KR20230174847A - Nano-theranostics for single-laser-activated phototherapy having mitochondria specificity - Google Patents

Abstract

The present invention relates to a photosensitizer containing a compound represented by the following [Chemical Formula 1]:
[Formula 1]
.

Description

{Nano-theranostics for single-laser-activated phototherapy having mitochondria specificity}

The present invention relates to nanotheranostics for mitochondria-specific light therapy.

Photodynamic therapy (PDT) is attracting attention as a non-invasive and safe therapeutic modality for the treatment of malignant tumors, a treatment that relies on light-mediated activation of photosensitizers (PS) to generate reactive oxygen species (ROS). am. The generated ROS causes serious oxidative damage to tumor tissue through DNA and protein damage and induces apoptosis of cancer cells. However, there is a problem that the therapeutic effect is limited by intrinsic limitations such as tumor hypoxia and the short lifespan of the generated ROS.

Photothermal therapy (PTT), another type of light therapy that converts light energy into heat by photothermal agents (PTAs) to induce irreversible cancer cell damage, is attracting attention as an alternative treatment option for cancer therapy. (Chu, K.F et al. (2014). Thermal ablation of tumors: biological mechanisms and advances in therapy. Nat. Rev. Cancer 14, 199-208). Like PDT, PTT also has limitations, such as low tissue penetration of the excitation light, low photothermal conversion efficiency, and stability of the PTT agent.

Additionally, due to the complementary characteristics of PDT and PTT, the combination of PDT and PTT has recently been attracting attention as an effective anticancer treatment strategy. For example, PDT acts to overcome resistance due to high heat shock protein-mediated resistance to PTT (Calderwood, S.K., and Ciocca, D.R. (2008). Heat shock proteins: stress proteins with Januslike properties in cancer. Int. J. Hyperthermia. 24, 31-39), PTT may play a role in overcoming hypoxia-mediated resistance of cancer cells (Zhang, J et al. (2020). Activatable molecular agents for cancer theranostics. Chem. Sci. 11, 618-630). Additionally, ROS generated during the PDT process can improve the tumor cell permeability of drug conjugates and make the PTT process more sensitive (Ye, S et al. (2018). Rational design of conjugated photosensitizers with controllable photoconversion for dually cooperative phototherapy. Adv. Mater. 30, 18012161). In addition, further integration with tumor diagnostics can achieve real-time monitoring of the entire treatment process. However, integrating multiple imaging and therapeutic agents into a single platform can be challenging due to low encapsulation efficiency, premature leakage, and lengthy preparation procedures. Additionally, PDT and PTT require different chemical agents and wavelength light sources, which may make clinical use more complicated. Therefore, developing a single-wavelength laser-activated diagnostic system consisting of both diagnostic and therapeutic functions could be a promising approach for image-guided coupled PDT/PTT for cancer treatment.

The present invention seeks to provide a novel single laser-activated nanotheranostics for phototherapy that specifically targets mitochondria and can exhibit excellent photodynamic and photothermal treatment effects.

In order to solve the above problems, the present invention

Provided is a photosensitizer containing a compound (MsPDTT) represented by the following [Chemical Formula 1]:

[Formula 1]

.

According to the present invention, the compound may be characterized as specifically accumulating in mitochondria.

According to the present invention, the compound may be characterized as having photodynamic and photothermal therapeutic effects.

In addition, the present invention provides nanoparticles (MsPDTT NPs) comprising a compound represented by the following [Formula 1] and a polymeric lipid, and wherein the compound is encapsulated by the polymeric lipid:

[Formula 1]

.

According to the present invention, the polymerized lipid may be DSPE-mPEG.

Additionally, the present invention provides a composition for treating or diagnosing cancer containing the photosensitizer or the nanoparticles.

According to the present invention, it is possible to provide a novel single laser-activated nanotheranostics for phototherapy that specifically targets mitochondria and can exhibit excellent photodynamic and photothermal treatment effects.

