A J-aggregation-induced emission photosensitizer, its pharmaceutical composition, preparation method, and application.

Abstract

This invention discloses a J-aggregation-induced emission photosensitizer emitting NIR-II fluorescence, its constructed pharmaceutical composition, preparation method, and applications. This photosensitizer generates cytotoxic type I reactive oxygen species under light irradiation, exhibiting excellent NIR-II region fluorescence imaging capabilities and phototherapy performance. Further co-assembly of this photosensitizer with sorafenib into a pharmaceutical composition possesses J-aggregation-induced secondary self-assembly characteristics, enabling long-term imaging at tumor sites and exerting a dual function of NIR-II region fluorescence imaging and tumor therapy. It exhibits good stability, low toxicity, and broad clinical application prospects.

Description

Technical Field

[0001] This invention relates to a J aggregation-induced emission photosensitizer, its pharmaceutical composition, preparation method, and application, and particularly to a J aggregation-induced emission photosensitizer, its pharmaceutical composition, preparation method, and application that possesses NIR-II region in vivo fluorescence imaging, J aggregation-induced secondary self-assembly, and hypoxic tumor therapy properties. Background Technology

[0002] Fluorescence imaging in the second near-infrared optical window (NIR-II, 1000–1700 nm) offers numerous advantages, including high spatial resolution, high sensitivity, strong tissue penetration, and low background interference, demonstrating great potential for deep tumor imaging. Currently discovered NIR-II fluorescent dyes mainly include quantum dots, carbon nanotubes, rare-earth-doped nanoparticles, and small organic molecules. Among these, small organic molecule dyes have attracted significant attention from researchers due to their better biocompatibility and more easily tunable molecular structures and properties.

[0003] J-aggregates are a common strategy for constructing NIR-II region fluorescent dyes. J-aggregates are formed due to the presence of intermolecular hydrogen bonds, van der Waals forces, exciton coupling, hydrophilic / hydrophobic interactions, or electrostatic interactions, leading to chromophore slip stacking alignment and constructive coupling of excited-state transition dipoles. The formation of J-aggregates results in a redshift in absorption emission, an enhanced absorption coefficient (ε), a shortened fluorescence lifetime, and an increased quantum yield.

[0004] Currently, most photosensitizers induce oxygen-dependent type II photodynamic processes in vivo. However, solid tumors present with a hypoxic environment, which severely weakens the oxygen-dependent photodynamic process. Furthermore, J aggregates exhibit poor stability in complex biological environments, easily decomposing into monomers or forming kinetically stable H aggregates. All of these factors limit the therapeutic efficacy of photodynamic therapy (PDT). Summary of the Invention

[0005] Objectives of the invention: The first objective of this invention is to provide a J-aggregation luminescent photosensitizer that emits fluorescence in the NIR-II region; the second objective is to provide a pharmaceutical composition with combined phototherapy and chemotherapy efficacy constructed from the J-aggregation luminescent photosensitizer; the third objective is to provide a method for preparing them; and the fourth objective is to provide their pharmaceutical applications.

[0006] Technical solution: The J-aggregation-induced emission photosensitizer of the present invention has the following structure:

[0007]

[0008] Compared to conventional dyes in the first near-infrared region (NIR-I, 700–900 nm), fluorophores in the second near-infrared window (NIR-II, 1000–1700 nm) promise deeper tissue penetration (approximately 5–20 mm) and higher spatial resolution due to reduced light scattering and autofluorescence. The AIE photosensitizer designed in this invention exhibits significant advantages in fluorescence imaging and phototherapy due to its J-aggregate state emission wavelength reaching the NIR-II region, high brightness, good photostability, and large Stokes shift, demonstrating potential for designing and applying drugs for NIR-II region fluorescence imaging. Furthermore, the photosensitizer is an oxygen-independent type I photosensitizer, utilizing photoreactions based on electron transfer or hydrogen extraction to generate reactive substances, such as superoxide anions (O2). ·- ) and hydroxyl radicals (OH·).

