Near-infrared PDT photosensitizer as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared PDT photosensitizer as well as a preparation method and application thereof, belongs to the technical field of PDT photosensitizers, and solves the problem that the application effect of a PDT photosensitizer in a near-infrared light area is not ideal in the prior art. The invention discloses an application of a non-fullerene oligomer as a PDT photosensitizer. The non-fullerene oligomer particles provided by the invention can effectively exert the synergistic effect of type I photodynamic therapy and type II photodynamic therapy under the irradiation of 808nm laser, and the acting effect of PDT is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of near-infrared PDT photosensitizers, and more specifically to a near-infrared PDT photosensitizer and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) refers to a treatment method in which photosensitizers (PSs) use light energy to generate reactive oxygen species (ROS) to eliminate cancer cells. It has attracted much attention in recent years due to its non-invasive, easy-to-operate, and highly specific characteristics. Specifically, PDT is divided into type I and type II, which work through two mechanisms. Type I PDT involves the charge transfer of the excited triplet state (T1) of PSs to the surrounding matrix, thereby generating free radicals, including hydroxyl radicals (·OH) and superoxide anion radicals (·O2 - Type II PDT involves energy transfer from the excited triplet state (T1) of PSs to surrounding oxygen molecules to form singlet oxygen ( 1 O2). The free radicals generated by PDT and 1 O2 kills cancer cells and eliminates tumors. Unfortunately, most commercial photosensitizers have short excitation wavelengths, mainly in the visible light region, and shallow penetration depths, which limits their further application in the biological field. In contrast, near-infrared (NIR) light has a deeper penetration depth and better biosafety than visible light, so the development of near-infrared PSs, especially those with absorption wavelengths exceeding 800nm, is promising in achieving deep-penetration PDT. For example, indocyanine green (ICG), an imaging agent approved by the US Food and Drug Administration (FDA), has a maximum absorption wavelength of 780nm and has been widely used in photodynamic therapy. However, ICG only shows tail absorption under 808nm laser irradiation, has a low ROS production rate, and is prone to photobleaching under near-infrared light excitation, which seriously hinders its further research and clinical practice. To address these problems, a series of near-infrared PSs, such as semiconductor polymers (SPs) and conjugated small molecules (CSMs), have been developed. However, the excitation wavelength of most near-infrared PSs is less than 800 nm, which is relatively short, and the ROS production rate is low, which makes them ineffective in clinical applications.

[0003] The intersystem crossing (ISC) process occurs between the singlet excited state (S1) and triplet excited state (T1) of a molecule, during which the excited state molecule undergoes a non-radiative transition from S1 to T1, laying the foundation for PDT. Currently, in order to promote the generation of ROS, a commonly used strategy is to add heavy atoms such as halogen atoms and heavy metal atoms to PSs to enhance the spin-orbit coupling of the molecules, thereby promoting the ISC process and improving the efficacy of PDT. However, heavy atoms may cause long-term toxicity issues, hindering their potential clinical application. Deciphering the ISC dynamics is crucial for exploring new and efficient PSs, and regulating the ISC process is another effective strategy to improve ROS generation. However, due to the significant instability of PSs and the high triplet energy level (T1) requirement (usually higher than the singlet energy level of O2), it is quite difficult to design responsive PSs exceeding 800nm, not to mention safe and useful strategies to improve their photodynamic therapy effects.

[0004] In recent years, non-fullerene organic semiconductors with tunable energy levels and strong near-infrared absorption have been widely used as acceptor structures for organic photovoltaic (OPVs) materials due to their excellent photovoltaic performance (A). Y6, as an emerging non-fullerene acceptor (NFA), has a central fused ring based on an electron-deficient core (benzothiadiazole, BT), which greatly improves the power conversion efficiency of OPVs devices to a record 15.7%. Inspired by Y6, a large number of Y-derivatives with ADA′-DA configuration have been designed, synthesized and applied in the field of OPVs. The performance of Y-derivatives can be easily optimized by engineering the BT core, end groups and alkyl chains. Considering the strong near-infrared absorption ability and easy modification of non-fullerene molecules, they also have great potential in applications outside of OPVs (such as biomedicine). However, to date, they have not been applied in the biomedical field, and their excitation dynamics are still unclear. Summary of the invention

[0005] The present invention provides a near-infrared PDT photosensitizer and a preparation method and application thereof, so as to solve the problem that the application effect of the PDT photosensitizer in the prior art in the near-infrared light region is not ideal.

[0006] In a first aspect, the present invention provides a use of a non-fullerene oligomer as a PDT photosensitizer, characterized in that the structural formula of the non-fullerene oligomer is as shown in any one of Formula I, Formula II and Formula III;

[0007]

[0008] As a possible implementation manner, the non-fullerene oligomer has an ADA′-DA structure.

[0009] In a second aspect, the present invention provides a non-fullerene oligomer, the structural formula of the non-fullerene oligomer being as described in any possible implementation manner of the first aspect.

[0010] In a third aspect, the present invention provides a method for preparing a non-fullerene oligomer according to any possible implementation of the second aspect, comprising the following steps: performing Knoevenagel condensation on a donor-acceptor'-donor fragment and an acceptor unit to design and synthesize the non-fullerene oligomer; wherein the structural formula of the donor-acceptor'-donor fragment is as shown in Formula IV; the structural formula of the acceptor unit is as shown in any one of Formula V, Formula VI and Formula VII;

[0011]

[0012] In a fourth aspect, the present invention provides a use of non-fullerene oligomer nanoparticles as a PDT photosensitizer, wherein the structural formula of the non-fullerene oligomer is as described in any possible implementation of the first aspect.

