An oxygen-independent aggregation-induced emission photosensitizer with mitochondrial targeting ability

Abstract

The present invention relates to an oxygen-independent aggregation-inducing luminescent photosensitizer having mitochondrial targeting ability. In this invention, anion-π+ AIE-PS, which possesses a potent donor-acceptor (DA) configuration and heavy atomic effects, can generate TPEPyTMB, a mixture of type 1 and type 2 ROS. TPEPyTMB exhibits excellent type-1 ROS generation efficiency and demonstrates effective PDT efficacy due to increased intracellular ROS generation efficiency and enhanced mitochondrial targeting ability under both normal oxygen and hypoxic conditions. Furthermore, in vivo studies indicate that TPEPyTMB can inhibit subcutaneous tumor growth in a manner that minimizes systemic damage upon exposure to light, exhibits excellent long-term in vivo imaging capabilities, and accumulates at the tumor site. Therefore, the present invention is expected to provide a novel approach to the development of type 1 AIE-PS by promoting the ISC effect through enhanced molecular aggregation.

Description

Technology Field

[0001] The present invention relates to an oxygen-independent aggregation-inducing luminescent photosensitizer having mitochondrial targeting ability, and more specifically, to the synthesis of a novel oxygen-independent aggregation-inducing luminescent photosensitizer having mitochondrial targeting ability embedded in its molecular structure for anticancer phototherapy with improved safety and efficacy, and a method for manufacturing nanoparticles for the in vivo application thereof. Background Technology

[0002] Cancer, one of the leading causes of mortality and morbidity worldwide, results in a significant number of deaths annually. While traditional treatments such as surgery, chemotherapy, and radiation therapy have been widely used to treat cancer, they are hampered by multidrug resistance, side effects, and low selectivity. Therefore, alternative treatment strategies offering optimal efficacy, minimal side effects, and high selectivity are crucial in cancer therapy.

[0003] In recent years, photodynamic therapy (PDT), which generates reactive oxygen species (ROS) to induce oxidative damage in tumor cells, has been regarded as an effective and promising approach in cancer treatment due to its high selectivity, minimal invasiveness, and broad applicability.

[0004] Photosensitizers (PS), oxygen, and light are three important components of PDT. Photosensitizers, which are a key component of PDT, must possess excellent luminescence properties, high ROS generation efficacy, and excellent biocompatibility. Traditional PSs, such as tetrapyrroles or aminolevulinic acid, exhibit an aggregation-caused quenching (ACQ) effect in an aggregated state due to π-π stacking in their rigid planar structures. The aggregation of these PSs extinguishes fluorescence and singlet oxygen ( 1 It limits application in PDT by reducing O2 generation efficiency.

[0005] Meanwhile, the synthesized photosensitizer exhibits aggregation-induced emission (AIE) behavior in the aggregate state, displaying strong fluorescence and high ROS generation efficiency due to the restriction of intramolecular motions (RIMs). Therefore, the AIE function of these synthetic photosensitizers differs from conventional photosensitizers, and AIE-based photosensitizers (PS) with high ROS generation efficiency and strong emission in the red / near infrared (NIR) region can be considered highly suitable for PDT.

[0006] In addition to high ROS generation efficiency, the therapeutic efficiency of PDT is singlet oxygen ( 1Due to the short half-life (<40ns) and limited range of action (<20nm) of O2, it depends on the intracellular location of PS. Among various subcellular targets, mitochondria hold particularly great potential as PDT targets because they play an essential role in supplying energy to cells and regulating cell apoptosis. For this reason, numerous studies are being conducted to develop novel mitochondria-specific PDT agents.

[0007] As prior art targeting mitochondria, Korean Published Patent No. 10-2020-0000079, "Photosensitizer for photodynamic therapy specific to mitochondria," discloses a photodynamic therapy photosensitizer that specifically targets mitochondria using a novel compound and can induce reactive oxygen species by intensively releasing heat from mitochondria; and Korean Published Patent No. 10-2021-0116155, "Compound, photosensitizer containing the same, composition for tumor diagnosis or treatment targeting mitochondria, and photodynamic therapy method using said composition," discloses a photosensitizer containing PY-BOD compound, PH-BOD compound, MeO-BOD compound, and DMA-BOD compound, a composition for tumor diagnosis or treatment targeting mitochondria, and a photodynamic therapy method using the same.

[0008] In recent years, many AIE-based photosensitizers targeting mitochondria have been developed to achieve better PDT effects, but oxygen-dependent aggregation-induced emission photosensitizers (AIE-PS) with the ability to produce reactive oxygen species (ROS), particularly type 1 ROS, which are specific to cellular organelles and efficient, are in high demand but remain a challenging task.

[0009] Accordingly, the inventors synthesized three new AIE-PSs with mitochondrial targeting ability and obtained nanoparticles (NPs) by encapsulating the synthesized AIE-PSs. Furthermore, the inventors confirmed the mitochondrial targeting ability of the AIE-PSs NPs under white light irradiation and completed the invention by confirming that they exhibit a PDT effect even under normal oxygen and hypoxia. The problem to be solved

[0010] Therefore, the technical problem to be solved by the present invention is to provide an oxygen-independent aggregation-induced luminescence photosensitizer having mitochondrial targeting ability. means of solving the problem

[0011] In order to solve the above-mentioned technical problem, the present invention provides a compound represented by the following chemical formula 1, chemical formula 2, or chemical formula 3:

[0012] [Chemical Formula 1]

[0013]

[0014] [Chemical Formula 2]

[0015]

[0016] [Chemical Formula 3]

[0017]

[0018] The present invention provides an aggregation-induced luminescence photosensitive agent comprising the above compound.

[0019] In addition, the present invention provides a photodynamic therapy composition for cancer treatment comprising the above-mentioned photosensitizer as an active ingredient.

[0020] The above cancer may be selected from breast cancer, kidney cancer, testicular cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, vaginal cancer, fallopian tube cancer, rectal cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, small intestine cancer, pancreatic cancer, oral cancer, melanoma, or sarcoma.

[0021] In addition, the present invention provides a photodynamic therapy method for cancer treatment characterized by including the following steps:

[0022] 1) A step of administering a composition containing the above-mentioned photosensitizer as an active ingredient to a subject;

[0023] 2) a step of allowing time for the administered composition to accumulate within the target cells of the subject; and

[0024] 3) A step of irradiating light onto the target cell area of ​​the above-mentioned object. Effects of the invention

[0025] As such, in the present invention, anion-π+ AIE-PS having a potent donor-acceptor (DA) configuration and heavy atomic effects can generate TPEPyTMB, a mixture of type 1 and type 2 ROS. TPEPyTMB exhibits excellent type-1 ROS generation efficiency and demonstrates effective PDT efficacy due to increased intracellular ROS generation efficiency and enhanced mitochondrial targeting ability under both normal oxygen and hypoxic conditions. Furthermore, in vivo studies indicate that TPEPyTMB can inhibit subcutaneous tumor growth in a manner that minimizes systemic damage when exposed to light, exhibits excellent long-term in vivo imaging capabilities, and accumulates at the tumor site. Therefore, the present invention is expected to provide a novel approach to the development of type 1 AIE-PS by promoting the ISC effect through enhanced molecular aggregation. Brief explanation of the drawing