Figure 1 shows the characterization and in vitro PDT/PTT properties of MsPDTT NPs and RsPDTT NPs, and (A) shows the absorbance spectrum and encapsulation efficiency of MsPDTT. (B) shows DLS and TEM (inset) images of MsPDTT NPs. Scale bar, 200 nm. (C) shows the photoacoustic (PA) spectrum of MsPDTT NPs and RsPDTT NPs. (D) shows the concentration-dependent photothermal properties of MsPDTT NPs when irradiated with a 690 nm laser at a power density of 0.897 W/cm ² for 300 seconds. Temperature was recorded every 10 seconds with an infrared thermal imaging camera (Fluke, Ti400, USA). (E) is a fluorescence spectrum showing the ROS generation ability of MsPDTT NPs and RsPDTT NPs in the presence of DCFH-DA, a ROS indicator. (F) shows the corresponding time-dependent fluorescence enhancement of DCFH formed under light irradiation conditions: 690 nm laser (0.897 W/cm ² ). The fluorescence spectra of DCF induced by MsPDTT NP- and RsPDTT NP-sensitized ROS were recorded ( λ _ex = 488 nm, λ _em = 525 nm).
Figure 2 shows the intracellular delivery and anticancer effect of MsPDTT NPs and RsPDTT NPs. (A) shows a confocal fluorescence microscopy image confirming ROS production (green) under normoxic and hypoxic conditions; Red indicates the fluorescence of NPs, and DAPI (blue) was used for nuclear staining. Mergerd image column shows intracellular delivery of NPs. MsPDTT NPs and RsPDTT NPs, λ _ex = 633 nm, λ _em = 680-800 nm; DCF, λ _ex = 488 nm, λ _em = 500-580 nm; DAPI, λ _ex = 405 nm, λ _em = 420-470 nm. Scale bar, 50 μm. BF, bright field. (B) shows the semi-quantified green fluorescence intensity corresponding to ROS generation (DCF formation) under PDT conditions of NPs. (C) shows the concentration-dependent cytotoxicity of MsPDTT NPs and RsPDTT NPs in 4T1 cells in a normoxic environment with or without laser irradiation (690 nm, 1.0 W/cm ² , 10 minutes). (D) shows the concentration-dependent cytotoxicity of MsPDTT NPs in 4T1 cells with or without laser irradiation (690 nm, 1.0 W/cm ² , 10 min) under normoxic and hypoxic conditions.
Figure 3 shows the light-induced cytotoxicity measurement and cell imaging results of MsPDTT NPs and RsPDTT NPs under normoxic (21% O ₂ ) and hypoxic conditions (0.1% O ₂ , 5% CO ₂ ). (A) and (B) show the cytotoxicity evaluation of MsPDTT NPs (10 μM) in 4T1 cells in hypoxic (A) and normoxic (B) environments, respectively. (C) shows the cytotoxicity evaluation of 4T1 cells treated with RsPDTT NPs (10 μM) under various conditions under normoxic environment (NAC, N-acetylcysteine, an effective scavenger for wiping out ROS; light, 690 nm laser irradiation, 1.0 W/cm ² , 10 min). ₍ D ₎ ^MsPDTT NPs ( ₁₀ The calcein-AM and PI-labeled images of 4T1 cells treated with RsPDTT NPs (10 μM) and 4T1 cells treated with RsPDTT NPs (10 μM) in the presence or absence of light irradiation under normoxic (21% O ₂ ) conditions are shown. Scale bar, 100 μm.
Figure 4 shows the in vivo and in vitro dual-mode photoacoustic and near-infrared fluorescence (PA/NIRF) imaging capabilities of MsPDTT NPs and RsPDTT NPs. (A) shows the PA imaging results of 4T1 tumors (0, 3, 6, 9, 12, and 24 hours) after intravenous injection of MsPDTT NPs and RsPDTT NPs. (B) shows the PA signal intensity of the 4T1 tumor-bearing mouse model after injection of MsPDTT NPs and RsPDTT NPs. (C) shows the fluorescence imaging results of 4T1 tumor-bearing mice after intravenous injection of MsPDTT NPs and RsPDTT NPs (150 μM, 200 μL). (D) shows the results of semi-quantitative analysis of the fluorescence signal shown in (C) over time. Fluorescence images were taken at different time points, with excitation at 640 ± 20 nm and emission wavelengths at 710 ± 30 nm, with an exposure time of 0.75 s. (E) shows the tumor-to-normal tissue ratio (T/NT) of fluorescence signals recorded at different time points in 4T1 tumor-bearing mice after injection of MsPDTT NPs and RsPDTT NPs. Data are expressed as mean ± SEM (n = 3). (F) shows the ex vivo NIRF imaging results of tumors and major organs (heart, liver, spleen, lung, and kidney) after injection of MsPDTT NPs and RsPDTT NPs in a tumor-bearing mouse model. (G) shows the results of semi-quantitative analysis of the fluorescence signal shown in (F).
Figure 5 shows the in vivo therapeutic effects of MsPDTT NPs and RsPDTT NPs under light irradiation. (A) and (B) show (A) photothermal imaging and (B) temperature of 4T1 tumor-bearing mice administered PBS, RsPDTT NPs, and MsPDTT NPs under 690 nm light irradiation, respectively. (C) shows the H&E staining results of tumor sections from various treatment groups (2 days after treatment) (scale bar, 50 μm), (D) shows the relative tumor volume of mice, and (E) shows the results of each group. Mouse body weight, (F) represents the survival rate of each group (n=3).
Figure 6 shows the photophysical properties of MsPDTT and RsPDTT, showing (A) absorption and (B) fluorescence spectra of MsPDTT and RsPDTT (5 μM, respectively) in DMSO (λ _ex = 660 nm).
Figure 7 shows the results of photothermal performance analysis of MsPDTT. (A) shows the photothermal heating curve for DMSO solution under various MsPDTT concentrations (0-0.25mM) and constant laser irradiation intensity (0.897 W/cm ² ), (A) B) shows the photothermal heating curve for DMSO solution under constant MsPDTT concentration (0.25 mM) and various laser irradiation intensities (0-0.897 W/cm ² ). (C) shows the photostability analysis results of MsPDTT (0.25mM), and the curve shows passive cooling after irradiation (0.897 W/cm ² , 660 nm) for 30 minutes and (E) IR thermal image of MsPDTT. It shows the temperature change of MsPDTT for several ON/OFF cycles including. (D) shows the photothermal heating curves of MsPDTT (0.25mM each) dispersed in 660 nm laser irradiation (0.897 W/cm ² ) and 50% DMSO buffer solution (pH = 7.4, 10 mM PBS).
Figure 8 shows the results of photothermal performance analysis of RsPDTT, (A) shows the photothermal heating curve for DMSO solution under various RsPDTT concentrations (0-0.25mM) and constant laser irradiation intensity (0.897 W/cm ² ), (B) ) shows the photothermal heating curve for DMSO solution under constant RsPDTT concentration (0.25 mM) and various laser irradiation intensities (0-1.6 W/cm ² ). (C) shows the results of photostability analysis of RsPDTT (0.25mM), and the curve shows passive cooling after irradiation (0.897 W/cm ² , 660 nm) for 30 minutes and (E) IR thermal image of RsPDTT. It represents the temperature change of RsPDTT for several ON/OFF cycles including . (D) shows the photothermal heating curves of RsPDTT (0.25mM each) dispersed in 660 nm laser irradiation (0.897 W/cm ² ) and 50% DMSO buffer solution (pH = 7.4, 10 mM PBS).
Figure 9 shows photothermal heating images of MsPDTT, (A) showing IR thermal images of DMSO solution under various MsPDTT concentrations (0-0.25 mM) and NIR laser irradiation (0.897 W/cm ² ) conditions, (B) shows IR thermal images of DMSO solution under constant MsPDTT concentration (0.25 mM) and various irradiation intensities (0-1.6 W/cm ² , 660 nm).
Figure 10 shows the results of ROS generation analysis, in the presence of (A) MsPDTT (5 μM), (B) RsPDTT (5 μM), (C) methylene blue (MB) (5 μM), and (D) at 660 nm. This shows the time-dependent absorption spectrum change of DCFH (20 μM) in the absence of photosensitizer after irradiation. All experiments were performed in PBS buffered solution (0.1 M, pH 7.4, containing 5% DMSO). (E) shows the absorption change plot at 504 nm for the experiment shown in (AC), and (F) summarizes the ROS generation results for the photosensitizers.
Figure 11 shows the capture efficiency of MsPDTT NPs and RsPDTT NPs. The amount of MsPDTT and RsPDTT encapsulated in DSPE-mPEG5000 (2.5-40 μg/mL) was measured by UV/vis spectrophotometry (A, C). The calibration curves of MsPDTT and RsPDTT were linear in the range of 2.5-40 μg/mL (B, D). The capture efficiencies of MsPDTT NPs and RsPDTT NPs were 58.3% and 49.7%, respectively.
Figure 12 shows the size distribution of RsPDTT NPs, and shows the hydrodynamic size of RsPDTT NPs measured by DSL analysis and TEM images (inset) (scale bar, 200 nm).
Figure 13 shows the measurement of the aqueous and physiological stability of MsPDTT NPs and RsPDTT NPs, and shows the results of DLS measurement of the hydrodynamic size distribution of MsPDTT NPs and RsPDTT NPs in PBS and 10% FBS after incubation for 6 days.
Figure 14 shows the PA signals of MsPDTT NPs and RsPDTT NPs, where (A) shows the PA spectra of MsPDTT NPs and RsPDTT NPs in DMSO, and (B) shows the PA intensity of MsPDTT NPs and RsPDTT NPs (0.5-10 μM).
Figure 15 shows the results of photothermal performance analysis of MsPDTT NPs and RsPDTT NPs. (A), (B) plots of temperature changes under laser irradiation at 690 nm (0.897 W/cm ² ) and various concentrations (0, 0.0078, 0.03125, 0.25 mM) of MsPDTT NPs (A) and RsPDTT NPs (B), respectively. It represents. (C) and (D) show laser irradiation at various power density conditions (0, 0.297, 0.697 and 0.897 W/cm 2 ) and constant concentration (0.25 W/cm ² ) at 690 nm for MsPDTT NPs (C) and RsPDTT NPs (D), respectively. It shows a plot of temperature change in mM).
Figure 16 shows the mitochondrial localization of MsPDTT NPs, (A) treated with MsPDTT NPs (10μM) for 2 hours in a hypoxic environment (0.1% O ₂ , 5% CO ₂ ), and MitoTracker Red (0.5%) μM) shows a confocal fluorescence image of 4T1 cells stained for 15 minutes (scale bar, 50 μm), and (B) is a scatter plot of green and red images for calculating Pearson's correlation coefficient (PC = 0.94). ), and (C) shows the fluorescence intensity of MsPDTT NPs and MitoTracker Red collected along the white dotted line in the merged image of (A).
Figure 17 is an image for confirming changes in tumor volume in 4T1 tumor-bearing mice (yellow dotted circle: tumor area, (L): laser irradiation)
Figure 18 shows the H&E staining results of mouse major organs (Scale bar, 50 μm, (L): laser irradiation).
Figure 19 shows the mechanism of action of the mitochondria-targeted nanotherapeutic agent containing MsPDTT.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Experimental method

Materials and Methods

All reagents and solvents were purchased from Merck, Samchun, TCI, and Thermofisher and used without further purification. UV/Vis and fluorescence spectra were recorded using a Jasco V-750 spectrophotometer and Shimadzu RF-5301PC spectrofluorometer, respectively. ESI-MS data was obtained using Shimadzu LCMS-2020. MALDI-TOF MASS was performed using the MALDITOF/TOF™5800 system (AB SCIEX). NMR spectra were collected on a Bruker 500MHz NMR spectrometer, and column chromatography was performed using silica gel 60 (70-230 mesh) as the stationary phase. Analytical thin layer chromatography was performed using 60 silica gel (0.25 mm thick pre-coated sheets). Photoacoustic (PA) spectra were recorded on a multispectral optoacoustic tomographic (MSOT) imaging system (inVision 128, iThera Medical GmbH, Neuherberg, Germany). Agar phantoms were filled with MsPDTT and RsPDTT at various DMSO concentrations and suspended in a water tank, and then the PA signals excited by a pulsed laser in the range of 600 to 950 nm were recorded. PA intensity was measured in regions of interest (ROIs) and a linear correlation between PA intensity and various concentrations was presented.