[0013] As a possible implementation manner, the absorption wavelength of the non-fullerene oligomer nanoparticles is ≥808 nm.

[0014] As a possible implementation, the non-fullerene oligomer nanoparticles have the ability of synergistic effects of type I PDT and type II PDT.

[0015] As a possible implementation, the particle size of the non-fullerene oligomer nanoparticles is 45-55 nm.

[0016] In a fifth aspect, the present invention provides a method for preparing the non-fullerene oligomer nanoparticles according to any possible implementation of the fourth aspect, comprising the steps of: dissolving the non-fullerene oligomer in tetrahydrofuran to obtain an oligomer solution, dissolving an encapsulating matrix in tetrahydrofuran to obtain an encapsulating matrix solution; uniformly mixing the oligomer solution and the encapsulating matrix solution, dripping them into deionized water under stirring conditions, removing the tetrahydrofuran, filtering and concentrating to obtain the non-fullerene oligomer nanoparticles.

[0017] As a possible implementation, the concentration of the oligomer solution is 0.8-1.2 mg / mL; and / or, the concentration of the encapsulation matrix solution is 3-8 mg / mL; and / or, the ratio of the oligomer solution to the encapsulation matrix solution is 1:8-13; and / or, the encapsulation matrix is ​​DSPE-PEG 2000 , any one of PEG-b-PPG-b-PEG, PSMA, and BSA.

[0018] The oligomer provided by the present invention has an ADA′-DA structure, and its photophysical properties, such as the excited triplet energy level (T1), energy shift (ΔE ST ) and oscillator strength (f), intersystem crossing efficiency (Φ ISC ) can be increased from 10% to 28%. Here, receptor engineering is used to regulate Φ ISC It has been unprecedentedly used as an effective strategy for developing high-performance near-infrared PSs.

[0019] The nanoparticles (ONPs 1 to ONPs 3) assembled by using the oligomers provided by the present invention show good water dispersibility and high absorption of 808nm photons. In addition, ONPs 1 to ONPs 3 show higher reactive oxygen species (ROS) generation capacity (1.7 to 6.8 times higher than ICG) and 1 O2 production rate (0.9-2.2% relative to 0.2% of ICG). The most efficient ONPs 1 can also produce ·OH at the same time, which greatly overcomes the problem of tumor hypoxia. With these advantages, non-fullerene oligomer nanoparticles (ONPs 1-ONPs3) achieved excellent type I and type II PDT synergistic therapeutic effects both in vitro and in vivo.

[0020] This study designed a controllable Φ ISC The novel near-infrared PSs can be used for efficient tumor photodynamic therapy, thereby promoting the rational design of high-performance near-infrared PSs. The non-fullerene oligomer nanoparticles provided by the present invention can effectively exert the synergistic effect of type I and type II photodynamic therapy under 808nm laser irradiation, thereby improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Schematic diagram of the synthesis route of non-fullerene oligomers O1-O3 provided in an embodiment of the present invention.

[0023] Figure 2 The non-fullerene oligomer O1 provided in the embodiment of the present invention is 1 H NMR spectrum.

[0024] Figure 3 The non-fullerene oligomer O1 provided in the embodiment of the present invention is13 C NMR nuclear magnetic resonance spectroscopy.

[0025] Figure 4 The non-fullerene oligomer O2 provided in the embodiment of the present invention 1 H NMR spectrum.

[0026] Figure 5 The non-fullerene oligomer O2 provided in the embodiment of the present invention 13 C NMR nuclear magnetic resonance spectroscopy.

[0027] Figure 6 The non-fullerene oligomer O3 provided in the embodiment of the present invention is 1 H NMR spectrum.

[0028] Figure 7 The non-fullerene oligomer O3 provided in the embodiment of the present invention is 13 C NMR nuclear magnetic resonance spectroscopy.

[0029] Figure 8 This is the optimized configuration of the non-fullerene oligomers O1-O3 provided in the embodiments of the present invention.

[0030] Fig. 9 The spatial distribution of HOMO and LUMO of the non-fullerene oligomers O1-O3 provided in the embodiments of the present invention.

[0031] Fig.10 The energy gaps of the non-fullerene oligomers O1-O3 provided in the embodiments of the present invention.

[0032] Fig.11 Absorption / emission spectra of non-fullerene oligomers O1-O3 dissolved in tetrahydrofuran (THF) provided in an embodiment of the present invention.

[0033] Fig.12 The two-dimensional femtosecond transient spectra of O1-O3 at different delay times provided in the embodiment of the present invention.

[0034] Fig.13 The fs-TA spectra of the non-fullerene oligomers O1-O3 provided in the embodiments of the present invention at different delay times, wherein the upper arrow indicates the increase of the triplet excited state, and the lower arrow indicates the decrease of the singlet excited state.

[0035] Fig.14 The singlet and triplet state evolution dynamics of the non-fullerene oligomers O1-O3 provided in the embodiments of the present invention.