[0009] The antitumor pharmaceutical composition of the present invention is obtained by assembling J aggregation-induced emission photosensitizer M with sorafenib into nanoparticles, and then modifying them with an amphiphilic PEG modifier.

[0010]

[0011] The pharmaceutical composition designed in this invention can emit fluorescence in the NIR-II region with negligible biotoxicity and is injected into healthy mice via tail vein. The photosensitizer within the composition emits NIR-II fluorescence, enabling NIR-II vascular imaging in healthy mice. Furthermore, when the composition is injected via tail vein into tumor-bearing mice, it accumulates at the tumor site due to the high-permeability long retention effect (EPR effect). Simultaneously, this invention utilizes biocompatible amphiphilic PEG to encapsulate the composition into nanomaterials. The nanostructure isolates and protects the payload, thereby enhancing the stability of J aggregates in vivo. This pharmaceutical composition can also undergo J-aggregate-induced secondary self-assembly, achieving long-term imaging at the tumor site. Under illumination, the photosensitizer within the composition generates cytotoxic type I reactive oxygen species for phototherapy of the tumor, making the pharmaceutical composition designed in this invention a multifunctional drug integrating NIR-II imaging and tumor treatment.

[0012] Preferably, the average particle size of the pharmaceutical composition of the present invention is 100-200 nm.

[0013] More preferably, the average particle size of the pharmaceutical composition of the present invention is 109.29 nm.

[0014] Preferably, the amphiphilic PEG modifier is DSPE-PEG or DSPE-PEG-RGD, wherein the molecular weight of PEG is 200 to 20000.

[0015] More preferably, the amphiphilic PEG modifier is DSPE-PEG or DSPE-PEG-RGD, wherein the molecular weight of PEG is 2000.

[0016] More preferably, the amphiphilic PEG modifier is DSPE-PEG and DSPE-PEG-RGD in a mass ratio of 3:1, wherein the molecular weight of PEG is 2000.

[0017] Preferably, the mass ratio of the J-aggregation-induced emission photosensitizer to sorafenib is (1-5):1.

[0018] Further preferably, the mass ratio of the J-aggregation-induced emission photosensitizer to sorafenib is 100:79.

[0019] Preferably, the sum of the amounts of the J-aggregation-induced emission photosensitizer and sorafenib, and the mass ratio of the amphiphilic PEG modifier, is 1:(3-10).

[0020] Further preferably, the sum of the amounts of the J-aggregation-induced emission photosensitizer and sorafenib, and the mass ratio of the amphiphilic PEG modifier, is 179:339.

[0021] The J-aggregation-induced emission photosensitizer of the present invention is prepared by reacting (Z)-2-(3-(cyano(isocyano)methylene)-2,3-dihydro-1H-inden-1-ylidene)malonitrile with 5-(4-(di-p-tolylamino)phenyl)furan-2-carboxaldehyde in an inert gas atmosphere.

[0022] Preferably, the molar ratio of (Z)-2-(3-(cyano(isocyano)methylene)-2,3-dihydro-1H-inden-1-ylidene)malonitrile to 5-(4-(di-p-tolylamino)phenyl)furan-2-carboxaldehyde is 3:1.

[0023] Preferably, the reaction solvent is acetic anhydride.

[0024] Preferably, the reaction time is 24 hours.

[0025] The method for preparing the pharmaceutical composition of the present invention includes the following steps:

[0026] (1) J aggregation-induced emission photosensitizer and sorafenib were dissolved separately, and both solutions were added to water. Under stirring, co-assembled nanoparticles were spontaneously formed.

[0027] (2) Dissolve the amphiphilic PEG modifier and add it to the co-assembled nanoparticles obtained in step (1). Remove the solvent to obtain the drug composition.

[0028] Preferably, the J-aggregation-induced emission photosensitizer, sorafenib, and amphiphilic PEG modifier are all dissolved in DMSO.

[0029] Further preferably, the aggregation-induced emission photosensitizer and sorafenib are dissolved, and both solutions are added to water, where co-assembled nanoparticles are spontaneously formed under stirring at 1500 rpm. An amphiphilic PEG modifier is dissolved and added to the prepared co-assembled nanoparticle solution, and the solvent is removed to obtain the pharmaceutical composition.