MsPDTT and RsPDTT's synthesis

The compound MsPDTT according to the present invention was synthesized according to the following synthetic route (Scheme 1).

[Scheme 1]

Synthesis of compounds a, b, and c

Compounds a, c according to procedures described in the previous literature (Tang, S., Ghazvini Zadeh, EH, Kim, B., Toomey, NT, Bondar, MV, and Belfield, KD (2017). Protein-induced fluorescence enhancement of twophoton excitable water-soluble diketopyrrolopyrroles. Org. Biomol. Chem. 15 , 6511-6519) and compound b (Zhang, W., Liu, P., Sadollahkhani, A., Li, Y., Zhang, B., Zhang, F ., Safdari, M., Hao, Y., Hua, Y., and Kloo, L. (2017). Investigation of Triphenylamine (TPA)-Based Metal Complexes and Their Application in Perovskite Solar Cells. ACS Omega 2 , 9231- 9240) was synthesized.

Synthesis of Compound 1

Compound a (2.00 g, 6.66 mmol) was dissolved in anhydrous DMF (50 mL), anhydrous K ₂ CO ₃ (3.68 g, 26.6 mmol) was added, and stirred at 60° C. under nitrogen for 3 hours. Next, bromoacetic acid tert -butyl ester (4.00 mL, 26.6 mmol) was added dropwise and stirred at 60°C overnight. After the reaction mixture was cooled to room temperature, the solution was removed under vacuum. Then, the mixture was extracted with DCM, the organic layer was dried with anhydrous Na ₂ SO ₄ , and after evaporation and vacuum drying, the crude product was purified using CH ₂ Cl ₂ /Hexane (v/v, 3:1) as an eluent. product) was purified using a silica gel column to obtain dark red solid Compound 1 (2.56 g, 73%).

¹ H NMR (CDCl ₃ , 500 MHz): δ 8.75 (dd, J = 3.9 Hz, 1.0 Hz, 2H), 7.62 (dd, J = 5.0 Hz, 1.1 Hz, 2H), 7.27 (dd, J = 4.4 Hz) , 3.4 Hz, 2H), 4.79 (s, 4H), 1.43 (s, 18H) ppm; ¹³ C NMR (CDCl ₃ , 125 MHz): δ 167.26, 161.16, 140.14, 135.10, 130.82, 129.88, 128.92, 107.74, 82.98, 44.33, 28.09 ppm.

Synthesis of Compound 2

Compound 1 (1.00 g, 1.89 mmol) was dissolved in chloroform (200 mL) and preheated to 60°C. N -Bromosuccinimide (841 mg, 4.72 mmol) was added to the mixture, and the flask was wrapped in aluminum foil to block light and stirred overnight. Then, the mixture was extracted with DCM, dried over anhydrous Na ₂ SO ₄ , evaporated and dried under vacuum, and the crude product was purified using a silica gel column using CH ₂ Cl ₂ /Hexane (v/v, 3:1) as an eluent. Compound 2 (0.85g, 65%) was obtained.

^1H NMR (CDCl ₃ , 500 MHz): δ 8.46 (d, J = 4.2 Hz, 2H), 7.22 (d, J = 4.2 Hz, 2H), 4.70 (s, 4H), 1.46 (s, 18H) ppm ; ¹³ C NMR (CDCl ₃ , 125 MHz): δ 167.2, 161.0, 142.5, 139.3, 135.3, 132.1, 131.5, 119.5, 83.6, 44.5, 28.3 ppm.

Synthesis of Compound 3

Compound b (2.00 g, 6.55 mmol) was dissolved in DMF (25.0 mL) and allowed to cool to 0°C. N -Bromosuccinimide (1.20 g, 6.55 mmol) was dissolved in 5mL DMF and added dropwise to the reactor. After removing the ice bath, the mixture was stirred overnight. The mixture was extracted with ethyl acetate and collected. The organic layer was dried over Na ₂ SO ₄ , and after evaporation and vacuum drying, the crude product was purified using a silica gel column using CH ₂ Cl ₂ /Hexane ( v / v , 1:10) as an eluent to obtain compound 3 (1.60 g, 64%) was obtained.

¹ H NMR (CDCl ₃ , 500 MHz): δ 7.23 (d, J = 8.9 Hz, 2H), 7.03 (d, J = 9.0 Hz, 4H), 6.83 (d, J = 9.0 Hz, 4H), 6.76 ( d, J = 8.9 Hz, 2H), 3.77 (s, 6H) ppm.

Synthesis of Compound 4

Compound 3 (1.50 g, 3.43 mmol) was reacted with 1,1'-bis( It was mixed with diphenylphosphino)ferrocene palladium dichloride (0.100 g). The mixture was heated to reflux overnight. The mixture was cooled to room temperature and then extracted with DCM. The collected organic layer was dried over anhydrous Na ₂ SO ₄ , evaporated and dried under vacuum, and the crude product was purified using a silica gel column using CH ₂ Cl ₂ /Hexane ( v / v , 1:1) as an eluent to obtain compound 4 ( 0.98g, 58%) was obtained.

¹ H NMR (CDCl ₃ , 500 MHz): δ 7.59-7.60 (m, 2H), 7.06-7.07 (m, 4H), 6.89-6.79 (m, 6H), 3.79 (s, 6H), 1.34 (s, 12H) ppm. MS (ESI): C ₂₆ H ₃₀ BNO ₄ [M + H] ⁺ , m / z calcd. 432.23, found 432.20.

Synthesis of compound 5

Compound 2 (0.500 g, 1.50 mmol) was mixed with compound 4 (0.540 g, 1.25 mmol), Pd(PPh ₃ ) ₄ (85.0 mg, 0.075 mmol), and K ₂ CO ₃ (2.10 mg) in 30 mL of degassed 1,4-dioxane. g, 15.0 mmol) and heated to reflux for 12 hours under a nitrogen atmosphere. After the mixture was cooled to room temperature, extracted with DCM, the collected organic layer was dried over anhydrous Na ₂ SO ₄ , evaporated and dried under vacuum, and then crudely purified using EtOAC/MeOH ( v / v , 1:1) as an eluent. The product was purified using a silica gel column to obtain compound 5 (0.610 g, 43%).

¹ H NMR (CDCl ₃ , 500 MHz): δ 8.81 (d, J = 4.32 Hz, 2H), 7.44 (d, J = 4.11 Hz, 4H), 7.32 (d, J = 4.20 Hz, 2H), 7.11- 7.06 (m, 8H), 6.89-6.83 (m, 12H), 4.84 (s, 4H), 3.81 (s, 12H), 1.44 (s, 18H) ppm. MS (ESI): C ₆₆ H ₆₂ N ₄ O ₁₀ S ₂ [M + H] ⁺ , m / z calcd. 1135.39, found 1135.45.

Synthesis of compound 6

Compound 5 (0.570 g, 0.500 mmol) was dissolved in DCM (20 mL) and cooled to 0°C. Then, trifluoroacetic acid (2.00 mL) was added to the mixture and stirred overnight. The solvent was evaporated under reduced pressure, and then any excess trifluoroacetic acid was removed by adding toluene (20 mL) and evaporating again to obtain compound 6 (0.370 g, 72%).

RsPDTT's synthesis

Compound 6 (0.300 g, 0.270 mmol), EDCI (63.0 mg, 0.320 mmol) and DMAP (66.0 mg, 0.540 mmol) were dissolved in anhydrous DMF (15 mL) and stirred under nitrogen for 10 min at room temperature. Propanol (5 mL) was then added to the mixture and the reaction mixture was stirred overnight. The solvent was evaporated and extracted with DCM (50 mL)/water (50 mL). The collected organic layer was dried over anhydrous Na ₂ SO ₄ , and after evaporation and vacuum drying, the crude product was purified using a silica gel column using EtOAC/MeOH ( v / v , 1:1) as an eluent, and the compound RsPDTT (0.110 g, 38%) was obtained.

