined effects of enhanced tumor penetration, photodynamic / chemodynamic effects, and photothermal effects. These results suggest that P-ZWNIR NP-assisted phototherapy using dual lasers has the potential to maximize therapeutic efficacy through deep tumor penetration and combined phototherapy.

[0207]

[0208] <Analysis Example 3> In vivo biodistribution and tumor penetration analysis of P-ZWNIR NPs in a rectal cancer (RC) model

[0209] The in vivo tumor-targeting ability of P-ZWNIR NPs was evaluated in an HT-29 tumor xenograft mouse model using near-infrared fluorescence (NIRF) imaging.

[0210]

[0211] 3-1. Mouse model

[0212] Male nude mice (5 weeks old) were purchased from Nara Biotech (Seoul, Republic of Korea). The animal experiment protocol (SNU-200630-1-1) was approved by the Animal Care and Use Committee of Seoul National University (Seoul, Republic of Korea). Mice were maintained at 22 ± 2°C in a light-controlled room with a relative humidity of 55 ± 5% (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University, Republic of Korea). Mice were anesthetized with 250 mg kg -1 It was performed by intraperitoneal (IP) injection of avertin or inhaled isoflurane (induction 3% and maintenance 1.5%).

[0213] The heterotopic (subcutaneous) HT-29 tumor xenograft mouse model was inoculated with 100 μL of HT-29 cell suspension (2 × 10 6 cells) were injected subcutaneously. The tumor volume (V) was calculated according to the above equation 1. For the in vivo tumor invasion experiment, an ear tumor transplantation mouse model was used, which was injected with 20 μL of HT-29 cell suspension (2 × 10 6 cells) were injected subcutaneously into the mouse ear.

[0214] The orthotopic (rectal) xenograft mouse model was performed by carefully exposing the rectum of the mouse and injecting 20 μL of HT-29 cell suspension (2 × 10 6cells) were inoculated into the submucosal layer of the rectal wall to establish tumor growth. For in vivo antitumor efficacy experiments, tumor growth was monitored by bioluminescence imaging using luciferase / green fluorescent protein-expressing HT-29 (HT-29 / Luc-GFP) cells. To establish the HT-29 / Luc-GFP cell line, HT-29 cells were transfected with a lentiviral plasmid encoding the Luc-GFP gene. Transfected HT-29 / Luc-GFP cells were then cloned by serial dilution in 96-well plates. Single clones were selected based on the GFP fluorescence signal observed by a biomolecular imaging system (Amersham™ ImageQuant™ 800, Cytiva Life Sciences, Marlborough, MA, US).

[0215]

[0216] 3-2. NIRF imaging analysis in the HT-29 tumor xenograft mouse model

[0217] Whole-body fluorescence images of mice were acquired using the ICG filter of a Visque InVivo Smart-LF imaging system (Vieworks, Gyeonggi-do, Republic of Korea) at 0, 2, 4, 6, 8, and 24 h after injection. Organs of interest were excised from experimental animals 24 h after nanoparticle administration, and ex vivo imaging was performed. The average radiant efficiency of the fluorescence images was measured using CleVue software version 3.1.3 (Vieworks, Gyeonggi-do, Republic of Korea).

[0218] NIR fluorescence intensity in the tumors was observed to increase up to 8 hours and then decrease in both orthotopic and ectopic tumor-bearing mice, indicating that NPs gradually accumulated in the tumors during the first 8 hours and were subsequently cleared from the tumors. At 8 and 24 hours post-injection, major organs and tissues were resected for ex vivo NIRF imaging, which revealed a higher mean fluorescence intensity at 8 hours than at 24 hours.

[0219] From the above results, since P-ZWNIR NP accumulation was highest at 8 hours, the biodistribution profile and tumor targeting ability of P-ZWNIR NP were confirmed at 8 hours after nanoparticle administration and compared with the control group administered ZWNIR NP.