[0036] Fig.15 Schematic diagram of the preparation process of non-fullerene oligomer nanoparticles ONPs 1 to ONPs 3 provided in an embodiment of the present invention.

[0037] Fig.16 This is a DLS distribution diagram of the non-fullerene oligomer nanoparticle ONPs 1 provided in an embodiment of the present invention, wherein the inset is a TEM image of ONPs 1.

[0038] Fig.17 Absorption and emission spectra of non-fullerene oligomer nanoparticles ONPs 1 to ONPs 3 provided in the embodiments of the present invention.

[0039] Fig.18 The embodiment of the present invention provides that in the presence of ONPs 1, the fluorescence spectrum of DCFH changes with the increase of illumination time.

[0040] Fig.19 The fluorescence spectrum of DCFH changes with the increase of irradiation time in the presence of different photosensitizers provided in the embodiments of the present invention, wherein the photosensitizer in a is ICG, the photosensitizer in b is ONPs2, and the photosensitizer in c is ONPs 3.

[0041] Fig. 20 The relationship between the fluorescence intensity of DCFH at 525 nm and the illumination time in the presence of ONPs 1, ONPs 2, ONPs 3 or ICG provided in the embodiments of the present invention.

[0042] Fig.21 The embodiment of the present invention provides that in the presence of ONPs 1, the absorption spectrum of DPBF changes with the increase of illumination time.

[0043] Fig. 22 The absorption spectrum of DPBF changes with the increase of illumination time in the presence of different photosensitizers provided in the embodiments of the present invention, wherein the photosensitizer in a is ICG, the photosensitizer in b is ONPs 2, and the photosensitizer in c is ONPs 3.

[0044] Fig.23 The embodiment of the present invention provides the variation of the absorption of DPBF at 415 nm with the illumination time in the presence of ONPs 1, ONPs 2, ONPs 3 or ICG.

[0045] Fig.24 ESR spectra of PBS and ONPs 1 provided in the embodiments of the present invention under conditions of illumination and no illumination, wherein the left figure is under the condition of TEMP, and the right figure is under the condition of DMPO.

[0046] Fig.25 The ESR spectra of PBS and ONPs 1 under the conditions of illumination and no illumination in the presence of DMPO provided in the embodiments of the present invention.

[0047] Fig.26The results of the cell application experiment of the non-fullerene oligomer nanoparticles provided in the embodiment of the present invention, wherein a is the detection of intracellular ROS, blue channel: Hoechst 33342, green channel: DCFH-DA; b is the cytotoxicity evaluation of ONPs 1 on 3T3 cells under dark conditions; c is the cytotoxicity evaluation of ONPs 1 on A549 cells under dark conditions or laser irradiation; d is the cytotoxicity evaluation of A549 cells incubated with ONPs 1 (12.5 μg / mL) under 808 nm laser (0.75 W / cm 2 , 15min) irradiation; e is AM / PI staining of A549 cells.

[0048] Fig. 27 The results of in vivo application experiments of non-fullerene oligomer nanoparticles provided in the embodiments of the present invention, wherein a is the in vivo PDT experimental process; b is the laser irradiation (808nm, 0.44W / cm 2 ) after intravenous injection of PBS or ONPs 1 on A549 tumor mice; c is the curve of tumor temperature with prolonged irradiation time; d is the curve of tumor volume change with time in different groups of mice (*p<0.05, **p<0.01); e is the change of mouse body weight within 14 days after different treatments; f is tumor weight measurement.

[0049] Fig.28 Representative images of dissected tumors of mice in different groups on day 14 after treatment in an in vivo application experiment of non-fullerene oligomer nanoparticles provided in an embodiment of the present invention

[0050] Fig.29 The therapeutic effect and biosafety test results of the non-fullerene oligomer nanoparticles provided in the embodiments of the present invention, wherein a is the H&E staining of the main organs and tumors of mice receiving various treatments; b is the result of blood analysis of different groups of mice. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] In order to solve the problem that the application effect of PDT photosensitizer in the near-infrared light region in the prior art is not ideal, the embodiment of the present invention provides a preparation experiment of a near-infrared photosensitizer, and a non-fullerene oligomer (O1-O3) is designed and synthesized by a simple one-step Knovenagel condensation reaction of "donor-acceptor'-donor" ("DA'-D") fragment with different acceptor (A) units to construct a narrow gap "ADA'-DA" structure. Based on density functional theory (DFT), the embodiment of the present invention analyzes the photophysical properties of O1~O3 molecules.

[0053] It can be seen that the preparation method provided by the present invention successfully prepared the target product non-fullerene oligomers O1-O3; and the non-fullerene oligomers O1-O3 have excellent near-infrared light capture / emission capabilities, and have relatively excellent T1 to O 2 The feasibility of energy transfer of S0 with a smaller ΔE ST , which promotes the intersystem crossing (ISC) process and is beneficial to the generation of reactive oxygen species; the mass extinction coefficients of O1~O3 at their maximum absorption are better than those of most reported molecules.

[0054] Furthermore, the embodiment of the present invention conducted an ISC dynamic analysis experiment on the prepared non-fullerene oligomers O1-O3. It can be seen that O1-O3 all have high Φ ISC This is attributed to the regulation of the photophysical properties of the acceptor unit; and it can be concluded that the weaker the electron-withdrawing strength of the acceptor unit of the non-fullerene oligomer, the smaller the ΔE ST , the smaller f is, the larger T1 is, Φ ISC The bigger.