[0026] The following drawings attached to this specification illustrate preferred embodiments of the present invention and serve to further enhance understanding of the technical concept of the present invention together with the aforementioned description; therefore, the present invention should not be interpreted as being limited only to the matters described in such drawings. Figure 1 shows TPEPyTMB-1's 1 This is the H NMR spectrum. Figure 2 shows TPEPyTMB-1's 13 This is the C NMR spectrum. Figure 3 is the LC-MS spectrum of TPEPyTMB-1. Figure 4 shows TPEPyTMB-2's 1 This is the H NMR spectrum. Figure 5 shows TPEPyTMB-3's 13 This is the C NMR spectrum. Figure 6 is the LC-MS spectrum of TPEPyTMB-3. Figure 7 shows TPEPyTMB-3's 1 This is the H NMR spectrum. Fig. 8 shows TPEPyTMB-3's 13 This is the C NMR spectrum. Figure 9 is the HRMS spectrum of TPEPyTMB-3. Figure 10 is the absorbance spectrum of anion-π+ AIE-PS in DMSO. Figure 11 shows the particle size distribution of (A) TPEPyTMB-1 NPs, (B) TPEPyTMB-2 NPs, and (C) TPEPyTMB-3 NPs in PBS (pH 7.4). Figure 12 shows the absorbance and PL spectra of TPEPyTMB-1 NPs, TPEPyTMB-2 NPs, and TPEPyTMB-3 NPs. Figure 13 shows the absorption spectrum before and after irradiation. Figure 14 shows the PL spectrum of DCFH +TPEPyTMB-3 NPs and the relative ROS generation efficiency of AIE-PS NPs and Ce6 under white light irradiation. Figure 15 shows the absorption spectrum of ABDA+TPEPyTMB-3 NPs and the relative values ​​of AIE-PSs NPs and Ce6 upon white light irradiation. 1 This represents the O2 generation efficiency. Figure 16 shows the absorption spectrum of DHR123+TPEPyTMB-3 NPs and the relative values ​​of AIE-PSs NPs and Ce6 upon white light irradiation. 1This represents the O2 generation efficiency. Figure 17 shows the results of ROS detection inside the cell. Figure 18 shows the results of the mitochondrial destruction assessment. Figure 19 shows the release profiles of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 PS from AIE-PSs NPs under various physiological conditions. Figure 20 shows the cell viability of MCF-7 cells treated with TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs under dark conditions. Figure 21 is a confocal microscopy image of apoptosis in MCF-7 cells after 24 hours of culture with TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs in a hypoxic environment under dark conditions. Figure 22 shows the in vivo imaging results of TPEPyTMB-3 NPs. Figure 23 shows the results of the in vivo phototherapy evaluation of TPEPyTMB-3 NPs. Figure 24 shows the H&E staining results of major organs of mice on day 14 after treatment with PBS and TPEPyTMB-3 NPs. Specific details for implementing the invention

[0027] The present invention will be explained in more detail below.

[0028] The present invention provides a compound represented by the following chemical formula 1, chemical formula 2, or chemical formula 3:

[0029] [Chemical Formula 1]

[0030]

[0031] [Chemical Formula 2]

[0032]

[0033] [Chemical Formula 3]

[0034]

[0035] The compound of the above chemical formula 1 is TPEPyTMB-1, which is tetraphenylethylene pyridinium trimethyl dibromobenzene.

[0036] The compound of the above chemical formula 2 is TPEPyTMB-2, which is Tetraphenylethylene pyridinium trimethyl bromobenzene.

[0037] The compound of the above chemical formula 3 is TPEPyTMB-3, which is tetraphenylethylene pyridinium trimethyl benzene.

[0038] The above compounds can be synthesized based on the donor-acceptor (DA) concept by connecting 1, 2, and 3 tetraphenylethene-vinylpyridine (TPEPy) cores with 1,3,5-tribromomethylbenzene (bridging unit). That is, 1, 2, and 3 molecules (TPEPy) were introduced into the structures of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3, respectively, using tetraphenylethene (TPE) as the electron donor unit and cation pyridinium as the acceptor unit to control the molecular aggregation of anion-π+ AIE-PS.

[0039] According to one embodiment of the present invention, all three anion-π+ AIE-PSs exhibited absorption peaks in the UV-Vis range at 422, 425, and 427, respectively, which means they can be excited under a white light source. The AIE performance of all anion-π+ AIE-PSs was investigated in DMSO / PBS mixtures with various PBS fractions, and all anion-π+ AIE-PSs exhibited weak photoluminescence (PL) intensity in DMSO, but the PL intensity of anion-π+ AIE-PSs gradually increased as the proportion of PBS in the DMSO / PBS mixture increased.

[0040] According to one embodiment of the present invention, after adding 90% fraction of PBS, the PL intensities of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 were improved by 34.7, 41.2, and 56.5 times, respectively, which demonstrates excellent AIE characteristics. In particular, TPEPyTMB-3 exhibited higher PL intensity and a superior AIE tendency, which may be attributed to a more twisted configuration that severely restricts molecular rotation in an aggregated state.

[0041] According to one embodiment of the present invention, all anion-π+ AIE-PSs exhibited a broad fluorescence spectrum in the 550-800 nm range, with maximum emissions located at 642, 645, and 648 nm, respectively. Additionally, all anion-π+ AIE-PSs exhibited a large Stokes shift (> 220 nm) and a long emission tail into the NIR region, which suggests suitability for image-induced PDT.

[0042] The present invention provides an aggregation-induced emission (AIE) photosensitizer comprising the above compound.

[0043] The term "aggregation-induced luminescence" in this invention refers to a fluorescence amplification phenomenon induced by the aggregation of a light-emitting body.

[0044] The photosensitizer of the present invention can be specifically accumulated in mitochondria.

[0045] The term "mitochondria" in this invention refers to organelles essential to cellular energy metabolism that play a crucial role in regulating cell function. Therefore, mitochondria have been considered effective as major intracellular targets for apoptosis or necrosis induced by PDT. Reactive oxygen species (ROS) generated during photodynamic processes can damage mitochondrial membranes. Since all three AIE-PSs NPs of this invention specifically accumulate in mitochondria, mitochondrial ROS levels increase, causing damage to the cell membrane.

[0046] The photosensitizer of the present invention can generate reactive oxygen species (ROS).

[0047] The term "reactive oxygen species" in this invention refers to chemically reactive molecules containing oxygen atoms. These are oxygen compounds generated within living organisms that possess strong oxidizing power, attacking biological tissues and damaging cells. The molecules contain oxygen ions and hydrogen peroxide, and their reactivity is very high due to unpaired electrons. Reactive oxygen species are known to be produced during the normal metabolic processes of oxygen and are known to play a role in cell signaling and homeostasis. However, there is a risk that the concentration of reactive oxygen species may surge due to environmental stress, such as exposure to ultraviolet rays or high heat, which can damage cell structures. Through this phenomenon known as oxidative stress, excessively increased reactive oxygen species can trigger uncontrolled reactions and act as substances harmful to the body. Reactive oxygen species can also be generated by exogenous factors, such as ionizing radiation.

[0048] The photosensitizer of the present invention can target mitochondria due to cationic pyridinium moiety and efficiently generated ROS under white light irradiation.

[0049] The photosensitizer of the present invention exhibits an efficient in vitro PDT effect in both normoxic and hypoxic environments. Furthermore, in vivo results demonstrate the potential of trimer TPEPyTMB NPs for long-lasting in vivo imaging and effective photodynamic therapy under hypoxia.

[0050] According to one embodiment of the present invention, the compound may be an encapsulated nanoparticle.

[0051] The above encapsulation can be performed using a bipolar block polymer, Poly(ethylene glycol)-distearoylphosphatidylethanolamine (DSPE-PEG).

[0052] The above nanoparticles may have a diameter of 10 to 200 nm, 50 to 150 nm, or 100 to 130 nm.

[0053] The present invention provides a photodynamic therapy composition for cancer treatment comprising the above-mentioned photosensitizer as an active ingredient.

[0054] The composition of the present invention may be formulated to include one or more pharmaceutically acceptable carriers in addition to the pharmaceutically effective amount of active ingredient described above for administration.

[0055] In the foregoing, "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. Other pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above-mentioned factors, and this can be easily determined by a person skilled in the art.

[0056] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, condition, body weight, absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and concomitant drugs. Generally, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of obesity, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0057] The above cancer may be selected from breast cancer, kidney cancer, testicular cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, vaginal cancer, fallopian tube cancer, rectal cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, small intestine cancer, pancreatic cancer, oral cancer, melanoma, or sarcoma.

[0058] Meanwhile, the present invention provides a photodynamic therapy method comprising: 1) administering the photodynamic therapy composition to a target; 2) allowing time for the administered composition to accumulate within the target cells of the target; and 3) irradiating light onto the target cell area of ​​the target.

[0059] The administering step of step 1) above can be administered orally or parenterally.