[0220] As shown in Fig. 12, ZWNIR NPs and P-ZWNIR NPs were injected into the tail vein of mice bearing rectal HT-29 tumors, and real-time NIRF imaging was performed for 8 hours. The ZWNIR NPs showed a similar accumulation trend to that of P-ZWNIR NPs, but the fluorescence intensity in the tumor region of P-ZWNIR NPs was 1.5 times higher than that of ZWNIR NPs (Figs. 12A and 12B). At 8 hours after injection, ex vivo NIRF imaging showed that the accumulation of P-ZWNIR NPs in the tumor was 1.6 times higher than that of ZWNIR NPs, but the off-target accumulation profiles of both NPs were similar except for the kidney (Fig. 12C). These results indicate that the introduction of the tumor-targeting functional group PBA did not induce off-target accumulation of the nanoparticles, but only enhanced the tumor-targeting efficiency.

[0221] As shown in Fig. 12(D), P-ZWNIR NPs exhibited near-infrared fluorescence imaging capabilities specific to rectal tumors. The tumor-to-background ratio (TBR) values ​​of P-ZWNIR NPs for adjacent normal tissues (muscle, small intestine, colon, and rectum) were 6.3, 8.0, 10.1, and 7.8, respectively, which were significantly higher than those of ZWNIR NPs (Fig. 12E). Since contrast agents with a TBR value of 2.5 or higher can be applied to tumor image-guided surgery under near-infrared light, P-ZWNIR NPs can be used to reduce the size of the resection margin in tumor resection.

[0222]

[0223] 3-3. Confirmation of photobleaching of P-ZWNIR NPs in the HT-29 tumor xenograft mouse model

[0224] In situ photobleaching of P-ZWNIR NPs was investigated by in vivo NIRF imaging and ex vivo matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) after intravenous injection of P-ZWNIR NPs into HT-29 rectal tumor-bearing mice.

[0225] Tumor volume approximately 250 mm 3 Upon reaching , P-ZWNIR NP (1.5 mg kg -1 Pheo-Fc) was intravenously injected into tumor-bearing mice. In vivo whole-body NIRF imaging was performed 8 hours after injection using a Visque InVivo Smart-LF imaging system (Vieworks, Gyeonggi-do, Republic of Korea). After NIRF imaging, the rectal tumor area of ​​the mice was exposed to an 808 nm laser (0.8 W cm -2) were investigated and the fluorescence intensity changes were monitored. Photobleaching of P-ZWNIR NPs in tumors was further evaluated using a MALDI-MSI system.

[0226] P-ZWNIR NPs were intravenously injected into tumor-bearing mice, and the mice were irradiated with 808 nm laser (0.8 W cm -2 , 5 min) and 8 h after irradiation. Rectal tumors were resected from the mice, immediately frozen, and stored at -70°C until use. The frozen tumor tissues were sectioned into 10 μm thick (CM3050S; Leica Biosystems) and attached to indium tin oxide (ITO)-coated glass slides. The samples were diluted with DHB matrix solution (40 mg mL -1 The tumor tissue sections were coated with DHB in MeOH / DDW (50:50, v / v) with 0.1% TFA. Mass spectrometry images of the tumor tissue sections were acquired using a RapifleX MALDI Tissuetyper (Bruker Daltonics, Billerica, MA, USA).

[0227] Referring to Figure 12(F), the fluorescence intensity of the tumor area was measured under 808 nm laser irradiation (0.8 W cm -2 ) gradually decreased during the first 4 minutes and maintained a similar level of fluorescence intensity thereafter. Next, the generation of ketone fragments (c in Fig. 5A) by photobleaching of P-ZWNIR NPs in the dissected tumor was observed using MALDI-MSI. As shown in Fig. 12(G), 5 minutes after laser irradiation, the ketone fragment signal in the tumor tissue significantly increased, suggesting that the photobleaching of P-ZWNIR NPs in the tumor followed the mechanism presented in Fig. 2(A). Moreover, in the 808 nm laser-treated tumor, the intratumoral ketone fragment signals were evenly distributed throughout the tumor mass, indicating that uniform charge conversion was achieved in the rectal tumor tissue.

[0228]

[0229] 3-4. In vivo penetration of P-ZWNIR NPs in an HT-29 tumor xenograft mouse model.