^1H NMR (CDCl ₃ , 500 MHz): δ 8.80 (d, J = 4.20 Hz, 2H), 7.43 (m, 4H), 7.31 (d, J = 4.20 Hz, 2H), 7.09-7.06 (m, 8H) ), 6.91-6.83 (m, 12H), 4.94 (s, 4H), 4.14 (t, J = 6.86 Hz, 4H), 3.81 (s, 12H), 1.66-1.62 (m, 4H), 0.88 (t, J = 7.33 Hz, 6H) ppm; ¹³ C NMR (CDCl ₃ , 125 MHz): δ 168.4, 161.0, 156.5, 151.2, 150.7, 149.6, 140.0, 139.0, 136.8, 129.2, 127.1, 127.0, 126.9, 124.4, 12 3.3, 119.6, 114.9, 107.4, 82.9, 67.4, 55.5, 53.4, 21.9, 10.3 ppm. MS (MALDI-TOF): C ₆₄ H ₅₈ N ₄ O ₁₀ S ₂ [M + H] ⁺ , m / z calcd. 1106.36, found 1107.2921.

MsPDTT's synthesis

Compound 6 (0.200 g, 0.180 mmol), EDCI (42.0 mg, 0.216 mmol) and DMAP (44.0 mg, 0.360 mmol) were dissolved in anhydrous DMF (10 mL) and stirred at room temperature under nitrogen for 10 min. Compound c (0.140 g, 0.360 mmol) was then added to the mixture and the reaction mixture was stirred overnight. The solvent was evaporated and extracted with DCM (50 mL)/water (50 mL). The collected organic layer was dried over anhydrous Na ₂ SO ₄ , and after evaporation and vacuum drying, the crude product was purified using a silica gel column using EtOAC/MeOH ( v / v , 1:1) as an eluent, and the compound MsPDTT (0.150 g, 47%) was obtained.

¹ H NMR (CDCl ₃ , 500 MHz): δ 8.47 (d, J = 4.20 Hz, 2H), 7.76-7.53 (m, 30H), 7.31-7.24 (m, 8H), 7.10-7.02 (m, 8H) , 6.87-6.77 (m, 10H), 4.90 (s, 4H), 4.33-4.31 (m, 4H), 3.81 (s, 12H), 3.19-3.17 (m, 4H), 1.97-1.96 (m, 4H) ppm. ¹³ C NMR (DMSO- d 6, 125 MHz): δ 170.8, 168.7, 160.4, 156.9, 150.9, 149.9, 139.5, 138.5, 136.5, 135.5, 134.0, 133.9, 130.8, 130.7, 1 27.9, 127.4, 126.6, 124.0, 123.5, 118.8, 118.6, 118.1, 115.6, 106.5, 60.2, 55.8, 55.4, 21.9, 14.5 ppm. MS (ESI): C ₁₀₀ H ₈₆ Br ₂ N ₄ O ₁₀ P ₂ S ₂ [M] ²⁺ , m / z calcd. 814.26, found 814.85.

MsPDTT and RsPDTT's fever Performance( Photothermal performance test

The photothermal performance of MsPDTT and RsPDTT at various concentrations (0-0.25mM) was measured by continuous exposure to a 660nm laser at a power density of 0.897 W/cm ² for 120 seconds. Similarly, the photothermal effect of photosensitizers at a concentration of 0.25mM was measured by continuous exposure to a 660nm laser at power densities of 0, 0.297, 0.697, and 0.897 W/cm ² for 120 seconds. Temperature was recorded every 10 seconds with an infrared thermal imaging camera (FLIR, E60, USA).

spectral data

For UV/Vis and fluorescence measurements, 1mM dimethyl sulfoxide (DMSO) stock solutions of MsPDTT and RsPDTT were made up and diluted to the appropriate concentration for the analysis of each solution.

reactive oxygen species, ROS ) Occurrence analysis

The production of ROS was measured using 2,7-dichlorodihydrofluorescein (DCFH). Due to the tendency of DCFH to autooxidize under long-term storage conditions, the more stable diacetate form of DCFH (DCFH-DA) was purchased commercially and used to produce DCFH. Specifically, 0.1 mL of a 1mM ethanol solution of DCFH-DA was mixed with 0.4 mL of 0.1M NaOH aqueous solution and stirred for 30 minutes in the dark. Then, 150 μL of the above mixture was diluted with 2700 μL of 0.1 M PBS solution and mixed with 15 μL of 1 mM DMSO solution of each PS. To obtain a consistent DMSO concentration (5%), 135 μL of additional DMSO was added to the mixture. Then, the sample was irradiated every minute at 660 nm (Xenon Lamp Monochromatic Light Source, slit width 15/1.5 nm) and the increase in DCF absorption was monitored at 504 nm. The ROS generation rate of PS was calculated using [Equation 1] below based on the performance of MB.

[Equation 1]

m is the slope of the plot between the absorption change of DCF at 504 nm and irradiation time in the presence of PS or MB, and F is the absorption correction factor determined by F = 1–10 ^-OD (OD at the irradiation wavelength).

Calculation details and theoretical calculations

For the study of non-radiative processes, in the framework of Kohn-Sham density functional theory (DFT) (Hohenberg, P., and Kohn, W. (1964). Inhomogeneous Electron Gas. Phys. Rev. B 136 , B864-B871), non-adiabatic molecular dynamics (NAMD) were simulated. During NAMD, the motions of light electrons were processed quantum mechanically and the motions of heavy nuclei were processed using the PYXAID software package (Akimov, A.V., and Prezhdo, O.V. (2014). Advanced Capabilities of the PYXAID Program: Integration Schemes, Decoherence Effects , Multiexcitonic States, and Field-Matter Interaction. J. Chem. Theory Comput. 10, 789-804). In this package, decoherence induced surface hopping (DISH) was implemented to describe the dynamics of charge in excited states (Jaeger, H.M., Fischer, S., and Prezhdo, O.V. (2012). Decoherence-induced surface hopping (DISH) hopping. J. Chem. Phys. 137, 22A545). Electronic states and adiabatic MD trajectories were performed by the Vienna Ab initio Simulation Package (VASP) program using the plane-wave basis set by Perdew, Burke and Ernzerhof (PBE) and Projector-Augmented Wave (PAW) pseudopotential theory. Obtained from initial molecular dynamics (AIMD). AIMD simulations were performed for 1 ps with a 1 fs time step.

To obtain the oscillator strength and excitation energy, time-dependent DFT (TDDFT) calculations were performed by applying the conductor-like polarizable continuum model (CPCM). Spin-orbit coupling (SOC) constants were calculated with the B3LYP hybrid function and 6-31G basis set using the Q-Chem 5.2 software package.

MsPDTT NPs and RsPDTT NPs preparation

9.00 mg DSPE-mPEG5000 was added to 9 mL double distilled water (DDW) containing 1.00 mg MsPDTT or 1.00 mg RsPDTT in 1 mL THF. The mixture was then sonicated in an ice bath for 2 min using an ultrasonic cell disruptor. After THF was completely evaporated with nitrogen gas, the solution was transferred to an ultrafiltration tube (Millipore, Mw=50 kDa) to remove excess DSPE-mPEG5000. Aqueous solutions of MsPDTT NPs and RsPDTT NPs were obtained by filtration through a 0.220 μm filter membrane.