[0055] The embodiment of the present invention also provides a preparation and characterization experiment of non-fullerene oligomer nanoparticles.

[0056] It can be seen that the target product non-fullerene oligomer nanoparticles ONPs 1 to ONPs 3 were successfully prepared. Moreover, ONPs 1 to ONPs 3 have good responsiveness to 808nm excitation light and have strong ROS generation ability and 1 O2 production rate; achieved significant in vitro and in vivo type I and type II PDT synergistic therapeutic capabilities, and verified its ability to have in vivo effects.

[0057] Furthermore, the detection experiment of intracellular ROS generation, the in vivo application experiment in mice and the biosafety evaluation experiment of the embodiments of the present invention show that the non-fullerene oligomer nanoparticles provided by the present invention have reliable biosafety and excellent PDT effect.

[0058] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0059] All chemical reagents related to molecular synthesis in the examples of the present invention were purchased from Ruixun Optoelectronic Materials Technology Co., Ltd. 2000 was purchased from Shanghai Yare Biotechnology Co., Ltd., 1,3-diphenylisobenzofuran (DPBF) and MTT powder were purchased from Sigma-Aldrich. Hoechst 33342, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and all other cell experiment-related reagents were purchased from Thermo Fisher Scientific.

[0060] Example 1

[0061] This example provides an experiment for the preparation and performance characterization of a non-fullerene oligomer.

[0062] according to Figure 1 The synthetic route shown in the figure was to react 12,13-di(2-ethylhexyl)-3,9-heneicosyl-12,13-dihydro-[1,2,5]thiadiazo[3,4e]thieno[2",3":4',5']thieno[2',3':4,5]pyrrolo[3,2-g]thieno[2',3':4,5]thieno[3,2-B]indole-2,10-dicarbaldehyde (0.15 g, 0.15 mmol), 2-(2-methoxyphenyl)- 1-(4-methoxyphenyl)-3-(trifluoromethyl)-4-(trifluoromethyl)-2-(trifluoromethyl)benzoic acid methyl ester (0.15 g, 0.15 mmol) and an acceptor unit (A, 0.6 mmol) were placed in a round-bottom flask to obtain a mixture; the mixture (0.16 g, 0.6 mmol) and pyridine (1 mL) were dissolved in chloroform (45 mL) under a nitrogen atmosphere, and the mixture was placed in an oil bath at 65° C. overnight. After cooling to room temperature, the mixture was poured into methanol and filtered. The residue was purified by silica gel column chromatography using dichloromethane / petroleum ether (1 / 1, v / v) as an eluent and recrystallized from dichloromethane / petroleum ether (1 / 1, v / v) to obtain a dark solid. Figure 1 As shown, in this example, three different acceptor units (A) were used for preparation experiments to obtain products O1, O2 and O3.

[0063] The non-fullerene oligomers O1-O3 were characterized by using a Bruker 600MHz AVANCEⅢ NMR system. 1 H and 13 C NMR, as Figure 2 to Figure 7 The results shown show that O1, O2 and O3 1 H spectrum and 13 The C spectra are shown below.

[0064] O1 (0.15 g, yield: 72%):

[0065] 1 HNMR (CDCl3, ppm): 9.12 (2H), 8.67 (2H), 7.98 (2H), 7.76 (4H), 4.81 (4H), 3.21 (4H), 2.18 (2H), 1.87 (4H), 1.50 (4H), 1.36 (4H), 1.25 (30H), 1.09 (10H), 0.99 (2H), 0.86, (6H), 0.79 (6H), 0.69 (6H).

[0066] 13 CNMR (CDCl3, ppm): 188.55, 160.81, 147.40, 144.89, 140.06, 137.85, 136.91, 135.35, 135.10, 135.03, 134.27, 133.51, 133.23, 129.76, 123.62, 121.45, 117.11, 114.94, 113.48, 31.15, 29.82, 29.65, 29.63, 29.53, 29.50, 29.34, 27.64, 22.88, 22.68, 14.11, 10.33.

[0067] O2 (0.15 g, yield: 66%):

[0068] 1 HNMR (CDCl3, ppm): 9.21 (2H), 8.82 (2H), 8.00 (2H), 4.80 (4H), 3.26 (4H), 2.11 (2H), 1.91 (4H), 1.54 (4H), 1.40 (4H), 1.28 (24H), 1.19 (5H), 1.04 (13H), 0.89 (7H), 0.78 (6H), 0.69 (6H).

[0069] 13 CNMR (CDCl3, ppm): 154.15, 147.55, 145.32, 139.58, 139.18, 138.78, 137.80, 136.24, 136.09, 135.86, 133.97, 133.60, 130.91, 126.94, 125.03, 119.99, 113.69, 40.40, 31.92, 31.24, 29.83, 29.69, 29.64, 29.62, 29.51, 29.44, 29.34, 22.79, 22.69, 14.11, 13.72.

[0070] O3 (0.15 g, yield: 72%):

[0071] 1 HNMR (CDCl3, ppm): 9.05(2H), 8.37(2H), 8.01(2H), 4.80(4H), 3.22(4H), 2.20(2H), 1.88(4 H), 1.51(4H), 1.38(4H), 1.27(29H), 1.12(11H), 0.98(3H), 0.88(6H), 0.83(6H), 0.69(6H).