[0030] Further optimization is achieved by using a DMSO total volume to water volume ratio of 1:50.

[0031] The aggregation-induced emission photosensitizer described in this invention, or a pharmaceutical composition thereof, is used in the preparation of drugs for NIR-II region fluorescence imaging.

[0032] Preferably, the drug is a drug for NIR-II region fluorescence imaging of blood vessels and tumors.

[0033] The pharmaceutical composition described in this invention is used in the preparation of drugs that induce J aggregation secondary self-assembly or in drugs for treating hypoxic tumors.

[0034] Preferably, the tumor is a liver cancer tumor.

[0035] The J-aggregation-induced emission photosensitizer or the pharmaceutical composition thereof described in this invention can achieve NIR-II region fluorescence imaging of blood vessels and tumors. Under light irradiation, the photosensitizer in the pharmaceutical composition can generate cytotoxic type I reactive oxygen species to perform PDT and photothermal therapy on tumors, which can effectively inhibit tumors.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0037] The photosensitizer designed in this invention can generate cytotoxic type I reactive oxygen species under light irradiation, exhibiting excellent NIR-II region fluorescence imaging capability and phototherapy performance. This photosensitizer is further co-assembled with sorafenib to form a pharmaceutical composition, possessing J-aggregation-induced secondary self-assembly characteristics, enabling long-term imaging at the tumor site and exerting dual functions of NIR-II region fluorescence imaging and tumor therapy. It exhibits good stability, low toxicity, and broad clinical application prospects. Attached Figure Description

[0038] Figure 1 Synthetic route of aggregation-induced emission photosensitizer M for J ((i) NaOAc, reflux; (ii) K2CO3, Pd(dppf)Cl2, Tol / MeOH; (iii) Ac2O, N2);

[0039] Figure 2The ultraviolet (a) and fluorescence (b) spectra of the aggregation-induced emission photosensitizer M (10 μM) in aqueous solution at an excitation wavelength of 600 nm are shown.

[0040] Figure 3 To differentiate the aggregation-induced luminescence photosensitizer M by different water fractions (f w Fluorescence spectra of DMSO-H2O mixtures;

[0041] Figure 4 The aggregation-induced emission photosensitizer M generates type I reactive oxygen species (O2) in aqueous solution. ·- The result;

[0042] Figure 5 J is an aggregation-induced emission photosensitizer M in a laser (0.6 W·cm) -2 The heating results under irradiation;

[0043] Figure 6 The particle size distribution of the pharmaceutical composition MSDPNPs is shown in (a), and the stability results in water are shown in (b).

[0044] Figure 7 TEM images of the pharmaceutical composition MSDPNPs in H2O at different times;

[0045] Figure 8 For illumination (660nm, 0.6W·cm) -2 Cytotoxicity results of HepG2 cells treated with different concentrations of the drug composition MSDPNPs under dark conditions (MSDPNPs represents dark conditions, MSDPNPs+L represents light conditions);

[0046] Figure 9 This is a confocal image (scale bar 50 nm) of a live-dead cell experiment under hypoxic conditions.

[0047] Figure 10 NIR-II region fluorescence imaging of the blood vessels of healthy mice 5 minutes after the drug composition MSDPNPs was injected into the tail vein of the mice.

[0048] Figure 11 In vivo NIR-II region fluorescence image of H22 tumor-bearing mice after intravenous injection of the drug composition MSDPNPs;

[0049] Figure 12 The tumor status of H22 tumor-bearing mice after laser irradiation (n=3). Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the embodiments.