Quantitative measurement of entrapment efficiency

A linear calibration curve was derived from the relationship between concentrations in the range 2.5-40 μg/mL and the corresponding absorbance through the Lambert-Beer law. Entrapment efficiency (EE) was defined as the ratio of the amount of MsPDTT and RsPDTT encapsulated in DSPE-mPEG5000 and the amount added during nanoparticle preparation. EE was calculated using the following formula: EE (%) = (weight of loaded MsPDTT or RsPDTT/weight of MsPDTT or RsPDTT in feed) × 100%. The EE calculation results of MsPDTT NPs and RsPDTT NPs were 58.3% and 49.7%, respectively. Considering the difference in molecular weight between MsPDTT and RsPDTT, the stock solution was diluted to obtain equal molar concentrations for MsPDTT NPs and RsPDTT NPs according to EE.

Size and morphology

The size distribution was analyzed through dynamic light scattering (DLS) measurements, and the shape was analyzed through TEM images obtained with a high-resolution transmission electron microscope.

size stability

The prepared MsPDTT NPs and RsPDTT NPs were incubated with PBS (0.01 M) or 10% FBS (Gibco, Australia). After 6 days of incubation, the size distribution of the solutions was performed by DLS.

MsPDTT NPs and RsPDTT NPs fever performance test

MsPDTT NPs and RsPDTT NPs at concentrations of 0, 0.0078, 0.03125, and 0.25mM were continuously exposed to a 690 nm laser at a power density of 0.897 W/cm ² for 300 seconds. Additionally, MsPDTT NPs and RsPDTT NPs at a concentration of 0.25mM were continuously exposed to a 690 nm laser at power densities of 0, 0.297, 0.697, and 0.897 W/cm ² for 300 seconds. Temperature was recorded every 10 seconds with an infrared thermal imaging camera (Fluke, Ti400, USA).

MsPDTT NPs and RsPDTT NPs ROS produce

The ROS generation efficiency of MsPDTT NPs and RsPDTT NPs was measured using DCFH-DA. 0.5 mL DCFH-DA (MedChem Express, USA, 1.0 mM) in DMSO was mixed with 2 mL NaOH (0.01 N) to hydrolyze to DCFH. Deacetylated DCFH-DA (DCFH, 20 μM) was added to the aqueous solution of MsPDTT NPs and RsPDTT NPs (5 μM), and then the solution was treated with a 690 nm laser (0.897 W/cm ² ) for various irradiation times. The fluorescence spectra of DCF ( λ ex = 488 nm, λ em = 525 nm) induced by MsPDTT NPs and RsPDTT NPs sensitized ROS were recorded.

cell culture

4T1 cells were grown in RPMI-1640 medium (Gibco) containing 10% FBS (Gibco, Australia) and 1% antibiotics (penicillin/streptomycin, HyClone) in an incubator containing 5% CO ₂ and 95% air at 37°C. Cultured.

Establishment of an intracellular hypoxic environment

For cell culture, hypoxic conditions (~0.1% O ₂ , 5% CO ₂ ) were established using AnaeroPack System™ sachets (Mitsubishi Gas Chemical Co., Tokyo, Japan). Unless otherwise specified, all cells were cultured under normoxic conditions (21% O2).

cell fluorescence imaging and intracellular ROS produce

4T1 cells were cultured in a confocal ^dish _{( 1 2} ) was cultured for an additional 8 hours. Replace 1 mL culture medium containing MsPDTT NPs/RsPDTT NPs (10 μM) in each dish, and culture 4T1 cells for an additional 2 h under normoxic (21% O ₂ ) or hypoxic conditions (~0.1% O ₂ ), respectively. did. DCFH-DA (20 μM) was then added and incubated for an additional 30 min under each condition. Afterwards, 4T1 cells were treated with 690 nm laser irradiation at a power density of 1.0 W/cm ² for 5 minutes. Subsequently, 4T1 cells were stained with 4',6-Diamidino-2-phenylindole (DAPI) for 5 minutes and washed three times with PBS buffer under each condition. Cells were imaged with a confocal microscope (Leica TCS SP8; Leica Camera, Solms, Germany). Cell images were analyzed with Image J. MsPDTT NPs and RsPDTT NPs; DCF: λ _ex = 488 nm, λ _em = 500-580 nm; DAPI: λ _ex = 405 nm, λ _em = 420-470 nm.

intracellular localization (Subcellular localization)

4T1 cells were cultured in a confocal ^dish _{( 1 2} ) was cultured for an additional 8 hours. The 1 mL culture medium containing MsPDTT NPs (10 μM) was replaced, and 4T1 cells were cultured for an additional 2 h under hypoxic conditions (~0.1% O ₂ ). 4T1 cells were stained with Mito-Tracker Red (0.5 μM) for an additional 15 min and washed three times with PBS buffer. Cells were imaged by confocal microscopy (Leica TCS SP8; Leica Camera, Solms, Germany). Cell images were analyzed with ImageJ. MsPDTT NPs: λ _ex = 633 nm, λ _em = 680-800 nm; Mito-Tracker Red: λ _ex = 561 nm, λ _em = 580-620 nm.

Cytotoxicity assay

Cytotoxicity was determined by MTT assay. ^4T1 _cells inoculated in a 96-well plate ( ₁ It was cultured for another 8 hours in 0.1% O ₂ ). Next, the culture medium was replaced with medium containing MsPDTT NPs/RsPDTT NPs (0, 1.25, 2.5, 5, 10 μM), and 4T1 cells were cultured under normoxic (21% O ₂ ) or hypoxic conditions (~0.1% O 2 ), respectively. ₂ ) was incubated for an additional 2 hours. The medium was removed and replaced with fresh RPMI-1640 medium (100 μL). Then, 4T1 cells were treated with 690 nm laser irradiation at a power density of 1.0 W/cm ² for 10 minutes (laser group) or treated in a dark environment without laser irradiation (without laser group). Afterwards, 4T1 cells were further cultured at 37°C for 12 hours. MTT solution (5 mg/mL) was added to each well and incubated for 4 hours, then 100 μL DMSO was added to dissolve formazan, and OD ₄₉₀ (absorbance at 490 nm) was measured using a microplate reader (BioTek, USA). .

The therapeutic contribution of PDT or PTT produced by MsPDTT NPs/RsPDTT NPs was investigated through ROS scavenger ( N -acetylcysteine = NAC) and heat suppression (ice bath). ^4T1 _cells inoculated in a 96-well plate ( ₁ It was cultured for another 8 hours in 0.1% O ₂ ). To test the therapeutic contribution of PDT, the culture medium was replaced with medium containing MsPDTT NPs/RsPDTT NPs (10 μM), MsPDTT NPs/RsPDTT NPs (10 μM) + NAC (10mM), and NAC (10mM), respectively, and 4T1 cells were cultured. The cells were cultured for an additional 2 hours each under normoxic (21% O ₂ ) or hypoxic conditions (~0.1% O ₂ ). The medium was removed and replaced with fresh RPMI-1640 medium (100 μL). Then, 4T1 cells were irradiated with a 690 nm laser at a power density of 1.0 W/cm ² for 10 minutes, or treated in a dark environment without laser irradiation. Afterwards, 4T1 cells were further cultured at 37°C for 12 hours. MTT analysis was performed in the same manner as described above. To test the therapeutic contribution of PTT, the culture medium was replaced with medium containing MsPDTT NPs/RsPDTT NPs (10 μM) and 4T1 cells were grown in normoxic (21% O ₂ ) or hypoxic conditions (~0.1% O ₂ ) for 2 days each. It was cultured for more time. The medium was removed and replaced with fresh RPMI-1640 medium (100 μL). Then, 4T1 cells were irradiated with a 690 nm laser at a power density of 1.0 W/cm ² for 10 minutes (laser+ice group: cells were kept in an ice bath during irradiation, laser group: cells were kept at room temperature during irradiation). . 4T1 cells were stored in the dark as a dark group. Afterwards, 4T1 cells were further cultured at 37º for 12 hours. MTT analysis was performed in the same manner as described above.

cell staining

Live cells and dead cells were stained with Calcein-AM (Acetoxymethyl ester of calcein) and PI (propidium iodide), respectively. 4T1 cells were cultured in a confocal ^dish _{( 2 2} ) was cultured for an additional 8 hours. Replace 1 mL culture medium containing MsPDTT NPs/RsPDTT NPs (10 μM) in each dish, and culture 4T1 cells for an additional 2 h under normoxic (21% O ₂ ) or hypoxic conditions (~0.1% O ₂ ), respectively. did. 1 mL culture medium containing MsPDTT NPs/RsPDTT NPs (10 μM) was replaced in each dish and 4T1 cells were cultured for an additional 2 h under normoxic (21% O2) or hypoxic conditions (~0.1% O2), respectively. The medium was removed and replaced with fresh RPMI-1640 medium (100 μL). Then, 4T1 cells were treated with 690 nm laser irradiation at a power density of 1.0 W/cm ² for 10 minutes. Then, 4T1 cells were further cultured at 37°C for 12 hours, then stained with Calcein-AM (2 μM) and PI (4.5 μM) for 15 minutes and washed three times with PBS buffer. Cells were imaged with an inverted fluorescence microscope (Olympus IX71, Japan). Calcein-AM: λ _ex = 488 nm, λ _em = 510-550 nm; PI: λ _ex = 530 nm, λ _em = 560-640 nm.