[0072] 13 CNMR (CDCl3, ppm): 181.72, 156.72, 153.24, 147.35, 145.08, 142.60, 142.34, 135.80, 133.27, 127.37, 127.05, 125.21, 113.61, 66.52, 40.31, 31.91, 31.15, 29.82, 29.64, 29.62, 29.52, 29.50, 29.34, 27.58, 27.55, 23.27, 22.83, 22.68, 14.11, 13.71, 10.36.

[0073] Based on density functional theory (DFT), the photophysical properties of non-fullerene oligomers O1-O3 were analyzed. The distribution of their optimized configuration, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are shown in Figure 2. Figure 8 and Fig. 9 The energy gap (Eg) calculation results are shown in Fig.10 As shown, the maximum absorption / emission wavelengths of O1-O3 when dissolved in tetrahydrofuran are as follows Fig.11 The key photophysical parameter values ​​of O1~O3 are shown in Table 1.

[0074] Table 1 Key photophysical parameter values ​​of O1~O3

[0075]

[0076] It can be seen that the optimized configurations of non-fullerene oligomers O1-O3 in the ground state (S0) show that the dihedral angles between the DA′-D segment and the two A parts are 43.8° / 0.58°, 40.82° / 1.37°, and 41.61° / 0.78°, respectively ( Figure 8). A smaller dihedral angle indicates a smaller steric hindrance, which facilitates the rotation of part A. On the contrary, a larger dihedral angle reveals a larger steric hindrance, which restricts intramolecular rotation but facilitates the formation of a distorted conjugated skeleton. From the orbital distribution of O1~O3 molecules, it can be seen that the HOMO of these molecules is mainly distributed on the main chain of O1~O3, while the LUMO is mainly distributed on the receptor unit of these molecules ( Fig. 9 The energy gaps (Eg) of O1 to O3 are calculated to be 2.058, 2.003 and 2.004 eV respectively ( Fig.10 ), which is beneficial to their near-infrared light capture / emission capabilities. The T1 energy levels of O1~O3 are 1294.5, 1249.6 and 1240.7 meV, respectively, which are higher than O 2 and 1 The energy difference between O2 (980meV) ensures that the T1 of O1~O3 molecules to O 2 The feasibility of energy transfer of S0. The energy shift of O1 to O3 (ΔE ST ) are 520.7, 527.7 and 549.1 meV respectively. Such a small ΔE ST The intersystem crossing (ISC) process is promoted, which is beneficial to the generation of reactive oxygen species. The oscillator strengths f of O1-O3 are 1.66, 1.96 and 1.84, respectively, which may be positively correlated with the overlap degree of HUMO and LUMO. Experimentally, O1-O3 show similar absorption and emission spectra, which is due to their comparable conjugated main chains composed of the same DA′-D segments and similar A units. Specifically, the absorption / emission spectra show that the maximum absorption / emission of O1-O3 molecules dissolved in tetrahydrofuran (THF) are located at 698 / 798, 725 / 809 and 709 / 781nm, respectively ( Fig.11 ), O2 shows the longest absorption and emission peaks, which is due to the strongest electron-withdrawing ability of the A part in O2, resulting in the smallest Eg. The mass extinction coefficients of O1~O3 at their maximum absorption are 107.2, 96.3 and 116.3 L / cm / g, respectively, which are better than most reported molecules.

[0077] Example 2

[0078] This example provides an ISC dynamic analysis experiment of a non-fullerene oligomer.

[0079] The generation process of ROS is directly related to the ISC of PS. The ISC dynamics of O1-O3 under 800nm ​​laser excitation were analyzed using ultrafast femtosecond transient absorption (fs-TA) spectroscopy, and the ISC lifetime was estimated based on the dynamic evolution trajectory of triplet excited state absorption (ESA). The dynamic evolution trajectory of ESA of O1-O3 is shown in Fig.13As shown in the figure, the singlet and triplet evolution dynamics of O1 to O3 are shown in the figure. Fig.14 The two-dimensional femtosecond transient spectra at different delay times are shown in Fig.12 As shown. Fig.13 It can be seen that O1 to O3 show similar ESA evolution trajectories, so the ESA analysis of O1 is taken as an example. Fig.14 It can be seen that the ESA of O1 at 415nm shows a fast singlet decay with a lifetime of 1.2ps, while the triplet ESA at 525nm shows a fast distribution with a lifetime within 4.24ps. Fig.14 The singlet and triplet evolution dynamics of O1-O3 in τ decay , τ rise and Φ ISC quantified, where the ISC efficiency can be calculated using the formula Φ ISC =[1 / (τ(T1,rise)] / [1 / (τ(S1,decay)] to obtain the results shown in Table 2.

[0080] Table 2τ of O1~O3 decay , τ rise and Φ ISC The quantitative results

[0081] <![CDATA[τ decay (ps)]]> <![CDATA[τ rise (ps)]]> <![CDATA[Φ ISC ]]> O1 1.2 4.24 28％ O2 0.43 4.47 10％ O3 1.3 5.4 24％

[0082] Depend on Fig.14 As shown in Table 2, O1~O3 all have higher Φ ISC , 28%, 10% and 24% respectively. This is attributed to the modulation of the photophysical properties of the acceptor unit, such as the triplet energy level (T1), energy shift (ΔE ST ) and oscillator strength (f), the introduction of different electron absorption strength acceptors (A) and heteroatoms (S) achieved T1, ΔE ST and f are adjustable, thus achieving the ISC control.

