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Effective photosensitizers are especially important for the widespread clinical use of phototherapy. However, conventional photosensitizers generally suffer from short wavelength absorption, insufficient photostability, low quantum yield of reactive oxygen species (ROS), and aggregation-induced quenching of ROS. Here we report a near-infrared (NIR) supramolecular photosensitizer (RuDA) mediated by self-assembly of Ru(II)-arene organometallic complexes in aqueous solution. RuDA can only generate singlet oxygen (1O2) in the aggregated state, and it exhibits obvious aggregation-induced 1O2 generation behavior due to a significant increase in the crossover process between the singlet-triplet system. Under the action of 808 nm laser light, RuDA exhibits an 1O2 quantum yield of 16.4% (FDA-approved indocyanine green: ΦΔ=0.2%) and a high photothermal conversion efficiency of 24.2% (commercial gold nanorods) with excellent photostability. : 21.0%, gold nanoshells: 13.0%). In addition, RuDA-NPs with good biocompatibility can preferentially accumulate at tumor sites, causing significant tumor regression during photodynamic therapy with a 95.2% reduction in tumor volume in vivo. This aggregation-enhancing photodynamic therapy provides a strategy for developing photosensitizers with favorable photophysical and photochemical properties.
Compared to conventional therapy, photodynamic therapy (PDT) is an attractive treatment for cancer due to its significant advantages such as accurate spatiotemporal control, non-invasiveness, negligible drug resistance, and minimization of side effects 1,2,3. Under light irradiation, the photosensitizers used can be activated to form highly reactive oxygen species (ROS), leading to apoptosis/necrosis or immune responses4,5. However, most conventional photosensitizers, such as chlorins, porphyrins, and anthraquinones, have relatively short-wavelength absorption (frequency < 680 nm), thus resulting in poor light penetration because of the intense absorption of biological molecules (eg, hemoglobin and melanin) in the visible region6,7. However, most conventional photosensitizers, such as chlorins, porphyrins, and anthraquinones, have relatively short-wavelength absorption (frequency < 680 nm), thus resulting in poor light penetration because of the intense absorption of biological molecules (eg, hemoglobin and melanin) in the visible region6,7. Однако большинство обычных фотосенсибилизаторов, таких как хлорины, порфирины и антрахиноны, обладают относительно коротковолновым поглощением (частота < 680 нм), что приводит к плохому проникновению света из-за интенсивного поглощения биологических молекул (например, гемоглобина и меланина) в видимая область6,7. However, most common photosensitizers such as chlorins, porphyrins and anthraquinones have relatively short wavelength absorption (< 680 nm) resulting in poor light penetration due to intense absorption of biological molecules (e.g. hemoglobin and melanin) into the visible region6,7.然而,大多数传统的光敏剂,如二氢卟酚、卟啉和蒽醌,具有相对较短的波长吸收(频率< 680 nm),因此由于对生物分子(如血红蛋白和黑色素)的强烈吸收,导致光穿透性差。然而 , 大多数 传统 的 光敏剂 , 二 氢 卟酚 、 卟啉 蒽醌 , 具有 相对 较 短 的 波长 吸收 (频率 频率 <680 nm) 因此 由于 对 分子 (血红 蛋白 和 黑色素) 的 , , , , 吸收 吸收 吸收 吸收 吸收 吸收 吸收 吸收 HI导致光穿透性差。 Однако большинство традиционных фотосенсибилизаторов, таких как хлорины, порфирины и антрахиноны, имеют относительно коротковолновое поглощение (частота < 680 нм) из-за сильного поглощения биомолекул, таких как гемоглобин и меланин, что приводит к плохому проникновению света. However, most traditional photosensitizers such as chlorins, porphyrins and anthraquinones have relatively short wavelength absorption (frequency < 680 nm) due to strong absorption of biomolecules such as hemoglobin and melanin resulting in poor light penetration. Visible area 6.7. Therefore, near-infrared (NIR) absorbing photosensitizers that are activated in the 700–900 nm “therapeutic window” are well suited for phototherapy. Since near infrared light is the least absorbed by biological tissues, it can lead to deeper penetration and less photodamage8,9.
Unfortunately, existing NIR-absorbing photosensitizers generally have poor photostability, low singlet oxygen (1O2) generating capacity, and aggregation-induced 1O2 quenching, which limits their clinical application10,11. Although great efforts have been made to improve the photophysical and photochemical properties of conventional photosensitizers, so far several reports have reported that NIR-absorbing photosensitizers can solve all these problems. In addition, several photosensitizers have shown promise for efficient generation of 1O212,13,14 when irradiated with light above 800 nm, since the photon energy decreases rapidly in the near-IR region. Triphenylamine (TFA) as an electron donor and [1,2,5]thiadiazole-[3,4-i]dipyrido[a,c]phenazine (TDP) as an electron acceptor group Donor-acceptor (DA) type dyes a class of dyes , absorbing near-infrared, which have been extensively studied for near-infrared bioimaging II and photothermal therapy (PTT) due to their narrow bandgap. Thus, DA-type dyes can be used for PDT with near-IR excitation, although they have rarely been studied as photosensitizers for PDT.