[0060] The step of irradiating light in step 3) above can be irradiated for 10 seconds to 5 hours, irradiated for 10 seconds to 4 hours, irradiated for 10 seconds to 3 hours, and irradiated for 10 seconds to 2 hours.

[0061] The step of irradiating light in step 3) above is 50 to 300 mW / cm² 2 It can irradiate light of 100 to 200 mW / cm² 2 It can irradiate light.

[0062] The light in step 3) above may be white light.

[0063] In specific examples, the inventors synthesized three new AIE-PSs with mitochondrial targets, TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3.

[0064] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0065] 1. Experimental Method

[0066] (1) ingredient

[0067] 6 g of 4,4'-dimethoxybenzophenone, palladium(II) acetate, 4-vinylpyridine, 4-bromobenzophenone, tri(o-tolyl)phosphine, trimethylamine, 1,3,5-tris(bromomethyl)benzene, and rhodamine were purchased from Tokyo Chemical Industry (Tokyo, Japan). Titanium tetrachloride, zinc, 9,10-anthracendiyl-bis(methylene)dimalonic acid (ABDA), 2'-7'-dichlorofluorescein diacetate (DCFH-DA), dihydrorhodamine 123 (DHR123), anhydrous tetrahydrofuran (THF), N,N-dimethylformamide (DMF), and chloroform-d6 (CDCl3) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dulbecco's Modified Eagle's Medium (DMEM) and Fetal Bovine Serum (FBS) were supplied by HyClone (Cytiva). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was supplied by Sigma-Aldrich (St. Louis, Missouri, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DSPE-PEG2000) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). All other chemicals were purchased from Sigma-Aldrich and used as received without further purification.

[0068] (2) Specialization

[0069] 1H NMR (600 MHz) and 13C NMR (150 MHz) spectra were measured in chloroform-d (CDCl3) at Bruker Analytik (Karlsruhe, Germany). Mass spectra were measured by liquid chromatography-mass spectrometry (LC-MS) using an Agilent 1260 Series HPLC system (Agilent Technologies Inc, Santa Clara, CA, USA) connected to an Agilent 6490 Triple Quadrupole mass spectrometer equipped with an electrospray ionizing Agilent Jet Stream ion source. UV-Vis absorption spectra were recorded using a Cary 60 UV-Vis spectrophotometer with a quartz cell (Agilent Technologies Inc, Santa Clara, CA, USA). Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies Inc, Santa Clara, CA, USA). Particles were analyzed by dynamic light scattering (ELSZ-1000, Otsuka Electronics Co, Osaka, 141 Japan). Photodynamic experiments were performed using a white light source (Fiber Illuminator, SWS-FO100).

[0070] (3) Synthesis of TPEBr

[0071] 4,4'-Dimethoxybenzophenone (0.243 g, 1 mmol) and 4-Bromobenzophenone (0.262 g, 1 mmol) were dissolved in 10 ml of dry THF, and then zinc powder (3.26 g, 5 mmol) was added. The suspension was cooled to 0°C, and then titanium tetrachloride (2.0 ml) was added dropwise. After addition, the mixture was slowly heated to room temperature and then refluxed at 80°C for 12 hours under an argon atmosphere. Afterward, the mixture was cooled in an ice bath, and a saturated sodium bicarbonate aqueous solution (50 ml) was slowly added. The mixture was extracted with ethyl acetate (100 ml × 3), the organic phase was washed with brine (100 ml × 2), and then dried with MgSO4. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography (hexane / ethyl acetate = 90 / 10) to obtain the desired product as a white solid (0.220 g, 46.8% yield).

[0072] 1 H NMR (600 MHz, CDCl3) δ 7.22 (d, 2H), 7.10 (m, 3H), 7.00 (m, 2H), 6.87- 6.94 (m, 6H), 6.67 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 8.8 Hz, 2H), 3.76 (s, 3H), 3.73 (3, 3H); 13 13C NMR (151 MHz, CDCl3) δ158.2, 158.0, 143.8, 143.1, 140.9, 137.8, 136.1, 135.9, 133.0, 132.5, 131.3, 130.8, 127.7, 126.2, 113.2, 113.0, 55.0; LC-MS, m / z: [M+H]+ Calculated 470.08, Found 470.30.

[0073] (4) Synthesis of TPEPy

[0074] TPE-Br (470 mg, 1 mmol), 4-vinylpyridine (108 mg, 1 mmol), palladium(II) acetate (22 mg, 0.1 mmol), and tri(o-tolyl)phosphine (31 mg, 0.1 mmol) were dissolved in 10 ml of dry DMF. Then, trimethylamine (0.2 ml) was added to the reaction mixture and stirred at 110°C for 12 hours under an argon atmosphere. After cooling the reaction mixture to room temperature, the reaction was quenched with water (30 ml), and the mixture was extracted with ethyl acetate. The collected organic layer was washed with brine, dried with Na2SO4, and concentrated under reduced pressure. The desired residue was purified by column chromatography using n-hexane / ethyl acetate (1 / 1 v / v) as the eluent to produce the desired product TPEPy as a yellow solid (282 mg, 56.8%).

[0075] 1 ¹H NMR (600 MHz, CDCl₃) δ 8.55 (d, 2H), 7.32 (d, 2H), 7.29 (s, 1H), 7.27 (s, 1H), 7.23-7.19 (m, 1H), 7.14-7.09 (m, 3H), 7.05-7.03 (m, 4H), 6.98-6.91 (m, 5H), 6.69 - 6.61 (m, 4H), 3.75 (s, 3H), 3.74 (s, 3H). 13 13C NMR (151 MHz, CDCl3) δ 158.4, 158.3, 150.2, 145.3, 145.0, 144.2, 141.0, 138.7, 136.4, 136.3, 133.9, 133.2, 132.8, 132.7, 132.0, 131.6, 127.9, 126.6, 126.4, 125.5, 120.9, 113.3, 55.2. LC-MS, m / z: [M+H]+ Calculated 496.2, Measured 496.2.

[0076] (5) Synthesis of TPEPyTMB-1

[0077] TPEPy (0.1 mmol, 49.6 mg) and 1,3,5-tris(bromomethyl)benzene (0.1 mmol, 35.6 mg) were dissolved in 10 ml of dry toluene, and the reaction mixture was stirred at 80°C for 12 hours under an argon atmosphere. The progress of the reaction was monitored using TCL. The red precipitate formed during the reaction was filtered, washed with diethyl ether, and dried at room temperature (42 mg, 54.4%).

[0078] 1 ¹H NMR (600 MHz, CDCl₃) δ 9.45 (d, 2H), 7.92-7.75 (m, 2H), 7.6 (d, 1H), 7.29 (s, 1H), 7.27 (s, 1H), 7.23-7.19 (m, 2H), 7.14-7.09 (m, 3H), 7.05-7.03 (m, 2H), 6.98-6.91 (m, 2H), 6.69-6.61 (m, 2H), 6.39 (m, 4H), 4.4 (s, 4H), 3.74 (s, 6H), 2.3. (s, 3H). 13 13C NMR (151 MHz, CDCl3) δ 158.4, 158.3, 152.2, 148.2, 144.6, 144.4, 142.2, 142.0, 140.0, 138.7, 136.4, 136.3, 133.9, 133.2, 132.8, 132.7, 129.4, 129.2, 127.9, 126.6, 124.4, 122.5, 113.9, 113.3, 62.0, 55.2, 32.2. LC-MS, m / z: [M+H]+ Calculated value 771.20, Measured value 772.20.

[0079] (6) Synthesis of TPEPyTMB-2

[0080] Synthesis of TPEPyTMB-2 was carried out in the same manner by varying the molar ratio of TPEPy (0.2 mmol, 99.2 mg), and pure TPEPyTMB-2 was isolated as a red powder (58.2 mg, 48.9%).