Subcutaneous tumor model

BALB/c nude mice (female, 5 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology. All animals were acclimatized to the conditions one week prior to the experiment. Next, 4T1 cancer cells (1×10 ⁷ cells per mouse) were injected subcutaneously into the right flank of each mouse. Additional experiments were performed after the tumor volume reached approximately 80 mm ³ .

In vivo vivo ) Neon imaging

After intravenous (i.v.) injection of MsPDTT NPs/RsPDTT NPs (150 μM, 200 μL), fluorescence images were taken using the IVIS Lumina XRMS III in vivo imaging system (PerkinElmer, U.S.A.) (excitation at 640 ± 20 nm, emission at 710±30 nm, taken at different viewpoints with 0.75 s exposure time). Imaging analysis was performed in native Living Image 4.0 software. Ex vivo biodistribution analysis was performed 24 hours after injection of MsPDTT NPs and RsPDTT NPs.

in vivo photoacoustic ( photoacoustic , PA) imaging

After intravenous (i.v.) injection of MsPDTT NPs/RsPDTT NPs (150 μM, 200 μL), PA images were taken at the same location and at different time points. During in vivo PA imaging experiments, mice were anesthetized with a vaporized isoflurane system. PA imaging was performed using a real-time multispectral optoacoustic tomographic (MSOT) imaging system.

In vivo therapeutic efficacy (therapeutic efficacy) ExamIn vivo therapeutic efficacy tests

4T1 cell-bearing nude mice were divided into 6 groups (5 mice per group): a. MsPDTT NPs (150 μM, 200 μL) with laser (690 nm, 1.0 W/cm ² , 5 min); b. MsPDTT NPs (150 μM, 200 μL) without laser; c. RsPDTT NPs (150 μM, 200 μL) with laser (690 nm, 1.0 W/cm ² , 5 min); d. RsPDTT NPs (150 μM, 200 μL) without laser; e. PBS (10 mM, 200 μL) with laser (690 nm, 1.0 W/cm ² , 5 min); f. PBS (10 mM, 200 μL) without laser. During laser irradiation, real-time thermal images of the mouse were recorded using an infrared camera (Fluke, Ti400, USA). Tumor size was recorded using a caliper every 3 days for 30 days. Tumor size was calculated using the following formula: Tumor volume (mm ³ ) = 0.5 × (tumor length) × (tumor width) ² . Body weight was recorded every 3 days for 30 days.

Histological analysis ( Histological analysis)

4T1 cell-bearing nude mice from each group were sacrificed and histological analysis was performed. Tumors were collected and fixed in 4% paraformaldehyde for histological analysis by hematoxylin-eosin (H&E) staining. After treatment for 30 days, 4T1 cell-bearing nude mice from each group were sacrificed and histological analysis was performed. Major organs including heart, liver, spleen, lungs and kidneys were collected for H&E staining.

statistical analysis

Data are expressed as mean ± standard deviation (sd) unless otherwise specified. Statistical comparisons were performed with unpaired Student's t-test (two-tailed) between the two groups. Significance for all experiments was **** P <0.0001; *** P <0.001; ** P <0.01; *Defined as P <0.05. P value>0.05 was considered not statistically significant (ns). All statistical calculations were performed using Stata software.

Results and Discussion

The compound MsPDTT represented by [Formula 1] according to the present invention and the comparative compound RsPDTT were synthesized according to Scheme 1 above. The characteristics of the synthesized compounds were confirmed through ¹ H NMR, ¹³ C NMR, and ESI-MS analysis. Both MsPDTT and RsPDTT exhibited a broad NIR absorption band centered at 650 nm and fluorescence emission at 718 nm due to the extended conjugation (Figure 6). Additionally, the bathochromic shift observed in the absorption maximum is due to the presence of a methoxy moiety in the donor core, resulting in improved intramolecular charge transfer interactions.

Next, the photothermal efficacy and ROS generation ability of MsPDTT and RsPDTT were investigated respectively to test their multifunctional roles. As shown in Figures 7 to 9, the two compounds showed concentration- and laser power-dependent heat generation ability under 660 nm laser irradiation conditions, and exhibited excellent heating and cooling response characteristics up to 3 cycles. In addition, the ROS generation ability of MsPDTT and RsPDTT was tested using DCFH-DA (ROS indicator) (FIG. 10). Upon light irradiation of MsPDTT at 660 nm, a time-dependent absorption enhancement was observed at 504 nm, indicating the formation of the product 2,7-dichlorofluorescein (DCF). Surprisingly, the ROS generation rate (△ _ROS ) of MsPDTT was calculated to be 1.41 times higher than that of methylene blue (MB, △ _ROS = 1), a representative photosensitizer (PS). In contrast, under the same conditions, RsPDTT showed 0.179 times △ _ROS compared to MB, showing no significant performance. Overall, these results confirmed that both compounds showed photothermal conversion efficiency under irradiation conditions, and that MsPDTT showed better ROS generation ability than the comparative compound RsPDTT under photosensitive conditions.

A density functional theory study was performed to understand the reasons for the differences in PDT and PTT properties of MsPDTT and RsPDTT. After photon-induced excitation, the excited energy can be released in two forms: radiative and non-radiative relaxation. Non-radiative relaxation is a photothermal effect that induces heat, releasing excitation energy. Therefore, non-radiative relaxation, which is relatively more dominant than radiative relaxation, is a prerequisite for PTT. To evaluate the dominance between the two relaxations, the timescale of each process was compared.

According to non-adiabatic molecular dynamics results, the time scales of the non-adiabatic exponential decay from the first excited state to the ground state (S ₁ →S ₀₎ were 1,428.6 and 125.0 fs for MsPDTT and RsPDTT, respectively. Considering that the rate constant (IC) is proportional to the square of the non-adiabatic coupling term, this large difference in the IC ratio is explained by the non-adiabatic coupling constant. The average non-adiabatic coupling constant is 1.65 × 10 ^- for MsPDTT and RsPDTT, respectively. ³ and 7.34×10 ^-3 eV. Meanwhile, the radiative decay rate in the excited state is determined by the Einstein coefficient A _ij related to the oscillator strength (f _0→1 ) and excitation energy (S ₀ →S ₁ ). It can be derived that the relationship between these factors is as follows:

[Equation 2]

Here, n and u are the refractive index and transition frequency of the solvent.

Table 1 below shows the oscillator strength, excitation energy, and lifetime of radiative decay from the first excited state to the ground state (S ₁ → S ₀ ) for the two systems. As shown in Table 1, MsPDTT and RsPDTT exhibit non-adiabatic decay, which is much faster than radiative decay. For example, the nonadiabatic decay of MsPDTT and RsPDTT is approximately 1.8 and 20 times faster than the radiative decay of the respective vertical transitions. These calculation results are consistent with experimental results showing the PTT effect in both systems.