[0230] 808 nm laser (0.8 W cm -2 ) The in vivo tumor penetration efficiency of P-ZWNIR NPs was evaluated by two-photon microscopy in HT-29 ear tumor-bearing mice before and after 5 min of irradiation.

[0231] HT-29 tumors in the mouse ear are approximately 50 μmm 3 When reached, P-ZWNIR NP (1.5 mg kg -1 Pheo-Fc) was injected into the tail vein of mice, and 4 h later, fluorescence from Pheo-Fc in tumor tissue was monitored at predetermined time points (0, 5, 10, 15, 30, 45, and 60 min) using a two-photon laser scanning microscope (FVMPE, Olympus Corporation, Tokyo, Japan). Then, the tumor site was illuminated with an 808 nm laser (0.8 W cm -2 ) for 5 min to induce photobleaching and tumor penetration of P-ZWNIR NPs. After irradiation, the fluorescence signal was recorded for an additional 1 h, and the fluorescence intensity was normalized to the vascular fluorescence intensity at the initial time point of each step. Graphs were plotted as a function of distance from the blood vessel and the normalized fluorescence intensity of the region of interest (ROI) using ImageJ (version 1.53t; National Institutes of Health, USA). During and after NIR irradiation, temperature changes in the ear tumor were recorded using an infrared thermal imaging camera (FLIR ONE Pro; Teledyne FLIR, Wilsonville, OR, USA).

[0232] As shown in Fig. 12(H), tumor tissues transplanted into the ear of mice were observed using a two-photon microscope 4 hours after intravenous administration of P-ZWNIR NPs. Since the near-infrared fluorescence of P-ZWNIR NPs disappears after 808 nm irradiation, the fluorescence signal of Pheo-Fc contained in P-ZWNIR NPs was observed before and after laser irradiation. Before 808 nm laser irradiation, the fluorescence signal of P-ZWNIR NPs was mainly confined to blood vessels (Fig. 12I), and a weak fluorescence signal was observed within a distance of approximately 30 μm (Fig. 12J). The fluorescence intensity around the blood vessels slightly increased over the next hour compared to 5 minutes. In contrast, the fluorescence signal around the blood vessels was confirmed to become stronger over time after 808 nm laser irradiation (Fig. 12I). The average fluorescence intensity after laser irradiation was significantly higher than before irradiation, and the fluorescence signal was observed up to approximately 100 μm from the blood vessels. These fluorescence imaging results indicate that photobleaching by the 808 nm laser promoted tumor penetration of P-ZWNIR NPs.

[0233] Referring to Fig. 10(D), the photothermal effect of P-ZWNIR NPs can influence the diffusion and penetration of NPs. However, since the tumor temperature only increases to approximately 41°C upon 808 nm laser irradiation and rapidly decreases to body temperature after 5 minutes, the enhanced tumor penetration of P-ZWNIR NPs can be attributed primarily to the transcytosis of photobleached cationic NPs rather than the hyperthermic effect.

[0234]

[0235] <Analysis Example 4> Phototherapy using P-ZWNIR NPs in an RC mouse model

[0236] 4-1. Confirmation of the phototherapeutic efficacy of P-ZWNIR NPs in mice bearing heterotopic (subcutaneous) HT-29 tumor xenografts.

[0237] Subcutaneous HT-29 tumor xenograft mouse models were prepared as described in the in vivo invasion and tumor invasion area.

[0238] Tumor volume approximately 250 mm 3 Upon reaching (day 0), mice were randomly divided into six groups: PBS, PBS + 808 nm + 660 nm, P-ZWNIR NP (darkroom), P-ZWNIR NP + 660 nm (photodynamic / chemodynamic effect), P-ZWNIR NP + 808 nm (photothermal effect and enhanced tumor penetration by photobleaching), and P-ZWNIR NP + 808 nm + 660 nm (photothermal effect combined with enhanced tumor penetration). PBS or P-ZWNIR NP was administered at a dose of 1.5 mg kg -1 Pheo-Fc was administered intravenously at a dose of 0.8 W cm at 808 nm and 660 nm, respectively. -2 and 2 mW cm -2 , and both lasers were irradiated to the tumor for 5 minutes. Eight hours after injection, the 808 nm laser (first laser) was irradiated for 5 minutes, and the temperature of the tumor site was monitored every minute during the irradiation. The 660 nm laser (second laser) was irradiated 10 hours after injection (Fig. 13A). Considering that the tumor volume in this experiment was approximately five times larger than that in the in vivo penetration experiment in the ear tumor-bearing mouse model (Fig. 12H-J), the treatment interval between the two lasers was set to 2 hours to ensure sufficient tumor penetration of P-ZWNIR NPs after 808 nm irradiation (photobleaching).