[0083] Figure 8 , Fig. 9 , Fig.13 , Fig.14 and Fig.12 The results together indicate that the weaker the electron-withdrawing strength of the acceptor unit of the non-fullerene oligomer, the smaller the ΔE ST , the smaller f is, the larger T1 is, Φ ISC For the non-fullerene oligomer O3, the introduction of heteroatoms (S) can also promote ISC because different orbitals are involved in the non-radiative transition from S1 to T1.

[0084] Example 3

[0085] This example provides a preparation and characterization experiment of non-fullerene oligomer nanoparticles.

[0086] according to Fig.15 The synthetic route shown uses the FDA-approved amphiphilic polymer DSPE-PEG 2000 , O1~O3 molecules were made into water-dispersible nanoparticles (ONPs 1~ONPs3) by a simple nanoprecipitation method. Taking O1 as an example, O1 was dissolved in THF at a final concentration of 1 mg / mL, and the encapsulation matrix DSPE-PEG 2000 Dissolve in THF at a final concentration of 5 mg / mL, then mix 1 mL of O1 solution and 2 mL of DSPE-PEG 2000 The solution was mixed evenly and quickly dropped into 27 mL of DI water under stirring. After removing THF, the mixture was filtered with a filter (0.22 μm) and then concentrated with an ultrafiltration device (100 kDa) to obtain nanoparticles ONPs 1. The size and morphology of nanoparticles ONPs 1 to ONPs 3 prepared using O1 to O3 as raw materials were studied by transmission electron microscopy (TEM, Philips Technai 12) and dynamic light scattering (DLS, Malvem Zetasizer Nano ZS). Fig.16 The particle size statistics shown, and Fig.16 Representative TEM images are inserted in Figure , showing the spherical morphology of the nanoparticles; Fig.17 Absorption and emission spectra (Shimadzu 1700 UV-Vis-NIR spectrophotometer) as shown; emission wavelength and extinction coefficient of each product as shown in Table 3.

[0087] Table 3 Emission wavelength and extinction coefficient of each product

[0088]

[0089] Depend on Fig.16 From Table 3, we can see that the average hydrodynamic diameters of ONPs 1 to ONPs 3 are 54.3, 47.1, and 51.23 nm, respectively, and the corresponding polydispersity indices are 0.28, 0.314, and 0.295. Fig.17 It can be seen that the absorption and emission spectra of ONPs 1 to ONPs 3 are greatly broadened and well extended. Specifically, the absorption and emission peaks of ONPs 1 to ONPs 3 are 764 / 908, 808 / 926, and 778 / 903 nm, respectively. The mass extinction coefficients of ONPs 1 to ONPs 3 at 808 nm are 30.1, 51.2, and 42.6 L / cm / g, respectively, enabling phototherapy under 808 nm laser excitation.

[0090] On this basis, 2′,7′-dichlorofluorescein (DCFH) was used as a fluorescent probe to study the reactive oxygen species production ability of ONPs 1 to ONPs 3. The specific steps were as follows: 700 μL DCFH (40 μM in PBS) was added to ONPs 1 to ONPs 3 respectively, and the final OD value at 808 nm was about 0.2; then the mixed solution was exposed to 808 nm laser (100 mW / cm 2 ) for discontinuous irradiation; after laser irradiation for 0, 1, 2, 3, 4 and 5 min, the fluorescence of DCFH was measured at 525 nm, and the following was obtained: Fig.18 , Fig.19 and Fig. 20 The results are shown. It can be seen that in the presence of ONPs 1 to ONPs 3, the fluorescence intensity of DCFH gradually increases with the extension of 808nm laser irradiation time. This fluorescence enhancement is much higher than the fluorescence enhancement of DCFH in the presence of ICG under the same irradiation conditions, indicating that ONPs 1 to ONPs 3 have good ROS production ability. In summary, it was found that the ROS production ability of ONPs 1 to ONPs 3 was 6.8 times, 1.7 times, and 4.3 times higher than that of ICG, respectively. This trend is consistent with the Φ ISC The relationship between the ROS-producing ability of ONPs 1 to ONPs 3 and their molecular structure was fundamentally clarified. ISC Positive correlation.

[0091] The 1,3-diphenylisobenzofuran (DPBF) probe was used to measure the 1 The O2 yield was determined by diluting the concentrated ONPs and ICG with 350 μL DI water and 350 μL DMSO, and mixing the solution with 10 μL DPBF (1 mg / mL, dissolved in DMSO), where the absorbance values ​​at 808 nm for ONPs 1 were 0.288, ONPs 2 were 0.278, ONPs 3 were 0.280, ONPs 4 were 0.282, and ICG was 0.310. Then, ONPs and ICG were exposed to 808 nm laser (100 mW / cm 2 ) to detect the absorption change of DPBF; the absorption spectrum was monitored every 10 seconds during laser irradiation; the singlet oxygen quantum yield of ONPs (ONPs) was measured according to the following equation:

[0092]