[0081] 1¹H NMR (600 MHz, CDCl₃) δ 9.78 (d, 4H,), 7.9 (d, 4H), 7.58 (d, 2H), 7.52 (m, 3H), 7.29 (m, 4H), 7.27 (m, 4H), 7.23-7.19 (m, 4H), 7.14-7.09 (m, 5H), 7.05-7.03 (m, 13H), 6.98-6.91 (m, 6H), 6.69 - 6.51 (m, 5H), 4.57 (s, 4H) 3.75 (s, 12H), 2.24 (s, 2H). 13 13C NMR (151 MHz, CDCl3) δ 158.4, 158.3, 150.2, 145.3, 145.0, 144.2, 141.0, 138.7, 136.4, 136.3, 133.9, 133.2, 132.8, 132.7, 132.0, 131.6, 127.9, 126.6, 126.4, 125.5, 120.9, 113.3, 65.2, 62.0, 55.2, 32.0. m / z: [M+H]+ Calculated value 1185.30, Measured value 1186.30.

[0082] (7) Synthesis of TPEPyTMB-3

[0083] Synthesis of TPEPyTMB-3 was carried out in the same manner by varying the molar ratio of TPEPy (0.3 mmol, 148.8 mg), and pure TPEPyTMB-3 was isolated as a red powder (68 mg, 42.5%).

[0084] 1 ¹H NMR (600 MHz, CDCl₃) δ 9.72 (m, 6H), 7.82 (d, 5H) 7.98 (m, 3H), 7.55 (d, 4H), 7.32 (d, 7H), 7.29 (m, 5H), 7.27 (m, 8H), 7.23-7.19 (m, 25H), 6.69 - 6.61 (m, 8H), 4.5 (s, 6H), 3.75- 3.74 (s, 18H). 13 13C NMR (151 MHz, CDCl3) δ158.4, 158.3, 150.2, 145.3, 145.0, 144.2, 141.0, 138.7, 136.4, 136.3, 133.9, 133.2, 132.8, 132.7, 132.0, 131.6, 127.9, 126.6, 126.4, 125.5, 120.9, 1132.8, 113.3, 62.2, 55.2. HRMS(ESI): m / z calculated value 1599.1014, measured value 1599.2042.

[0085] (8) Synthesis of homologous anion-π+ AIE-PS

[0086] Homologous anion-π+ AIE-PS was synthesized according to Reaction Scheme 1 below. The chemical structure of anion-π+ AIE-PS was confirmed by 1H NMR, 13C NMR, and mass spectrometry.

[0087] Reaction Equation 1

[0088]

[0089] (9) Preparation of Anion-π+ AIE-PSs Nanoparticles (AIE-PSs NPs)

[0090] 0.5 mg of DSPE-PEG was dissolved in 50 μl of ethanol and transferred to a vial containing 0.05 mg of anion-π+ AIE-PS. Then, the solution was thoroughly mixed and vortexed for 1 minute, after which 500 μl of PBS (7.4) was added. The solution was further mixed and placed under vacuum overnight to evaporate the ethanol from the solution. The particle size of the resulting AIE-PSs NPs was measured using a DLS instrument and stored at 4°C for further use.

[0091] (10) In vitro release study

[0092] A 50 μl suspension of AIE-PSs NPs was transferred to a dialysis tube (MWCO = 8 kD). The dialysis tube was immersed in 3 ml of phosphate buffer (pH 7.4) or citrate buffer (pH 5.5) while maintaining a sink condition of 37°C. At predetermined time intervals, the dialysis tube was transferred to an equal volume of fresh buffer. The amount of released photosensitizer was analyzed by UV-Vis spectroscopy with an absorbance of 425 nm and calculated according to the calibration curve.

[0093] (11) ROS generation efficacy evaluation

[0094] The ROS production efficiency of AIE-PSs NPs was investigated using DCFH modified from DCFH-DA as an indicator. 2 ml of a 10 mM NaOH solution was mixed with 0.5 ml of 1 mM DCFH-DA and hydrolyzed at room temperature for 30 minutes. An additional neutralization step was performed using 10 ml of PBS (pH 7.4). A mixture containing 5 μM DCFH and 10 μM AIE-PSs NPs (or Ce6) in PBS (pH 7.4) was exposed to a white light source (100 mW / cm²) for various durations. DCF fluorescence signals were observed in the 500–600 nm region, with an excitation wavelength of 425 nm. The rate of ROS generation was determined by recording the change in fluorescence intensity at 525 nm.

[0095] (12) Singlet oxygen ( 1 O 2 ) Generative research

[0096] The ABDA indicator was used to perform O2 generation measurements. White light (100 mW / cm²) was applied to a mixture of 10 μM ABDA and 10 μM (AIE-PSs NPs or Ce6) dissolved in PBS (pH 7.4) for various durations. 2) was applied. The absorption spectrum of the mixture was tracked in the 300–700 nm region. A decrease in the absorbance of ABDA at 378 nm was observed at various irradiation times. The rate of ABDA degradation was indicated by the decrease in absorbance relative to the original value at 380 nm.

[0097] (13) Peroxide anion (O 2 ·- ) Production efficacy

[0098] DHR123 is O2 ·- It was used as an indicator for measuring generation. A mixture containing 10 μM DHR123 and 10 μM (AIE-PSs NPs or Ce6) in PBS (pH 7.4) was subjected to white light (100 mW / cm²). 2 The mixture was exposed to ) for various periods, the excitation wavelength was set to 488 nm, and the emission spectrum of the mixture was observed in the range of 500–600 nm. Peroxide anion generation was indicated by the intensity of fluorescence observed at 525 nm.

[0099] (14) cell culture

[0100] ATCC (American Type Culture Collection) supplied MCF-7 breast cancer cells. Cells were cultured at 37°C in a 5% CO2 atmosphere using a DMEM culture mixture containing 10% FBS and 1% penicillin-streptomycin.

[0101] (15) Research on ROS generation within cells

[0102] The ability of AIE-PS NPs to generate intracellular ROS in MCF-7 cells was evaluated using DCFDA probes. 5 x 10⁴ per well in confocal culture plates 3After seeding at cell density, a 24-hour growth window was created in MCF-7 cells. The cells were washed before treatment with 1 μM AIE-PSs NPs. Subsequently, the cells were washed for 2 hours and cultured for an additional 30 minutes using fresh culture medium containing 10 μM DCFDA. Then, the cells were washed twice with PBS and cultured under white light (100 mW / cm²) for 20 minutes. 2 Cells that were not treated with laser were used as a control. Using a confocal microscope (Nikon Eclipse Ti-S, Nikon, Tokyo, Japan), green images were captured between 500 and 560 nm with an excitation of 488 nm.

[0103] (16) Confocal joint localization nine

[0104] Confocal co-localization studies were performed to evaluate the enrichment ability of AIE-PSs NPs in mitochondria. 5×10 cells were placed in a glass-bottom dish (35 mM). 5 Cells were seeded at a certain density and cultured overnight in a CO2 incubator. Cells were treated with 1 μM AIE-PSs NPs in a CO2 incubator for 2 hours and washed three times with PBS. Subsequently, cells were stained with Mito-Tracker (0.5 μM) at 37°C for 20 minutes. For AIE-PSs NPs, excitation was 425 nm and emission was 600–800 nm. In contrast, the excitation and emission of MitoTracker Green were 488 nm and 505–530 nm, respectively. Fluorescence images were analyzed using NIS-E software, and the co-locations between AIE-PSs NPs and organelle probes were calculated using Mander's overlay coefficient (MOC).

[0105] (17) Mitochondrial dysfunction research

[0106] To conduct a study on mitochondrial dysfunction, MCF-7 cells were used in a 12-well plate (3 x 10 5Cells were cultured in wells for 24 hours. After removing the cell culture medium, cells were treated with various concentrations of AIE-PSs NPs (0.1–5.0 μM) at 37°C for 4 hours. Then, they were rinsed with PBS buffer. Under white light (100 mW / cm²) -2 The solution was applied to the cells for 20 minutes. JC-1 solution (10 μg / mL) was added to PBS, and the cells were stained in the dark for 10 minutes, followed by two washes with PBS buffer. Finally, a fluorescence microscope (Nikon Eclipse Ti-S) was used to convert the cell fluorescence images.