[0239] On the last day of the experiment, blood (~0.5 mL) was collected from each mouse, 50 μL of blood sample was used for complete blood count, and the remainder was centrifuged at 16,000 × g for 5 min to obtain plasma samples. Complete blood count was analyzed using Scil Vet abc Plus (HORIBA, Kyoto, Japan), and the following were measured: white blood cell count, lymphocyte count, monocyte count, basophil count, eosinophil count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width, platelet count, and mean platelet volume. Plasma samples were analyzed using Fuji Dri-Chem 3500s (Fujifilm Holdings Corp., Tokyo, Japan), and aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CRE), total protein (TP), and lactate dehydrogenase (LDH) were recorded.

[0240] As a result, the maximum temperature observed during 808 nm laser irradiation (P-ZWNIR NP + 808 nm and P-ZWNIR NP + 808 nm + 660 nm) was less than 45℃, which did not cause significant thermal damage to surrounding normal tissues. Referring to Figures 13(B) to 13(D), the tumor growth rate of the PBS + 808 nm + 660 nm group was similar to that of the PBS group, suggesting that the laser treatment itself did not have a significant inhibitory effect on tumor growth. P-ZWNIR NP (without laser treatment) and P-ZWNIR NP-adjuvanted single laser treatment (808 nm or 660 nm) significantly inhibited tumor growth compared to the PBS group, but did not lead to tumor shrinkage. In contrast, in contrast to the marginal tumor growth inhibition of single laser therapy, P-ZWNIR NP + 808 nm + 660 nm treatment significantly inhibited tumors of clinically relevant size (~250 mm) after single treatment on day 0. 3 ) was completely eliminated. Five of the six mice in the group achieved complete remission, and only one showed very small tumor recurrence after 14 days. These results suggest that 808 nm laser-induced deep tumor penetration significantly enhanced the therapeutic efficacy of 660 nm-induced photodynamic / chemodynamic therapy. In addition to spatially delivering NPs to deep tumor sites, utilizing transcytosis for NP penetration may contribute to superior efficacy because it can deliver photosensitizers into the intracellular space, which can overcome major drawbacks of photodynamic / chemodynamic therapy (e.g., short half-life and diffusion distance of ROS).

[0241] Meanwhile, compared with the other groups, there was no significant weight loss in the P-ZWNIR NP + 808 nm + 660 nm treatment group. Blood biochemical parameters and complete blood count analysis also showed no significant differences between treatment groups, except for the lowest serum lactate dehydrogenase (LDH) level in the P-ZWNIR NP + 808 nm + 660 nm group. Because high serum LDH levels are positively correlated with tumor growth and necrosis, the difference in LDH levels contributed to the extensive tumor necrosis in the PBS group and the tumor remission in the P-ZWNIR NP + 808 nm + 660 nm group. Histological analysis of major organs, including the heart, lungs, liver, spleen, and kidney, showed no notable changes in the P-ZWNIR NP + 808 nm + 660 nm group compared with the other groups. These results collectively suggest the safety of phototherapy using P-ZWNIR NP.

[0242]

[0243] To investigate whether the potent efficacy of P-ZWNIR NP + 808 nm + 660 nm was due to the combined effect of photothermal and photodynamic / chemodynamic therapy or the synergistic effect of the combination through deep tumor penetration, the following antitumor efficacy study was performed in the same tumor model.

[0244] ZWNIR NPs or P-ZWNIR NPs were intravenously injected on day 0 and two different laser radiation schedules were applied as follows:

[0245] 1) First 660 nm laser irradiation 8 hours after injection, followed by second 808 nm laser irradiation 10 hours after injection.