[0051] Example 1: Preparation of NIR-II emission J aggregation-induced emission photosensitizer M

[0052] (1) Preparation of (Z)-2-(3-(cyano(isocyano)methylene)-2,3-dihydro-1H-inden-1-ylidene)malononitrile

[0053] Indanone (1.46 g, 10 mmol) and malononitrile (1.32 g, 20 mmol) were dissolved in 15 mL of anhydrous ethanol (EtOH) and stirred at room temperature for 15 minutes. Then, sodium acetate (NaOAc, 2.46 g, 30 mmol) was added, and the mixture was refluxed and stirred for 5 hours. After the experiment, the mixture was cooled to room temperature, and a large amount of water was added. Simultaneously, the pH was adjusted to 2 using HCl. The residue obtained by filtration was washed several times with water to obtain an intermediate crude product, a gray solid, in 80% yield. NMR characterization is as follows: 1 H NMR (600MHz, DMSO-d6) δ7.11-7.09(m,2H),6.62-6.61(m,2H),4.89(s,2H).

[0054] (2) Preparation of compound 5-(4-(di-p-tolylamino)phenyl)furan-2-carboxaldehyde

[0055] 4-Bromo-N,N-Di-p-Tolylaniline (0.527 g, 1.5 mmol), (5-formylfuran-2-yl)boronic acid (0.21 g, 1.5 mmol), K₂CO₃ (0.759 g, 7.5 mmol), and Pd(dppf)Cl₂ (0.120 g, 0.15 mmol) were dissolved in 30 mL of a toluene-methanol-MeOH mixture (V / V = 1:1), and all air was removed from the reaction system. The reaction was carried out at 85 °C for 12 hours. After the reaction was complete, the reaction solvent was removed, and the crude product was purified by rapid column chromatography (silica gel) eluting with petroleum ether-ethyl acetate (V / V = 10:1) to give a yellow solid in 86% yield. NMR characterization is as follows: 1 HNMR (600MHz, CDCl3) δ9.58 (s, 1H), 7.63-7.60 (m, 2H), 7.29 (d, J = 3.7Hz, 1H), 7.10 (d, J = 8.1Hz, 4H ),7.04-7.03(m,2H),7.02(d,J=1.8Hz,2H),7.01-6.99(m,2H),6.67(d,J=3.7Hz,1H),2.33(s,6H).

[0056] (3) Preparation of NIR-II emission J aggregation-induced emission photosensitizer M

[0057] Compound (Z)-2-(3-(cyano(isocyano)methylene)-2,3-dihydro-1H-inden-1-ylidene)malononitrile (0.363 g, 1.5 mmol) and compound 5-(4-(di-p-tolylamino)phenyl)furan-2-carboxaldehyde (0.184 g, 0.5 mmol) were dissolved in 12.5 mL of acetic anhydride (Ac₂O) and stirred at 90 °C for 24 hours under a nitrogen atmosphere. When the solution cooled to room temperature, 25 mL of water was added and the mixture was stirred for 2 hours. Next, the mixture was cooled to room temperature and filtered to obtain the crude product, which was then washed several times with ethanol to obtain the product, a gray-blue solid, in 65% yield. The structure was characterized as follows: 1 H NMR (600MHz, CDCl3) δ8.55(dd,J=5.0,3.2Hz,2H),8.20(s,1H),7.72-7.69(m,2H),7.59(d,J=8.9Hz,2H),7.40(d,J= 4.0Hz, 1H), 7.15 (d, J = 8.2Hz, 4H), 7.07 (d, J = 8.3Hz, 4H), 7.01 (d, J = 8.9Hz, 2H), 6.84 (d, J = 4.0Hz, 1H), 2.36 (s, 6H). 13 C NMR (151MHz, CDCl3) δ164.84,151.67,148.00,143.48,135.02,134.02,130.40,127.08,126.17,1 25.12,125.03,122.88,119.76,119.04,114.10,113.73,110.56,20.97.HR-MS(m / z)(APCI):calcd for C 40 H 26 N5O + [M+H] + :592.2131; found:m / z 592.21206.

[0058] Example 2: Evaluation of the photophysical properties of J aggregation-induced emission photosensitizer M emitted by NIR-II

[0059] like Figure 2 As shown, an aqueous solution (10 μM) of photosensitizer M exhibits a main absorption peak at 710 nm and a fluorescence emission peak at 1003 nm. Furthermore, as... Figure 3 As shown, photosensitizer M exhibits significant J-aggregation properties in a mixed solution of DMSO and water.