[0107] (18) In vitro PDT evaluation under normoxic and hypoxic conditions

[0108] The in vitro PDT efficacy of AIE-PSs NPs in MCF-7 cells was measured by the MTT assay. For oncogenicity, cells were plated in a 96-well plate (5x10 3 Cells were seeded into wells and cultured for 12 hours. Cells were treated with various concentrations of AIE-PSs NPs (0.1–5.0 μM) for 24 hours. Subsequently, medium containing 10% EZ-cytox solution was added to 100 μl of serum-free medium containing each cell. Afterward, absorbance at 450 nm was measured using a microplate reader, and cell viability (%) was calculated using Equation 1 below. Additionally, MCF-7 cells were seeded into 96-well plates and PDT was performed under normal oxygen or hypoxic conditions. After applying various concentrations of AIE-PSs NPs (0.1–5.0 μM) to the cells, they were cultured for 8 hours under normal oxygen conditions (21% O2 and 5% CO2) or hypoxic conditions (1% O2 and 5% CO2). Next, the cells were exposed to a white light source (100 mW / cm-2) for 20 minutes and then cultured for an additional 24 hours. Finally, relative cell viability was estimated by applying Equation 1.

[0109]

[0110] (19) Analysis of live / dead cell co-staining under normoxia and hypoxia

[0111] MCF-7 cells were grown in a confocal imaging dish at 37°C. Then, after treatment with AIE-PSs NPs (5 μM), the cells were maintained in a normoxic and hypoxic environment for 8 hours. Subsequently, the cells were irradiated with a white light source (100 mW / cm-2) for 20 minutes and cultured for an additional 24 hours. Afterward, the cells were stained with FITC (1 μM) at 37°C for 20 minutes, and the images were inverted using CLSM. For FITC, the excitation was 488 nm and the emission was 500–540 nm.

[0112] (20) Animal and Tumor Models

[0113] All animal procedures followed the guidelines established by the Animal Ethics Committee of Gachon University and were approved by the Korean Animal Care and Use Committee (Approval No. LCDI-2020-0076). Male BALB / c mice weighing 20–25 g were used in the experiment. The mice were 5 weeks old. The animals used in the in vivo study were divided into four groups: (1) PBS, (2) PBS + light, (3) AIE-PSs NPs, and (4) AIE-PSs NPs + light (n = 4). All experimental procedures were performed starting the day after randomization.

[0114] (21) In vivo fluorescence imaging

[0115] To evaluate the accumulation of TPEPyTMB-3 NPs in the tumor region, 5 μl of TPEPyTMB-3 NPs were injected intratumorally into MCF-7 tumor-bearing mice. In vivo fluorescence images were recorded at various time points (1, 3, 6, 24, 36, 48, 72, 96, and 168 hours) using an IVIS Spectrum imaging system (excitation: 420 nm and emission: 650 nm). To investigate the in vitro fluorescence distribution of major organs and tumors, mice were slaughtered after 168 hours, and major organs (heart, liver, spleen, lung, and kidney) and tumors were excised.

[0116] (22) In vivo photodynamic therapy

[0117] To investigate the in vivo anticancer efficacy of TPEPyTMB-3 NPs, four groups consisting of MCF-7 mice harboring four tumors—PBS, PBS + light, TPEPyTMB-3 NPs, and TPEPyTMB-3 NPs + light—were constructed. The tumor volume was approximately 100 mm³ 3 Upon reaching [the threshold], 2.5 mg / ml of TPEPyTMB-3 NPs and 5 μl of PBS were injected intratumorally into the mice. After 24 hours, a white light source (100 mW cm²) was applied to the tumors of the groups (PBS + light and TPEPyTMB-3 NPs + light). -2 ) was irradiated for 20 minutes. Tumor volume and body weight of the mice were measured daily for 15 days. Tumor size is calculated using the formula V = (tumor length x tumor width) 2 It was measured using ) / 2. In addition, to visualize the efficient anti-tumor effect, photos of mice with tumors were taken from day 0 to day 14.

[0118] (23) In vivo biosafety measurement

[0119] After a 14-day treatment period, the mice were euthanized and the tumors were removed. The tumors were then embedded in paraffin, incised to a thickness of 5 μm, and fixed overnight in 4% (v / v) formalin. The slices were then stained using immunohistochemical TUNEL, H&E staining, and CD31 labeling to determine the degree of tumor cell apoptosis. Finally, the stained slices were imaged using an inverted optical microscope and a fluorescence microscope. To investigate the biological safety of the treatment, major organs (heart, liver, spleen, lungs, and kidneys) were excised from each mouse, and the same techniques were used for H&E staining.

[0120] 3. Results

[0121] (1) Synthesis and Characterization of AIE-PS

[0122] Targeted homologous anion-π+ AIE-PS (TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3) were synthesized according to the synthesis pathway presented in Reaction Scheme 1. The most widely accepted DA strategy utilized TPE and cationic pyridinium (Py) as the donor and acceptor units, respectively, to synthesize the desired anion-π+ AIE-PS. To control the molecular aggregation of the anion-π+ AIE-PS, one, two, and three molecular rotors (TPEPy) were introduced into the structures of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3, respectively. Higher molecular aggressiveness and atomic effects ((Br) collectively promote an effective ISC process. - By utilizing the benefits of the presence of anions, the ROS generation efficiency of anion-π+ AIE-PS can be significantly improved. The chemical structures of TPETMB-1, TPETMB-2, and TPETMB-3 were confirmed by 1H NMR, 13C NMR, and mass spectrometry.

[0123] Figure 1 shows TPEPyTMB-1's 1 This is the H NMR spectrum, and Figure 2 is of TPEPyTMB-1. 13This is the C NMR spectrum, and Figure 3 is the LC-MS spectrum of TPEPyTMB-1.

[0124] Figure 4 shows TPEPyTMB-2's 1 This is the 1H NMR spectrum, and Fig. 5 is of TPEPyTMB-3. 13 This is the C NMR spectrum, and Figure 6 is the LC-MS spectrum of TPEPyTMB-3.

[0125] Figure 7 shows TPEPyTMB-3's 1 This is the H NMR spectrum, and Fig. 8 is of TPEPyTMB-3. 13 This is the C NMR spectrum, and Figure 9 is the HRMS spectrum of TPEPyTMB-3.

[0126] Figure 10 is the absorbance spectrum of anion-π+ AIE-PS in DMSO. Here, (A) absorbance spectrum of anion-π+ AIE-PS in DMSO; (B) fluorescence spectrum of anion-π+ AIE-PS in a DMSO / PBS mixture; (CE) PL intensity of anion-π+ AIE-PS in DMSO / PBS mixtures in various PBS fractions; (F) plot of relative PL intensity (I / I0) versus PBS fractions; (G) ROS generation of anion-π+ AIE-PS NPs (10 μM) using DCFH; (H) of anion-π+ AIE-PS NPs (10 μM) using ABDA 1 O2 generation; (I) Light irradiation (100 mW / cm²) 2 O2 of anion-π+ AIE-PSs NPs (10 μM) using DHR123 at ) ·- Indicates creation.

[0127] As shown in Fig. 10, all three anion-π+ AIE-PSs exhibited absorption peaks in the UV-Vis range at 422, 425, and 427, respectively, which can be excited under a white light source. The AIE performance of all anion-π+ AIE-PSs was investigated in DMSO / PBS mixtures with various PBS fractions. While all anion-π+ AIE-PSs exhibited weak photoluminescence (PL) intensity in DMSO, the PL intensity of anion-π+ AIE-PSs gradually increased as the proportion of PBS in the DMSO / PBS mixture increased. As can be seen in the CF of Fig. 10, after adding a 90% fraction of PBS, the PL intensities of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 improved by 34.7, 41.2, and 56.5 times, respectively, indicating an excellent AIE trend. Among them, TPEPyTMB-3 exhibited higher PL intensity and a superior AIE tendency, which can be attributed to a more twisted configuration that severely restricts molecular rotation in the aggregated state. All anion-π+ AIE-PSs displayed broad fluorescence spectra in the 550–800 nm range, with maximum emissions located at 642, 645, and 648 nm, respectively (Fig. 10B). Additionally, all anion-π+ AIE-PSs exhibited a large Stokes shift (> 220 nm) and a long emission tail into the NIR region, suggesting suitability for image-induced PDT.