[0093] In equation (a), represents the singlet oxygen yield of the reference (ICG), k and k ref Refers to the photodegradation rate constants of DPBF treated with ONPs and ICG, respectively.a and I represents the light absorbance of ONPs and ICG at 808 nm, respectively. a and The ratio of can be determined by equation (b). In equation (b), A 808 and are the absorbance values ​​of ONPs and ICG at 808 nm, respectively, and the results are as follows: Fig.21 , Fig. 22 and Fig.23 The results shown. It can be seen that the absorption of DPBF at 415nm (Abs 415) is detected 1 In the presence of ONPs 1 to ONPs 3, the Abs 415 of DPBF decreased significantly under laser irradiation, while in the presence of ICG, a slight decrease in the Abs 415 of DPBF was observed. 1 The O2 production rates were 2.2%, 0.4% and 0.9%, respectively. These results demonstrate that ONPs 1 to ONPs 3 have higher ROS production capacity and higher 1 O2 production rate. Further experiments were conducted using ONPs1 as an example.

[0094] The generation of ROS was detected by electron spin resonance (ESR, Bruker E500 spectrometer), and the specific steps were as follows: A549 cells were incubated with DMEM containing ONPs 1 (final concentration: 12.5 μg / mL) for 4 h; the culture medium was removed and the cells were washed three times with PBS; 16 μM Hoechst 33342 and 20 μM DCFH-DA were added; the cells were incubated for 30 min and exposed to 808 nm laser irradiation (0.75 W / cm 2 )5min. In order to distinguish the reactive oxygen species, 2,2,6,6-tetramethylpiperidine (TEMP), 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2-diethoxyphosphoryl-2-methyl-1-oxido-3,4-dihydropyrrol-1-ium (DEPMPO) were selected as spin trapping agents, respectively, to obtain Fig.24 and Fig.25 The mixture of ONPs1 and TEMP was irradiated with 808nm laser for 5 minutes (ONPs1+Laser), and the following Fig.24 The characteristic 1:1:1 triple resonance signal is shown in the left figure of Figure 1. No obvious characteristic signal was observed when there was no laser irradiation (ONPs 1) or when ONPs 1 was absent (Laser) or when both were absent (PBS). These results demonstrate that ONPs 1 can effectively generate 1After the mixture of ONPs 1 and DMPO was irradiated with 808 nm laser for 5 minutes (ONPs 1+Laser), the following Fig.24 The obvious resonance signal is shown in the right figure of the figure. No obvious signal was observed in the absence of laser excitation (ONPs 1) or in the absence of ONPs 1 (Laser) or both (PBS). These results confirm that ONPs 1 can generate hydroxyl radicals (·OH) under 808 nm laser irradiation. In addition, Fig.25 It can be seen that no resonance signal was observed in the mixture of ONPs 1 and DEPMPO, proving that there was no superoxide radical (O2 ·- )generate. 1 The generation of O2 and ·OH enables type I and type II synergistic PDT.

[0095] In summary, ONPs 1 to ONPs 3 have good responsiveness to 808 nm excitation light and strong ROS generation ability (1.7 to 6.8 times higher than ICG) and 1 O2 yield (0.9% to 2.2%, relative to 0.2% for ICG), the most efficient ONPs 1 was also confirmed to produce ·OH. With these properties, ONPs 1 was able to generate ·OH under 808 nm laser irradiation (0.44 W / cm 2 ) achieved significant in vitro and in vivo type I and type II PDT synergistic therapeutic capabilities.

[0096] To examine whether ONPs 1 produces ROS in cells, human lung cancer cells (A549) were incubated with ONPs 1 (12.5 μg / mL) and then confocal imaging of A549 cells was performed (confocal laser scanning microscopy, CLSM, Leica SP 5 with IR laser); the fluorescent dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used as a probe to detect intracellular ROS; the detection mechanism is that DCFH-DA is first deacetylated by cellular esterases to a non-fluorescent compound and then oxidized by ROS to 2',7'-dichlorofluorescein (DCF) with a fluorescence emission peak at 525 nm; the detection steps are as follows: A549 cells were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin in a 37°C cell culture incubator; the cells were seeded into 96-well plates (5000 cells per well) and cultured overnight; the indicated concentrations of ONPs 1 were used to detect ROS. 1DMEM solution (100 μL per well) replaced the original culture medium; to measure phototoxicity, cells were exposed to laser irradiation (0.75 W / cm 2) for 15 minutes; after incubation for 24 hours, the ONPs 1 solution was removed, and the MTT solution (0.5 mg / mL) diluted in DMEM was added to each designated cell for incubation for 4 hours, and then 100 μL DMSO was added to dissolve the result. The cell viability was then measured using a microplate reader, and the results were as follows: Fig.26 The results shown. Fig.26 As shown in a, after incubation with DCFH-DA, green fluorescence was only observed in A549 cells incubated with ONPs 1 and irradiated with 808 nm laser (ONPs 1+Laser), while no green fluorescence was found in cells treated with PBS, Laser, or ONPs 1, indicating that ONPs 1 has a good ROS production ability under 808 nm laser irradiation. To evaluate the PDT effect of ONPs 1 on cells, the toxicity of ONPs 1 was determined by the MTT method. Fig.26 As shown in Figures b and c, in the absence of laser irradiation, ONPs 1 showed little toxicity to normal cells (3T3 cells) and A549 cells. Under laser irradiation, ONPs 1 showed significant PDT effect and concentration-dependent cell death. In addition, AM / PI staining was performed to confirm the PDT efficacy of ONPs 1. Fig.26 As shown in the figure, laser or ONPs 1 itself has very little toxicity to A549 cells, but under 808nm laser irradiation, almost all cells are killed by ONPs 1. Fig.26 As shown in d, during laser irradiation, the temperature was kept below 43 °C, which is the threshold temperature for initiating cell apoptosis, thereby minimizing the photothermal effect of ONPs 1. These results indicate that ONPs 1 is an effective near-infrared PSs.