[0060] like Figure 4 As shown, by using dihydrorhodamine 123 (DHR123) as a type I reactive oxygen species (O2)·- Indicator, using 10mW·cm -2 Spectra of a mixture of photosensitizer M (10 μM) and DHR123 (10 μM) were recorded immediately after irradiation with white light for different durations. This showed that photosensitizer M exhibited excellent type I reactive oxygen species (O2). ·- Generation capability.

[0061] like Figure 5 As shown, the photothermal behavior of photosensitizer M was evaluated using a 660 nm laser. The laser wavelength was 660 nm, and the laser pressure was 0.6 W·cm⁻¹. -2 A solution of photosensitizer M (100 μM) was irradiated with light, and the temperature was gradually increased from 27.8 °C to a maximum of 67.8 °C. The photothermal conversion efficiency (η) of photosensitizer M was 21.42%, indicating that photosensitizer M has a photothermal effect in vitro.

[0062] Example 3: Preparation of MSDPNPs, a dual-function antitumor drug composition for NIR-II region imaging therapy

[0063] 1 mg of photosensitizer M, 0.79 mg of sorafenib, 2.54 mg of DSPE-PEG2000, and 0.85 mg of DSPE-PEG-RGD were dissolved in 200 μL of DMSO. The photosensitizer M solution and the sorafenib solution were added to 10 mL of water, and the mixture was stirred at 1500 rpm for 2 hours. Then, the DSPE-PEG2000 and DSPE-PEG-RGD solutions were added for modification to obtain the MSDPNPs solution.

[0064] The particle size and particle size distribution of the prepared pharmaceutical composition MSDPNPs were determined by dynamic light scattering. For example... Figure 6 As shown, the average particle size of the MSDPNPs in the pharmaceutical composition is approximately 109.29 nm, indicating that it may have good EPR activity in vivo. The pharmaceutical composition maintains good stability in water, and the UV absorption spectrum did not change significantly over 6 days.

[0065] Example 4: Evaluation of the J-aggregation-induced secondary self-assembly capability of the pharmaceutical composition MSDPNPs

[0066] J aggregates of the drug composition MSDPNPs were observed using transmission electron microscopy (TEM). Figure 7 As shown, the nanoparticles in water are nano-sized needle-like particles, satisfying the EPR effect. After 24 hours of observation, the needle-like nanoparticles self-assembled a second time into micro-sized spindle-shaped fiber bundles, indicating that the drug composition MSDPNPs has good secondary self-assembly ability and can perform long-term imaging at the tumor site.

[0067] Example 5: Phototoxicity evaluation of the drug composition MSDPNPs against cancer cells

[0068] The killing effect of the drug composition MSDPNPs on HepG2 tumor cells was detected using the MTT assay (methyltetrazolyl blue colorimetric method). The assay was performed under light conditions (660 nm, 0.6 W·cm⁻¹). -2 HepG2 cells were treated with different concentrations (0, 10, 20, 30, 40, 50 μg / mL) of the drug combination MSDPNPs under two conditions: 1 min (MSDPNPs+L group) and darkness (MSDPNPs group), and their cytotoxicity was determined. Each experiment should be repeated at least three times. Figure 8 As shown, after laser irradiation, cell viability decreased significantly with increasing concentration of the drug composition MSDP NPs, and the mortality rate reached over 85%, indicating that the drug composition MSDP NPs has significant phototoxicity on HepG2 cells.

[0069] Example 6: Evaluation of the killing effect of the drug composition MSDPNPs on hypoxic cancer cells

[0070] The efficacy of the drug combination MSDPNPs in treating hypoxic tumors was evaluated using a live / dead cell staining assay. HepG2 cells were cultured with MSDPNPs (40 μg / mL) in confocal culture dishes for 9 hours, followed by a further 3 hours of hypoxic incubation. The cells were then treated with a 660 nm laser (0.6 W·cm²). -2 Irradiate cells for 5 minutes, then discard the culture medium and wash cells with PBS. Co-culture HepG2 cells with working solution containing calcein-AM (2 μM) and propidium iodide (PI, 4 μM) for 30 minutes, wash three times with PBS, and then observe the fluorescence of Calcein-AM and PI using a confocal microscope. Figure 9 As shown, almost all cells in the MSDPNPs+L group were stained red by PI dye, indicating a good cancer cell killing effect. This demonstrates that the MSDPNPs drug composition has excellent killing ability against cancer cells under hypoxic conditions.