[0128] (2) Development and Characterization of Anion-π+ AIE-PSs NPs

[0129] To maximize the therapeutic effect of anion-π+ AIE-PS in PDT, biocompatible anion-π+ AIE-PS NPs were fabricated using DSPE-PEG polymer via nanoprecipitation. The particle size of the anion-π+ AIE-PS NPs was confirmed through dynamic light scattering (DLS) analysis.

[0130] Figure 11 shows the particle size distribution of (A) TPEPyTMB-1 NPs, (B) TPEPyTMB-2 NPs, and (C) TPEPyTMB-3 NPs in PBS (pH 7.4). As shown here, the average particle sizes of TPEPyTMB-1 NPs, TPEPyTMB-2 NPs, and TPEPyTMB-3 NPs were 107.0 ± 1.22 nm, 111.9 ± 2.14 nm, and 115.2 ± 1.48 nm, respectively, which indicates suitability for passive cell accumulation.

[0131] Figure 12 shows the absorbance and PL spectra of TPEPyTMB-1 NPs, TPEPyTMB-2 NPs, and TPEPyTMB-3 NPs. As can be seen here, TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 were almost similar to PS.

[0132]

[0133] As listed in Table 1 above, the fluorescence quantum yields (ΦF) for TPEPyTMB-1 NPs, TPEPyTMB-2 NPs, and TPEPyTMB-3 NPs were determined to be 8.49%, 11.81%, and 15.08%, respectively. The ΦF value of TPEPyTMB-3 NPs was significantly higher than that of TPEPyTMB-1 and TPEPyTMB-2 NPs due to their excellent aggregation tendency. The photostability of AIE-PSs NPs was [assumed] under visible light irradiation (100 mW / cm² 2 It was evaluated under , 20 minutes.

[0134] Figure 13 shows the absorption spectra before and after irradiation. Here, (A) the absorption spectra of TPEPyTMB-1 NPs; (B) TPEPyTMB-2 NPs; and (C) TPEPyTMB-3 NPs. As can be seen in Figure 13, the absorbance of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs remained almost constant, which demonstrates excellent photostability for image-induced PDT applications.

[0135] (3) Observation of ROS generation

[0136] ROS generation by anion-π+ AIE-PSs NPs was studied using 2',7'-dichlorodihydrofluorescein (DCFH) as a ROS indicator.

[0137] Figure 14 shows the PL spectrum of DCFH + TPEpyTMB-3 NPs and the relative ROS generation efficiency of AIE-PS NPs and Ce6 under white light irradiation. Specifically, white light (100 mW cm⁻¹) -2 (A) PL spectra of pure DCFH (5μM); (B) DCFH + Ce6 (10μM); (C) DCFH + TPEPyTMB-1 NPs (10μM); (D) DCFH + TPEPyTMB-2 NPs (10μM); (E) DCFH + TPEPyTMB-3 NPs (10μM) upon irradiation; and (F) the relative ROS generation efficiency of AIE-PS NPs and Ce6. As shown here, DCFH alone exhibited a very weak PL intensity upon white light irradiation. However, the PL intensity of DCFH gradually increased as it was excited by anion-π+ AIE-PSs NPs. The PL intensity of DCFH reached approximately 6.6 times, 14.6 times, and 27.7 times, respectively, in the presence of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs, which represent ROS production efficiency. Additionally, AIE-PS NPs demonstrated better ROS generation capabilities compared to commercially available PS (Chlorin e6, Ce6).

[0138] To better distinguish ROS, 9,10-anthracendiyl-bis(methylene)-dimalonic acid (ABDA) and dihydrorhodamine 123 (DHR123) were used, respectively 1 O2 and O2 ·- The generation of was evaluated.

[0139] Figure 15 shows the absorption spectrum of ABDA+TPEPyTMB-3 NPs and the relative values ​​of AIE-PSs NPs and Ce6 upon white light irradiation. 1 This indicates the O2 generation efficiency. Specifically, white light (100mW cm⁻¹) -2 ) Absorption spectra of (A) pure ABDA (5μM); (B) ABDA+Ce6 (10μM); (C) ABDA+TPEPyTMB-1 NPs (10μM); (D) ABDA+TPEPyTMB-2 NPs (10μM); (E) ABDA+TPEPyTMB-3 NPs (10μM) and; (F) relative to AIE-PSs NPs and Ce6 upon irradiation 1 This represents the O2 generation efficiency. As shown here, the absorption peak of blank ABDA (378 nm) hardly changed upon irradiation, whereas the absorption peak decreased upon irradiation in the presence of anion-π+ AIE-PSs NPs, which is 1 This indicates the generation of O2. Among these, TPEPyTMB-3 NPs were 1.2 times, 1.5 times, and 1.7 times compared to TPEPyTMB-1, TPEPyTMB-2, and Ce6, respectively. 1 O2 generation efficiency was shown. This can be explained based on molecular aggregation, which can improve the ROS generation efficiency of TPEPyTMB-3 NPs by amplifying the ISC rate.

[0140] Figure 16 shows the absorption spectrum of DHR123+TPEPyTMB-3 NPs and the relative values ​​of AIE-PSs NPs and Ce6 upon white light irradiation. 1 This indicates the O2 generation efficiency. Specifically, white light (100mW cm⁻¹) -2) Absorption spectra of (A) pure DHR123 (5 μM); (B) DHR123+Ce6 (10 μM); (C) DHR123+TPEPyTMB-1 NPs (10 μM); (D) DHR123+TPEPyTMB-2 NPs (10 μM); (E) DHR123+TPEPyTMB-3 NPs (10 μM) and; (F) relative to AIE-PSs NPs and Ce6 upon irradiation 1 This represents the O2 generation efficiency. As shown here, in the absence of anion-π+ AIE-PSs NPs, blank DHR 123 and DHR123 + Ce6 exhibited negligible PL intensity under white light irradiation. However, the PL intensity of DHR123 was significantly enhanced in the presence of anion-π+ AIE-PSs NPs, indicating that the anion-π+ AIE-PSs NPs [assist] O2 ·- This demonstrates that it can generate O2 1.5 times and 2.8 times that of TPEPyTMB-1 NPs and TPEPyTMB-2 NPs, respectively. ·- Generation efficiency was demonstrated. This can be explained based on polymer aggregation, which enhances ROS generation efficiency by further promoting the ISC process through AI-ISC (crossing between aggregation-inducing systems). Therefore, all anion-π+ AIE-PS NPs are Type 1 (O2) capable of overcoming the hypoxia problem of PDT. ·- ) and Type 2( 1 O2) Can generate all ROS.

[0141] (4) Detection of ROS inside cells

[0142] To detect ROS production in MCF-7 cells, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a ROS indicator. DCFH-DA is a cell-permeable substance that is oxidized to 2',7'-dichlorodihydrofluorescein (DCF), which emits green fluorescence upon contact with ROS. To analyze ROS production inside MCF-7 cells, the PL intensity of DCFH-DA was observed after 5 minutes of irradiation, regardless of the presence or absence of anion-π+ AIE-PSs NPs.

[0143] Figure 17 shows the results of ROS detection inside the cell. Here, (A) detection of ROS inside MCF-7 cells after treatment with anion-π+ AIE-PSs NP (5 μM) (with or without light irradiation, scale bar (50 μm)); (B) PL intensity corresponding to ROS generation after treatment with anion-π+ AIE-PSs NP (*** p < 0.001); (C) co-localization image of MCF-7 cells stained with mitotracker green or anion-π+ AIE-PSs NP, scale bar (20 μm); (D) fluorescence intensity and (E) Mander overlap factor of cells treated with anion-π+ AIE-PS NP (*** p < 0.001). As shown here, cells treated with DCFH + anion-π+ AIE-PSs NPs + light exhibited stronger PL intensity compared to the light-free control (DCFH + anion-π+ AIE-PSs NPs). Cells treated with TPEPyTMB-3 NPs exhibited the highest PL intensity (B), which implies that TPEPyTMB-3 NPs generate a greater amount of ROS inside MCF-7 cells than TPEPyTMB-1 and TPEPyTMB-2 NPs.