[Table 1]

During the photodynamic process, the generation of cytotoxic ROS that induce cell death is a critical step. These ROS are obtained by charge transfer in the T ₁ state of the photosensitive molecules used in PDT. As shown in Figure 19 below regarding the mechanism of action of MsPDTT and RsPDTT, the T ₁ state begins through an intersystem crossing (ISC) process in the S ₁ state of the RsPDTT and MsPDTT systems. Therefore, ISC rate can be considered an important factor in evaluating the photodynamic effect of a specific molecule.

To evaluate the ISC rate, Fermi's golden rule was referred to by considering only the direct spin-orbit coupling (SOC) constant, which is generally used to evaluate the ISC rate for organic compounds (Marian, CM (2012). Spin-orbit coupling and intersystem crossing in molecules. Wires. Comput. Mol. Sci. 2, 187-203). Then, the ISC rate constant ( _kISC ) was derived from the SOC constant directly between the singlet and triplet states:

[Equation 3]

(where a and b denote electronic states with multiplicity singlet or triplet, and j denotes vibrational state, a designates a fine-structure component, and q ₀ is a reference point, which is an optimized structure of the initial state. In this equation, is the vibrational density of state (VDOS) at the energy of the initial state, ie, the j-th vibrational state of excited singlet electronic states.

Since VDOS depends on the energy difference between electronic states (S ₁ and T ₁ states), and the two systems exhibit similar energy differences (RsPDTT, 0.580 eV and MsPDTT, 0.575 eV), their VDOS do not differ significantly in the ISC rate estimates. It may not be possible. Therefore, the SOC constant between the S ₁ and T ₁ states can have a dominant effect on the ISC rate of these systems. From these results, we confirmed that the two systems showed the minimum difference in SOC constant values (RsPDTT, 0.253 cm ^{- 1} and MsPDTT, 0.249 cm ^{- 1} ), which was only 1.04 times the difference in SOC constant in terms of equation (3). . In other words, the ISC rate of the system may be similar, but RsPDTT's IC tate is approximately 11.4 times faster than MsPDTT. In general, IC processes are similar to or faster than ISC rates. If the ISC rates of the two systems are similar to the IC rate of RsPDTT, the ISC rate will be relatively faster than the IC rate in the case of MsPDTT. In this context, we assumed that the ISC rates of both systems are similar to the IC rates of MsPDTT by an order of magnitude (~10 ³ fs lifetime). Assuming that the IC rate of RsPDTT is much faster than the ISC rate, the population of T ₁ states may not be sufficient to transfer charge to O ₂ molecules because the competitive IC process dominates over the ISC process. This is also in accordance with the experimental results that PDT is mainly observed only in MsPDTT.

To improve the water solubility of MsPDTT and RsPDTT for bioapplication, the components were encapsulated in the FDA-approved amphiphilic copolymer DSPE-mPEG5000 via nanoprecipitation method. A linear calibration curve was derived from the relationship between concentrations ranging from 2.5 to 40 μg/mL (MsPDTT) and the corresponding absorbance through the Lambert-Beer law. Entrapment efficiency (EE) was defined as the ratio of the amount of MsPDTT and RsPDTT encapsulated in DSPEmPEG5000 to the amount added during NP preparation. The calculated EE of MsPDTT NPs and RsPDTT NPs were 58.3% and 49.7%, respectively (Figure 1A and Figure 11). Through dynamic light scattering (DLS) results, it was confirmed that the average hydrodynamic size of MsPDTT NPs and RsPDTT NPs was about 122 nm and about 142 nm (FIGS. 1B and 12). In addition, through TEM images, it was confirmed that MsPDTT NPs and RsPDTT NPs showed a uniform spherical shape and size (Figure 11B, inset and Figure 12 inset). These size results suggest that the prepared NPs have the potential ability to target tumor sites. Additionally, the hydrodynamic size distribution of MsPDTT NPs and RsPDTT NPs showed negligible small changes even after the NPs were stored in PBS and 10% FBS (Figure 13). After dispersing in PBS buffer for 6 days, the EE of MsPDTT NPs and RsPDTT NPs was measured to be 52.8% and 41.0%, respectively, which were not significantly different from the freshly prepared samples. These results indicate excellent aqueous and physiological stability of the NPs.

The PA spectra of MsPDTT NPs and RsPDTT NPs ranged from 630 to 750 nm (Figure 1C and Figure 14A), and the PA intensity depended linearly on the concentration of MsPDTT NPs and RsPDTT NPs (Figure 14B). Additionally, the photothermal effect of MsPDTT NPs and RsPDTT NPs was monitored using an infrared thermal imaging camera (Figure 15). Concentration/laser power density dependent temperature changes of MsPDTT NPs and RsPDTT NPs in aqueous solution were performed in the range of 0-0.25mM and 0-0.897W/cm ² . As shown in Figure 1D and Figure 15, the temperature of MsPDTT NPs and RsPDTT NPs showed a similar upward trend with increasing concentration/laser power density, showing almost identical photothermal conversion behavior. The in vitro ROS generation ability of MsPDTT NPs and RsPDTT NPs was evaluated by the ROS indicator DCFH-DA. As shown in Figure 1E and Figure 1F, the fluorescence intensity of DCFH mixed with MsPDTT NPs (5 μM) gradually increased from 525 nm in a time-dependent manner from 0 to 120 s under laser irradiation (690 nm) conditions, through which , it was found that ROS were efficiently generated by MsPDTT NPs under laser irradiation conditions.

In contrast, the fluorescence intensity of DCFH mixed with RsPDTT NPs (5 μM) showed no noticeable change under similar conditions. These results indicate that MsPDTT NPs can generate heat and ROS under laser irradiation in solution, making them good candidates for PTT and PDT, whereas RsPDTT NPs can only generate heat under laser irradiation in solution and can only be used for PTT. will be.

Next, the intercellular ROS generation ability of MsPDTT NPs was evaluated in living cells. As shown in Figure 2A, 4T1 murine breast cancer cells were pre-incubated with DCFH-DA and NPs (10 μM). Upon laser irradiation (690 nm, 1W/cm ² , 5 min, 21% O ₂ ), bright green fluorescence was observed in cells treated with MsPDTT NPs (Figure 2A). This green fluorescence was not observed in cells treated with RsPDTT NPs. Additionally, the ROS generation ability of NPs was evaluated under hypoxic conditions. For this purpose, an in vitro hypoxic environment was established using the Anaero Pack System. 4T1 cells under hypoxic conditions (0.1% O ₂ ) showed a weaker fluorescence signal compared to normoxic environments (21% O ₂ ) under similar experimental conditions (Figures 2A and 2B). These results indicate that MsPDTT NPs can generate more ROS under normoxic conditions than under hypoxic conditions, while RsPDTT NPs do not generate ROS under normoxic or hypoxic conditions. In addition, as a result of an additional colocalization experiment using commercial MitoTracker red, it was confirmed that MsPDTT NPs (2-hour culture) were preferentially localized in mitochondria with a Pearson's correlation coefficient of 0.94 (Figure 16). These results confirmed that the mitochondrial targeting ability of NPs could be improved when TPP cations were incorporated into the MsPDTT scaffold.

Introducing TPP into RsPDTT enables the dual phototherapy properties of MsPDTT. To evaluate the efficacy of light-induced treatment, the antitumor effects of MsPDTT NPs and RsPDTT NPs on 4T1 cells were compared in vitro under normoxic conditions ( Figure 2C ). No obvious decrease in cell viability was observed when 4T1 cells were incubated with MsPDTT NPs and RsPDTT NPs without laser irradiation, indicating good biocompatibility in vitro. Under light irradiation (690 nm, 1W/cm ² , 10 min), a superior therapeutic effect was observed in MsPDTT NPs-treated cells (~97% cell death) compared to RsPDTT NPs (~67% cell death) at the same concentration of 10 μM. . As a result, despite the similar PTT effects of MsPDTT NPs and RsPDTT NPs, MsPDTT NPs showed better anticancer ability than RsPDTT NPs because MsPDTT NPs have both PDT effect and PTT effect. Additionally, MsPDTT NPs showed effective anticancer effects under hypoxia and normoxia (69% cell death under hypoxic conditions and 90% cell death under normoxic conditions), indicating that dual phototherapy would be advantageous for light-induced treatment ( Figure 2D ).