[0097] Example 4

[0098] This example provides an in vivo application experiment of non-fullerene oligomer nanoparticles.

[0099] according to Fig. 27 The performance of ONPs 1 for in vivo PDT was evaluated using A549 tumor-bearing mice, and the results were as follows: Fig. 27 b~f and Fig.28 The result of Fig. 27 As shown in Figures b and c, ONPs 1 was injected into the tail vein for 12 hours and then illuminated with 808 nm laser at 0.44 W / cm 2 The mouse tumors were irradiated with a power density of 100 μg / mL for 30 min, keeping the maximum temperature below 43 °C, the threshold temperature for cell apoptosis, thereby minimizing the thermal effect. To evaluate the PDT efficacy of ONPs 1, the tumor volume of the mice was measured every two days after treatment. Fig. 27As shown in the figure, the tumor growth rate of mice treated with PBS, Laser or NPs was very fast, while the tumor growth rate of mice treated with NPs+Laser was effectively inhibited. Body weight was monitored 14 days after treatment. Fig. 27 As can be seen from the figure, no significant weight loss was found in all mice, revealing that the applied treatment had minimal side effects. In addition, on the 14th day after PDT treatment, the tumors of mice receiving different treatments were weighed and recorded. Fig. 27 f and Fig.28 Compared with the control group, the tumors in mice treated with NPs+Laser were almost eliminated, demonstrating the excellent PDT effect of ONPs 1.

[0100] To further confirm the therapeutic efficacy and biosafety of ONPs 1, hematoxylin and eosin (H&E) staining was performed, followed by histological analysis of major organs (heart, liver, spleen, lung, and kidney) and tumor tissues. Fig.29 The results shown. Fig.29 As shown in a, significant nuclear dissociation was observed in tumor tissues from mice treated with NPs+Laser, but not in tumors from mice treated with PBS, Laser, or NPs, confirming that ONPs 1 has a good PDT effect under 808 nm laser irradiation. No obvious pathological abnormalities were found in the major organs of mice receiving different treatments, indicating that ONPs 1 has little toxicity. Fig.29 As shown in b, routine blood tests and blood biochemical analysis further proved that ONPs 1 had good biocompatibility. These results together demonstrated the reliable biosafety and excellent PDT effect of ONPs 1.

[0101] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0102] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A use of a non-fullerene oligomer as a PDT photosensitizer, characterized in that: The structural formula of the non-fullerene oligomer is shown in any one of Formula I, Formula II and Formula III; 2. The use according to claim 1, characterized in that: The non-fullerene oligomer has an ADA'-DA structure.

3. A non-fullerene oligomer, characterized in that The structural formula of the non-fullerene oligomer is as described in claim 1.

4. The method for preparing the non-fullerene oligomer according to claim 3, characterized in that: The following steps are involved: The non-fullerene oligomer is designed and synthesized by Knoevenagel condensation of the donor-acceptor′-donor fragment and the acceptor unit; Wherein, the structural formula of the donor-acceptor′-donor fragment is shown in Formula IV; the structural formula of the acceptor unit is shown in any one of Formula V, Formula VI and Formula VII; 5. A use of non-fullerene oligomer nanoparticles as a PDT photosensitizer, characterized in that: The structural formula of the non-fullerene oligomer is as described in claim 1.

6. The use according to claim 5, characterized in that: The absorption wavelength of the non-fullerene oligomer nanoparticles is ≥808 nm.

7. The use according to claim 5, characterized in that: The non-fullerene oligomer nanoparticles have the ability of synergistic effect of type I PDT and type II PDT.

8. The use according to claim 7, characterized in that: The particle size of the non-fullerene oligomer nanoparticles is 45-55 nm.

9. The method for preparing non-fullerene oligomer nanoparticles according to any one of claims 5 to 8, characterized in that: The following steps are involved: Dissolving the non-fullerene oligomer in tetrahydrofuran to obtain an oligomer solution, and dissolving an encapsulation matrix in tetrahydrofuran to obtain an encapsulation matrix solution; The oligomer solution and the encapsulation matrix solution are uniformly mixed, and dropped into deionized water under stirring conditions. After removing tetrahydrofuran, the mixture is filtered and concentrated to obtain the non-fullerene oligomer nanoparticles.

10. The preparation method according to claim 9, characterized in that: The concentration of the oligomer solution is 0.8 to 1.2 mg / mL; and / or, the concentration of the encapsulation matrix solution is 3 to 8 mg / mL; and / or, the ratio of the oligomer solution to the encapsulation matrix solution is 1:8-13; And / or, the encapsulating matrix is ​​DSPE-PEG 2000 , any one of PEG-b-PPG-b-PEG, PSMA, and BSA.

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