[0144] (5) Mitochondrial target specificity

[0145] This is because all anion-π+ AIE-PSs exhibit a cationic pyridinium moiety that demonstrates mitochondrial target specificity. Therefore, co-localization analysis was performed using a commercially available bioprobe (Mito-Tracker Green). The co-localization study was conducted after treating MCF-7 cells with anion-π+ AIE-PSs NPs (1 μM) for 2 hours. The co-localization images (Fig. 17C,D) showed that the red fluorescence of all three anion-π+ AIE-PSs efficiently overlapped with Mito-Tracker Green.

[0146] Pearson correlation coefficients (Rr) for TPEPyTMB-1 NPs, TPEPyTMB-2 NPs, and TPEPyTMB-3 NPs were obtained as 0.54, 0.57, and 0.60, respectively (Fig. 17E). These results demonstrate that all three anion-π+ AIE-PSs exhibit mitochondrial targeting capabilities. Among these anion-π+ AIE-PS NPs, TPEPyTMB-3 NPs, in particular, exhibited higher mitochondrial specificity due to the availability of a greater number of cationic pyridinium residues, which induces greater electrostatic interactions with the negative transmembrane potential of mitochondria.

[0147] (6) Mitochondrial destruction assessment

[0148] Since AIE-PSs NPs exhibited mitochondrial target specificity, the effects of ROS on mitochondria were evaluated by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining analysis. As probe JC-1 exhibited different colors in different states (monomer and aggregate), changes in mitochondrial membrane potential (MMP) could be qualitatively detected based on color changes. Additionally, the ratio of red to green fluorescence intensity was used to quantify detections resulting from mitochondrial depolarization. The dye JC-1 emitted red fluorescence upon forming an aggregate state in mitochondria. As MMP decreases, the fluorescence of JC-1 changes from red to green due to the transition from the aggregate to the monomer state.

[0149] Figure 18 shows the results of the mitochondrial disruption assessment. Here, (A) confocal image of treated cells stained with JC-1 under white light irradiation for 20 minutes (scale bar = 50 μm); (B) red / green fluorescence ratio of JC-1 (*p< 0.05, **p< 0.01); (C) cell viability of AIE-PSs NPs against MCF-7 cells in Normoxia (21% O2); (D) hypoxic (1% O2) environment under white light irradiation; and (E) confocal microscopy image of apoptosis in MCF-7 cells 24 hours after treatment with anion-π+ AIE-PSs NPs in a hypoxic environment (scale bar = 100 μm). As shown in Figure 18, the control group + experimental group emits red fluorescence without light irradiation, which indicates a minor change in MMPs. However, cells treated with AIE-PSs NPs (upon light irradiation) emitted green fluorescence, indicating mitochondrial dysfunction caused by ROS generation. As can be seen in Fig. 18B, TPEPyTMB-3 NPs exhibited the lowest red / green fluorescence ratio compared to TPEPyTMB-1 NPs and TPEPyTMB-2 NPs, demonstrating that TPEPyTMB-3 NPs potentially damage mitochondria and possess higher mitochondrial targeting ability.

[0150] (7) Emission profile of AIE-PS

[0151] The release behavior of anion-π+ AIE-PS from anion-π+ AIE-PSs NPs was performed using a dialysis tube (MWCO = 8000) immersed in 3 ml of phosphate buffer (pH 7.4) or citrate buffer (pH 5.5). The dialysis tube was transferred to the same volume of fresh buffer at different time intervals. The amounts of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 PS released from the nanoparticles were estimated by observing the absorbance at 425 nm.

[0152] Figure 19 shows the release profiles of (A) TPEPyTMB-1, (B) TPEPyTMB-2, and (C) TPEPyTMB-3 PS from AIE-PSs NPs under various physiological conditions. Anion-π+ AIE-PS exhibited a slow release rate at physiological pH (7.4), and approximately 44.26%, 41.67%, and 31.67% of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 were released from anion-π+ AIE-PSs NPs for more than 60 hours. However, at acidic pH (5.5), the release rate was higher than at physiological pH (7.4), and approximately 82.38%, 79.88%, and 76.96% of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 were released from AIE-PSs NPs for more than 60 hours. The release of anion-π+ AIE-PS from anion-π+ AIE-PSs NPs is accelerated by the degradation of anion-π+ AIE-PSs NPs at acidic pH (5.5). Overall, the results demonstrated that AIE-PS is effectively released in the acidic environment of tumor cells.

[0153] (8) Evaluation of cell viability under hypoxic and normal oxygen conditions

[0154] Since all three anion-π+ AIE-PSs NPs have the ability to generate type 1 and type 2 ROS, considering these advantages, the in vitro PDT effect of anion-π+ AIE-PSs NPs was investigated under normal oxygen (21% O2) and hypoxia (1% O2) conditions as determined by MTT analysis.

[0155] Figure 20 shows the cell viability of MCF-7 cells treated with TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs under dark conditions. As shown here, the anion-π+ AIE-PSs NPs exhibited excellent biocompatibility, as they did not show carcinogenicity to MCF-7 cells even when treated at high concentrations. TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs were irradiated with white light (100 mW / cm²). 2 During 20 minutes, significant dose-dependent cytotoxicity was exhibited in both normal oxygen (21% O2) and hypoxia (1% O2). As shown in Fig. 18C, cell viability under normal oxygen conditions (21% O2) decreased to 63, 67, and 78% when treated with 5 μM of TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs, respectively. However, under hypoxic conditions (1% O2), cell viability decreased to 52, 58, and 66%, respectively.

[0156] Compared to TPEPyTMB-1 and TPEPyTMB-2 NPs, in which the PDT effect was inhibited by hypoxia, TPEPyTMB-3 NPs exhibited a better PDT effect under hypoxia, which is due to higher type-1 (O2 ·- This may be due to ROS generation efficiency (Fig. 18D). The PDT effect of anion-π+ AIE-PSs NPs under hypoxic conditions (1% O2) was further visualized using a viable / dead cell co-staining assay (Annexin V / PI) by CLSM. As shown in Fig. 18E, compared to TPEPyTMB-1 NPs and TPEPyTMB-2 NPs, TPEPyTMB-3 NPs exhibited significant red fluorescence from PI in a hypoxic (1% O2) environment upon white light irradiation, while some green fluorescence showed low PDT efficiency, which differs from TPEPyTMB-3 NPs.

[0157] Figure 21 is a confocal microscopy image (scale bar = 100 μm) of apoptosis in MCF-7 cells after 24 hours of culture with 1 μM TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3 NPs in a hypoxic environment under dark conditions. As shown here, among the three anion-π+ AIE-PS NPs, TPEPyTMB-3 NPs, in particular, exhibited greater cytotoxicity under hypoxia compared to TPEPyTMB-1 and TPEPyTMB-2 NPs. The cytotoxicity of TPEPyTMB-3 NPs was also evaluated in a hypoxic environment (1% O2), and no distinct cytotoxicity was observed.

[0158] In summary, these results confirmed that TPEPyTMB-3 NPs can generate ROS not only in a normal oxygen environment but also under hypoxic conditions for efficient PDT.

[0159] (9) In vivo imaging

[0160] Inspired by the excellent photophysical properties and in vitro therapeutic performance of TPEPyTMB-3 NPs, the in vivo fluorescence imaging capabilities of TPEPyTMB-3 NPs were investigated in nude mice carrying breast cancer tumors (MCF-7).

[0161] Figure 22 shows the in vivo imaging results of TPEPyTMB-3 NPs. Here, (A) real-time in vivo images of mice after intratumoral injection of TPEPyTMB-3 NPs (5 µl); (B) time course of the standardized average fluorescence intensity of the tumor region at each time point determined by IVIS fluorescence imaging; (C) in vitro images of each organ at each time point after intratumoral injection of TPEPyTMB-3 NPs.