[0246] 2) First 808 nm laser irradiation 8 hours after injection, followed by second 660 nm laser irradiation 10 hours after injection.

[0247] Specifically, the tumor volume was approximately ~250 mm 3Upon reaching the target, mice were randomly divided into five groups: PBS, ZWNIR NP + 808 nm (8 hours after injection) + 660 nm (10 hours after injection), ZWNIR NP + 660 nm (8 hours after injection) + 660 nm (10 hours after injection), P-ZWNIR NP + 808 nm (8 hours after injection) + 660 nm (10 hours after injection), P-ZWNIR NP + 660 nm (8 hours after injection) + 660 nm (10 hours after injection), and tumor volume and body weight were monitored every other day. On the 20th day, organs and tissues of interest, including tumors, heart, lungs, liver, kidneys, and spleen, were dissected. Tumor weights were measured, and normal organs were stained with hematoxylin and eosin, and tissue images were observed using Vectra (v3.0.5).

[0248] Referring to Figures 13(E) to 13(G), the P-ZWNIR NP phototherapy group showed a higher antitumor effect in both irradiation schedules compared to ZWNIR NPs under the same irradiation schedule, which can be attributed to the greater accumulation of P-ZWNIR NPs in the tumor site compared to ZWNIR NPs. Moreover, in the ZWNIR NP and P-ZWNIR NP groups, the 808 nm laser (first) + 660 nm (second) irradiation schedule showed significantly higher antitumor efficacy compared to the 660 nm laser (first) + 808 nm (second) irradiation schedule. These results suggest that the potent antitumor efficacy of the P-ZWNIR NP + 808 nm (first) + 660 nm (second) regimen is a result of the enhanced penetration of NPs into the deep tumor.

[0249]

[0250] 4-2. Confirmation of the phototherapeutic efficacy of P-ZWNIR NPs in orthotopic (rectal) HT-29 tumor xenograft mice.

[0251] Finally, the phototherapeutic effect of phototherapy using P-ZWNIR NPs was evaluated in an orthotopic rectal tumor model.

[0252] An orthotopic rectal tumor model was prepared by inoculating 5-week-old male BALB / c nude mice with HT-29 cells expressing luciferase / green fluorescent protein (HT-29 / Luc-GFP). Seven days after inoculation, the mice were randomly divided into three groups: PBS, P-ZWNIR NPs (dark room), and P-ZWNIR NPs + 808 nm + 660 nm, and were administered 1.5 mg kg -1 Pheo-Fc was injected intravenously at a dose of 10 mg / kg. The laser treatment schedule was the same as for the ectopic tumor model. Tumor growth (via bioluminescence) and body weight were measured on days 0, 4, 7, 11, 14, and 20, and to monitor tumor growth, D-luciferin (150 mg kg -1 ) was injected intraperitoneally, and the bioluminescence intensity was measured using the IVIS Spectrum In Vivo Imaging System (PerkinElmer). After the experiment, the mouse rectal tissue was dissected, and 300 μg mL -1 After adding the D-luciferin solution and incubating, the rectal tissue was transferred to the stage and imaged using the IVIS Spectrum In Vivo Imaging System.

[0253] Referring to Figures 13(H) and 13(I), the bioluminescence intensities of the PBS and P-ZWNIR NP-treated groups gradually increased over the experimental period, and multiple signal foci were observed in some mice, indicating tumor growth with metastasis in the orthotopic rectal tumor model. However, the bioluminescence intensity of the P-ZWNIR NP + 808 nm + 660 nm group decreased to the autoluminescence level on day 4 after treatment, which remained constant throughout the experimental period. These results suggest that phototherapy using P-ZWNIR NPs achieved complete remission of the tumor. The body weight profiles of the three groups did not differ significantly over the experimental period, indicating that the systemic dark toxicity and phototoxicity of P-ZWNIR NPs can be ignored (Figure 13J).