[0071] Example 7: Evaluation of the NIR-II region fluorescence imaging capability of the pharmaceutical composition MSDPNPs

[0072] The drug composition MSDPNPs was injected intravenously into healthy mice, and whole-body vascular system imaging was performed on the healthy mice using a 980nm LP filter. Figure 10 As shown, after injection of MSDPNPs, both abdominal and hind limb vessels could be clearly identified and imaged. The vascular imaging results demonstrate that MSDPNPs possess excellent in vivo NIR-II region fluorescence imaging capabilities.

[0073] Example 8: Evaluation of the therapeutic effect of the pharmaceutical composition MSDPNPs

[0074] An H22 tumor-bearing mouse model (mouse liver cancer tumor) was established using intratumoral injection to evaluate the therapeutic effect of the drug composition MSDPNPs. The MSDPNPs solution (10 mg / kg; 10 mg per kg of mouse body weight) was injected intravenously into H22 tumor-bearing BALB / c mice. Figure 11 As shown, the fluorescence intensity of the MSDPNPs drug composition reached its maximum at the tumor site 24 hours after injection, thus serving as the optimal time point for phototherapy. Furthermore, fluorescence signals were still observable at the tumor site 72 hours after injection of the MSDPNPs solution, demonstrating that MSDPNPs achieved long-stay imaging at the tumor site.

[0075] To further demonstrate the antitumor activity of the drug composition MSDPNPs, a mouse unilateral axillary H22 tumor model was established to evaluate whether injecting the drug composition MSDPNPs could effectively inhibit the tumor. Figure 12 As shown, the tumor was effectively suppressed after 9 days of treatment, thus the drug composition MSDPNPs showed excellent anti-tumor effects.

Claims

1. A J-aggregation-induced emission photosensitizer, characterized in that, It has the following structure: 。 2. An antitumor pharmaceutical composition, characterized in that, The nanoparticles are obtained by co-assembling the J aggregation-induced emission photosensitizer M as described in claim 1 with sorafenib and then modifying them with an amphiphilic PEG modifier.

3. The pharmaceutical composition according to claim 2, characterized in that, Its average particle size is 100~200 nm.

4. The pharmaceutical composition according to claim 2, characterized in that, The amphiphilic PEG modifier is selected from DSPE-PEG and DSPE-PEG-RGD, wherein the molecular weight of PEG is 200~20000.

5. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio of the J-aggregation-induced luminescence photosensitizer to sorafenib is (1~5):

1.

6. The pharmaceutical composition according to claim 2, characterized in that, The sum of the amounts of the J-aggregation-induced emission photosensitizer and sorafenib, and the mass ratio of the amphiphilic PEG modifier, is 1:(3~10).

7. A method for preparing the J-aggregation-induced emission photosensitizer according to claim 1, characterized in that, The J aggregation-induced emission photosensitizer is obtained by reacting 1,3-bis(dicyanomethylene)indane with 5-(4-(di-p-tolylamino)phenyl)furan-2-carboxaldehyde under an inert gas with stirring.

8. A method for preparing the pharmaceutical composition according to claim 2, characterized in that, Includes the following steps: (1) J aggregation-induced emission photosensitizer and sorafenib were dissolved separately, and both solutions were added to water. Under stirring, co-assembled nanoparticles were spontaneously formed. (2) Dissolve the amphiphilic PEG modifier and add it to the co-assembled nanoparticles obtained in step (1). Remove the solvent to obtain the drug composition.

9. The use of the J aggregation-induced emission photosensitizer of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for NIR-II region fluorescence imaging.

10. The use of the pharmaceutical composition of claim 2 in the preparation of J aggregation-induced secondary self-assembly drugs and drugs for treating hypoxic tumors.

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