[0162] As shown in Fig. 22, in vivo real-time fluorescence images of the tumor (A) were collected at different time intervals using an IVIS fluorescence imaging system after intratumoral injection of TPEPyTMB-3 NPs (5 μl, 2.5 mg / ml). The standardized fluorescence intensity of the tumor site gradually increased with increasing culture time and reached a maximum value within 24 hours. After 24 hours, it began to decrease as TPEPyTMB-3 NPs gradually accumulated in the tumor site (B). TPEPyTMB-3 NPs gradually accumulated in the tumor site and showed maximum tumor accumulation at 168 hours after injection. Importantly, the tumor site exhibited strong NIR fluorescence on day 7 after injection, demonstrating the long-term imaging capability of TPEPyTMB-3 NPs. Subsequently, the mice were sacrificed, and major organs were removed for in vitro fluorescence imaging at 168 hours after injection. As indicated in Fig. 22C, the fluorescence signal was primarily located in the tumor tissue. These results show that TPEPyTMB-3 NPs effectively accumulate in the tumor site and can serve as a promising imaging agent during PDT treatment.

[0163] (10) In vivo phototherapy efficacy

[0164] After confirming in vitro cytotoxicity and in vivo fluorescence imaging capabilities, the in vivo phototherapeutic efficacy of TPEPyTMB-3 NPs was investigated in MCF-7 tumor-carrying mice. Initial tumor volume (100 mm³) 3Mice with (1) TPEPyTMB-3 NPs were divided into four groups: PBS, PBS + light, TPEPyTMB-3 NPs, and TPEPyTMB-3 NPs + light, and various treatments were performed for 14 days. As controls, mice administered TPEPyTMB-3 NPs without white light irradiation or mice administered saline regardless of white light irradiation were used. For the PDT group, TPEPyTMB-3 NPs (5 μl, 2.5 mg / mL in PBS) were injected into each mouse via intratumoral injection, and white light was irradiated for 20 minutes 24 hours after injection. For the control group, the same dose of TPEPyTMB-3 NPs and PBS were injected into the tumor without light irradiation. Tumor size was monitored every 2 days during the 15-day treatment period to obtain relative growth curves.

[0165] Figure 23 shows the results of the in vivo phototherapy evaluation of TPEPyTMB-3 NPs. As shown here, (A) in vivo tumor growth rates for various treatment groups over 14 days; (B) a photograph of the resected tumor on day 14; (C) body weight change over 14 days (n=4); (D) a representative LSCM image of a tumor slice from an MCF-7 tumor-bearing mouse after treatment with TPEPyTMB-3 NPs on day 14 (scale bar: 20 µm); (E) H&E staining and apoptosis analysis of the TUNEL stained image of the tumor slice from an MCF-7 tumor-bearing mouse.

[0166] As shown in Figure 23, the tumor volume of the control group increased rapidly during the study period, reaching nearly 10 times its initial size, whereas the group treated with TPEPyTMB-3 NPs + light showed significant inhibition of tumor progression. Furthermore, a tumor inhibition rate of 75% was achieved after 14 days of treatment with TPEPyTMB-3 NPs, suggesting potent efficacy of photodynamic therapy. To visually demonstrate the treatment results more directly, tumor tissues were collected from each mouse after photodynamic therapy.

[0167] As shown in Figure 23, the tumor volume of the photodynamic therapy group was much lower than that of the other control group, indicating that ROS generated by TPEPyTMB-3 NPs during irradiation can effectively inhibit tumor development. Furthermore, there was no noticeable difference in weight loss between the treatment group and the control group, as well as between the TPEPyTMB-3 NPs + light group, suggesting that TPEPyTMB-3 NPs do not cause serious side effects.

[0168] In addition, LSCM images of tumor fragments after treatment with TPEPyTMB-3 NPs showed that TPEPyTMB-3 NPs were still observed in the tumors even on day 14 after treatment (Fig. 23D). To further investigate the treatment process, dissected tumors were evaluated histologically (hematoxylin and eosin staining, H&E staining) and immunohistochemically (TUNEL) (Fig. 23E). Compared to the PBS, light, and TPEPyTMB-3 NPs groups, H&E staining of the TPEPyTMB-3 NPs + light group showed low cell proliferation and the highest levels of apoptosis. To investigate the systemic toxicity of TPEPyTMB-3 NPs, NPs were injected into healthy nude mice, and major organs were taken for histological examination.

[0169] Figure 24 shows the H&E staining results of major organs in mice on day 14 after treatment with PBS and TPEPyTMB-3 NPs. As seen here, no severe organ damage was found, which demonstrates the biocompatibility of TPEPyTMB-3 NPs. These findings support the remarkable anticancer activity of TPEPyTMB-3 NPs upon irradiation.

[0170] As described above, in the present invention, homologous anion-π+ AIE-PS (TPEPyTMB-1, TPEPyTMB-2, and TPEPyTMB-3) equipped with a potent donor-acceptor (DA) configuration and mitochondrial targeting function were successfully synthesized by controlling molecular aggregation. All anion-π+ AIE-PSs exhibited emission in the red-shift region with a tail in the NIR region. Furthermore, all anion-π+ AIE-PSs exhibited excellent ROS(O2 · , 1 O2 generation efficiency was demonstrated. Among them, trimer TPEPyTMB-3 NPs, which exhibit superior AIE characteristics and excellent Type 1 and Type 2 ROS generation efficiencies, demonstrated efficient in vitro PDT effects in both normoxic and hypoxic environments. Furthermore, in vivo results demonstrate the potential of trimer TPEPyTMB-3 NPs for long-term in vivo imaging and effective photodynamic therapy under hypoxic conditions. Therefore, the present invention provides a new paradigm for the future development of Type-1 AIE-PSs for long-term in vivo imaging and effective PDT under hypoxic conditions.

[0171] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 Compound represented by the following chemical formula 1: [Chemical Formula 1] Claim 2 Oxygen-independent aggregation-induced luminescence photosensitizer comprising the compound of claim 1. Claim 3 In claim 2, the oxygen-independent aggregation-induced luminescence photosensitizer is characterized by specifically accumulating in mitochondria and causing damage to mitochondria. Claim 4 An oxygen-independent aggregation-induced luminescence photosensitizer, characterized in that, in claim 2, the compound is an encapsulated nanoparticle. Claim 5 A photodynamic therapy composition for cancer treatment comprising a photosensitizer according to any one of claims 2 to 4 as an active ingredient. Claim 6 A composition according to claim 5, wherein the cancer is selected from breast cancer, kidney cancer, testicular cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, vaginal cancer, fallopian tube cancer, rectal cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, small intestine cancer, pancreatic cancer, oral cancer, melanoma, or sarcoma. Claim 7 Compound represented by the following chemical formula 2: [Chemical Formula 2] Claim 8 Oxygen-independent aggregation-induced luminescence photosensitizer comprising the compound of claim 7. Claim 9 In claim 8, the oxygen-independent aggregation-induced luminescence photosensitizer is characterized by specifically accumulating in mitochondria and causing damage to mitochondria. Claim 10 An oxygen-independent aggregation-induced luminescence photosensitizer, characterized in that, in claim 8, the compound is an encapsulated nanoparticle. Claim 11 A photodynamic therapy composition for cancer treatment comprising a photosensitizer of any one of claims 8 to 10 as an active ingredient. Claim 12 Compound represented by the following chemical formula 3: [Chemical Formula 3] Claim 13 Oxygen-independent aggregation-induced luminescence photosensitizer comprising the compound of claim 12. Claim 14 In claim 13, the oxygen-independent aggregation-induced luminescence photosensitizer is characterized by specifically accumulating in mitochondria and causing damage to mitochondria. Claim 15 An oxygen-independent aggregation-induced luminescence photosensitizer, characterized in that, in claim 13, the compound is an encapsulated nanoparticle. Claim 16 A photodynamic therapy composition for cancer treatment comprising a photosensitizer of any one of claims 13 to 15 as an active ingredient. Claim 17 A photodynamic therapy method comprising: 1) administering the composition of claim 16 to a subject other than a human; 2) allowing time for the administered composition to accumulate within a target cell of the subject other than a human; and 3) irradiating light to a target cell site of the subject other than a human. Claim 18 A method according to claim 17, wherein the administering step of step 1) is characterized by administering orally or parenterally. Claim 19 In claim 17, the step of irradiating light in step 3) is 100 to 300 mW / cm² for 10 seconds to 2 hours. 2 A method characterized by irradiating light. Claim 20 A method according to claim 17, characterized in that the light in step 3) is white light.

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