[0254] Referring to Fig. 13(K), bioluminescence signals were observed only in the PBS and P-ZWNIR NP groups, whereas no bioluminescence signal was observed in the rectal tissue in the P-ZWNIR NP + 808 nm + 660 nm group. The bioluminescence intensities of the rectal tissue were similar between the PBS and P-ZWNIR NP groups, indicating that P-ZWNIR NPs alone did not inhibit rectal tumor growth. In contrast, the bioluminescence intensity of the P-ZWNIR NP-assisted phototherapy group was at the same level as autobioluminescence, suggesting complete remission of rectal tumors after phototherapy (Fig. 13L). Toxicity studies, including blood biochemistry and complete blood count analysis, showed no significant differences among the three groups, suggesting that the toxicity of phototherapy using P-ZWNIR NPs in the orthotopic rectal tumor mouse model was negligible.

[0255]

[0256] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A cyclodextrin derivative represented by the following chemical formula 1, to which a heptamethine cyanine-based zwitterionic near-infrared fluorophore is bound: <Chemical Formula 1> In the above formula, X and Y can be the same or different, ASO3 - , ACO2 - , or a salt thereof, wherein A is aromatic or (CH2) a is selected from alkyl corresponding to , and a is selected from an integer from 1 to 8, m is selected from the integers 6 to 8, n is an integer selected from 1 to 40.

2. A cyclodextrin derivative represented by the following chemical formula 2, wherein a heptamethine cyanine-based zwitterionic near-infrared fluorophore and a tumor-targeting moiety are bound to cyclodextrin: <Chemical Formula 2> In the above formula, X and Y can be the same or different, ASO3 - , ACO2 - , or a salt thereof, wherein A is aromatic or (CH2) a is selected from alkyl corresponding to , and a is selected from an integer from 1 to 8, R is a tumor targeting moiety, selected from compounds having a molecular weight of less than 10,000 Da used for active targeting, m is an integer selected from 6 to 8, n is an integer selected from 1 to 40.

3. In paragraph 2, The above tumor targeting moiety is, A cyclodextrin derivative characterized by being at least one compound selected from the group consisting of phenylboronic acid (PBA), peptides containing Arginine-Glycine-Aspartate (RGD), IL-4 receptor targeting peptide (IL4RPep-1), biotin, folate, glucose, galactose, mannose, fructose, sorbitol, N-acetylglucosamine, N-acetylmannosamine, adenosine, thiamine, riboflavin, methotrexate, anisamide, and anacardic acid.

4. In paragraph 2, The above derivatives are, A cyclodextrin derivative characterized in that the tumor-targeting moiety and the zwitterionic near-infrared fluorophore are conjugated to each terminal of a pegylated cyclodextrin derivative at a molar ratio of 1:

1.

5. A compound in which a photosensitizer is incorporated into a cyclodextrin derivative according to claim 1 or 2.

6. In paragraph 5, The above photosensitizer is, A compound characterized by being pheophorbide or a derivative thereof.

7. In paragraph 6, The above photosensitizer is, A compound characterized by being a pheophorbide a to which ferrocene is bound.

8. In paragraph 7, The above compound is, A compound characterized in that ferrocene is bound in a host-guest form within the above cyclodextrin.

9. In paragraph 5, The above compound is, A compound characterized in that the cyclodextrin derivative and the photosensitizer are incorporated in a molar ratio of 1:

1.

10. Nanoparticles formed by self-assembly of a compound according to Article 5.

11. In paragraph 10, The above nanoparticles are, A nanoparticle characterized by photobleaching upon near-infrared irradiation, thereby converting the nanoparticle from a zwitterionic nanoparticle to a positively charged nanoparticle.

12. A composition for photodynamic therapy of cancer, comprising nanoparticles according to Article 10 as an active ingredient.

13. In paragraph 12, The above composition, A composition for photodynamic therapy of cancer, characterized by enhanced tumor penetration ability.

14. In paragraph 12, The above cancer, A composition for photodynamic therapy of cancer, characterized in that it is a solid tumor.

15. A composition for combined photodynamic and chemotherapy treatment of cancer, comprising nanoparticles according to Article 10 as an active ingredient.

16. A composition for near-infrared fluorescence imaging for cancer diagnosis, comprising nanoparticles according to Article 10 as an active ingredient.

Images