As a result of the MTT assay to evaluate the degree of PDT or PTT-mediated cytotoxicity, in the case of cells irradiated with MsPDTT NPs and laser under hypoxic conditions (0.1% O ₂ ), cells pretreated with NAC (~35% cell viability) and NAC pretreatment A similar cytotoxic effect was observed in cells that did not (~33% cell viability). However, under ice bath conditions, some cells were damaged (~14% cell death), which means that the anticancer effect of MsPDTT NPs under hypoxic conditions is mainly due to the PTT effect (Figure 3A). More 4T1 cells survived when treated with MsPDTT NPs and laser irradiated in an ice bath (~41% cell survival) than with NAC pretreatment (~31% cell survival) under normoxic conditions (21% O ₂ ). This suggests that the PTT effect contributed more than the PDT effect to killing tumor cells (Figure 3B). Likewise, RsPDTT NPs cultured with cells in an ice bath during laser irradiation showed little cell damage in the MTT assay, indicating that the anticancer effect of RsPDTT NPs is mainly due to the PTT effect (Figure 3C). As a result of simultaneous Calcein-AM/propidium iodide (PI) staining, more dead cells (red) were observed compared to live cells (green) after laser irradiation in both normoxic and hypoxic conditions (Figure 3D). Collectively, these results indicate that under laser irradiation, MsPDTT NPs can generate ROS in normoxic environment for efficient PDT as well as heat under hypoxic conditions for efficient hypoxic cell PTT destruction. In addition, it was confirmed that RsPDTT NPs are an effective material for photothermal destruction of normoxic cells.

Next, we evaluated the in vivo dual-mode PA/NIR imaging capabilities of MsPDTT NPs and RsPDTT NPs by intravenous (iv) administration in a 4T1 tumor-bearing nude model. PA imaging and NIRF imaging were performed on a real-time multispectral photoacoustic tomography imaging system and an IVIS Lumina XRMS III in vivo imaging system, respectively. Semiquantitative analysis of the PA signal and fluorescence intensity of the region of interest showed that the signal changes in dual-mode imaging were consistent with each other (Figures 4A-4D). Both MsPDTT NPs and RsPDTT NPs gradually accumulated in the tumor area and showed maximum tumor accumulation at 12 hours after injection. The tumor-to-normal tissue ratio gradually improved over time and reached a maximum of 6.8 and 6.0 at 12 h after administration of MsPDTT NPs and RsPDTT NPs, respectively ( Figure 4E ). For ex vivo NIRF imaging, dissected tumors and major organs (heart, liver, spleen, lungs, and kidneys) were collected 24 hours after injection. Tumors showed the highest fluorescence intensity, meaning that MsPDTT NPs and RsPDTT NPs could successfully target and accumulate at the tumor site (Figures 4F and 4G). Next, the therapeutic ability of MsPDTT NPs on nude mice bearing xenografted 4T1 murine mammary tumors was further evaluated (Figure 5). When the tumor volume reached approximately 80 mm ³ , BALB/c nude mice (female, 4 weeks old) were randomly separated into 6 groups (n = 5 each): (1) MsPDTT NPs (150 μM, 200 μL); ) + laser; (2) MsPDTT NPs (150 μM, 200 μL); (3) RsPDTT NPs (150 μM, 200 μL) + laser; (4) RsPDTT NPs (150 μM, 200 μL); (5) PBS (10 mM, 200 μL) + laser; (6) PBS (10 mM, 200 μL). Groups (1), (3), and (5) were irradiated with a 690 nm laser (1 W/cm ² , 5 minutes) 12 hours after intravenous injection. Accumulation and temperature changes for each tumor were recorded with an infrared thermal imaging camera. As shown in Figures 5A and 5B, the temperature of the tumor area in groups (1) and (3) showed a rapid increase from 35.8°C to 53.7°C and from 36.0°C to 54.8°C, respectively, during laser treatment. These results suggest that not only MsPDTT NPs and RsPDTT NPs have similar photothermal effects in vivo, but also that the sustained high temperature induced by accumulated NPs exceeds the needs of tumor hyperthermia. Mice in each group were sacrificed 2 days after treatment, and tumor pieces were collected for hematoxylin and eosin (H&E) staining (Figure 5C). For group (1), after administering MsPDTT NPs and irradiating with laser, obvious damage, including tumor cell necrosis and apoptosis, was observed. In group (3), fewer necrotic areas were distinguished after RsPDTTs nanoparticle administration and laser irradiation compared to group (1). In the case of the other control group (PBS), almost no damage was observed in the pathological part. Through these results, it was confirmed that MsPDTT NPs, which combine PDT and PTT properties, showed much better antitumor treatment ability in 4T1 tumor-bearing mice than RsPDTT NPs, which only have PTT effects. Evaluation of treatment efficiency was further performed by monitoring tumor volume and body weight every 3 days for 30 days after treatment. As shown in Figure 5D, tumor growth was partially inhibited after RsPDTT NPs and laser irradiation in group (3). Surprisingly, more significant tumor growth inhibition was confirmed in group (1) administered MsPDTT NPs and irradiated with laser than group (3), suggesting a synergistic therapeutic effect of MsPDTT NPs. Additionally, photographs were taken to record changes in tumor volume in each group (Figure 17). In each group, body weight changed within the normal range, confirming the excellent biological safety of both MsPDTT NPs and RsPDTT NPs (Figure 5E). Mice in each group were sacrificed 30 days after treatment and major organs (heart, liver, spleen, lungs, and kidneys) were collected for H&E staining (Figure 18). No abnormal damage was distinguished by histological analysis in all tested groups, suggesting that both MsPDTT NPs and RsPDTT NPs were biocompatible in living systems. Next, statistics on the survival rate of the mouse model were created (Figure 5F). As a result, only the mice that received intravenous injection of MsPDTT NPs and laser irradiation had a significant extension of lifespan after 50 days. In contrast, all mice in group (3) administered RsPDTT NPs and irradiated with laser died within 50 days. These results confirmed that MsPDTT NPs, which combine PDT and PTT properties, had a much better therapeutic effect in tumor-bearing mice than RsPDTT NPs, which only had PTT properties.

The present invention provides a compound MsPDTT with a mitochondria-targeted donor-acceptor-donor (D-A-D) molecular structure for dual-mode imaging (PA/fluorescence) and phototherapy (PDT/PTT), and a photosensitizer containing the same, and is also biocompatible. Nanoparticles MsPDTT NPs encapsulated by polymerized lipids are provided for formulation. The practicality of MsPDTT NPs as an efficient PS was confirmed through theoretical calculations, preliminary solutions, and cell-based experiments. Overall, due to the enhanced permeability, retention effect and TPP-mediated targeting, MsPDTT NPs have improved tumor tissue uptake ability, amplify strong fluorescence signal to effectively image the presence of tumor in the whole body, and also specifically target tumor tissue through PA imaging. High-resolution images can be provided. In addition, MsPDTT NPs showed significantly improved tumor growth inhibition ability and therapeutic efficacy according to the strategy of utilizing maximum photon usage through PDT and PTT, suggesting that the present invention is a useful strategy to circumvent traditional PDT-related hypoxia-mediated resistance. It is expected that it will be.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

A photosensitizer containing a compound represented by the following [Chemical Formula 1]:
[Formula 1]
. According to paragraph 1,
A photosensitizer characterized in that the compound accumulates specifically in mitochondria. According to paragraph 1,
The compound is a photosensitizer characterized in that it has photodynamic and photothermal therapeutic effects. Contains a compound and a polymerized lipid represented by the following [Chemical Formula 1],
Nanoparticles characterized in that the compound is encapsulated by the polymeric lipid:
[Formula 1]
. According to paragraph 4,
Nanoparticles, characterized in that the polymerized lipid is DSPE-mPEG. A composition for treating or diagnosing cancer comprising the photosensitizer of claim 1 or the nanoparticle of claim 4.