It is well known that the high efficiency of intersystem crossing (ISC) of photosensitizers promotes the formation of 1O2. A common strategy for advancing the ISC process is to enhance the spin-orbit coupling (SOC) of photosensitizers by introducing heavy atoms or special organic moieties. However, this approach still has some disadvantages and limitations19,20. Recently, supramolecular self-assembly has provided a bottom-up intelligent approach for the fabrication of functional materials at the molecular level,21,22 with numerous advantages in phototherapy: (1) self-assembled photosensitizers may have the potential to form ribbon structures. Similar to electronic structures with a denser distribution of energy levels due to overlapping orbits between building blocks. Therefore, the energy match between the lower singlet excited state (S1) and the neighboring triplet excited state (Tn) will be improved, which is beneficial for the ISC process 23, 24 . (2) Supramolecular assembly will reduce non-radiative relaxation based on the intramolecular motion limitation mechanism (RIM), which also promotes the ISC process 25, 26 . (3) The supramolecular assembly can protect the inner molecules of the monomer from oxidation and degradation, thereby greatly improving the photostability of the photosensitizer. Given the above advantages, we believe that supramolecular photosensitizer systems can be a promising alternative to overcome the shortcomings of PDT.
Ru(II)-based complexes are a promising medical platform for potential applications in the diagnosis and therapy of diseases due to their unique and attractive biological properties28,29,30,31,32,33,34. In addition, the abundance of excited states and the tunable photophysicochemical properties of Ru(II)-based complexes provide great advantages for the development of Ru(II)-based photosensitizers35,36,37,38,39,40. A notable example is the ruthenium(II) polypyridyl complex TLD-1433, which is currently in Phase II clinical trials as a photosensitizer for the treatment of non-muscle invasive bladder cancer (NMIBC)41. In addition, ruthenium(II)arene organometallic complexes are widely used as chemotherapeutic agents for cancer treatment due to their low toxicity and ease of modification42,43,44,45. The ionic properties of Ru(II)-arene organometallic complexes can not only improve the poor solubility of DA chromophores in common solvents, but also improve the assembly of DA chromophores. In addition, the pseudooctahedral half-sandwich structure of the organometallic complexes of Ru(II)-arenes can sterically prevent H-aggregation of DA-type chromophores, thereby facilitating the formation of J-aggregation with redshifted absorption bands. However, inherent disadvantages of Ru(II)-arene complexes, such as low stability and/or poor bioavailability, can affect the therapeutic efficacy and in vivo activity of arene-Ru(II) complexes. However, studies have shown that these disadvantages can be overcome by encapsulating ruthenium complexes with biocompatible polymers by physical encapsulation or covalent conjugation.
In this work, we report DA-conjugated complexes of Ru(II)-arene (RuDA) with an NIR trigger via a coordination bond between the DAD chromophore and the Ru(II)-arene moiety. The resulting complexes can self-assemble into metalosupramolecular vesicles in water due to non-covalent interactions. Notably, the supramolecular assembly endowed RuDA with polymerization-induced intersystem crossing-over properties, which significantly increased ISC efficiency, which was very favorable for PDT (Fig. 1A). To increase tumor accumulation and in vivo biocompatibility, FDA-approved Pluronic F127 (PEO-PPO-PEO) was used to encapsulate RuDA47,48,49 to create RuDA-NP nanoparticles (Figure 1B) that acted as a highly efficient PDT/ Dual-mode PTT proxy . In cancer phototherapy (Figure 1C), RuDA-NP was used to treat nude mice with MDA-MB-231 tumors to study the efficacy of PDT and PTT in vivo.
Schematic illustration of the photophysical mechanism of RuDA in monomeric and aggregated forms for cancer phototherapy, synthesis of B RuDA-NPs and C RuDA-NPs for NIR-activated PDT and PTT.
RuDA, consisting of TPA and TDP functionality, was prepared according to the procedure shown in Supplementary Figure 1 (Figure 2A), and RuDA was characterized by 1H and 13C NMR spectra, electrospray ionization mass spectrometry, and elemental analysis (Supplementary Figures 2-4). The RuDA electron density difference map of the lowest singlet transition was computed by time-dependent density functional theory (TD-DFT) to study the charge transfer process. As shown in Supplementary Figure 5, the electron density drifts mainly from triphenylamine to the TDP acceptor unit after photoexcitation, which can be attributed to a typical intramolecular charge transfer (CT) transition.
Chemical structure of Ore. B Absorption spectra of Ore in mixtures of various ratios of DMF and water. C Normalized absorption values ​​of RuDA (800 nm) and ICG (779 nm) versus time at 0.5 W cm-2 of 808 nm laser light. D The photodegradation of ABDA is indicated by RuDA-induced formation of 1O2 in DMF/H2O mixtures with different water contents under the action of laser radiation with a wavelength of 808 nm and a power of 0.5 W/cm2.
Abstract—UV-visible absorption spectroscopy was used to study the self-assembly properties of Ore in mixtures of DMF and water in various ratios. As shown in fig. 2B, RuDA exhibits absorption bands from 600 to 900 nm in DMF with a maximum absorption band at 729 nm. Increasing the amount of water led to a gradual red shift of the Ore absorption maximum to 800 nm, which indicates J-aggregation of Ore in the assembled system. The photoluminescence spectra of RuDA in different solvents are shown in Supplementary Figure 6. RuDA appears to exhibit typical NIR-II luminescence with a maximum emission wavelength of ca. 1050 nm in CH2Cl2 and CH3OH, respectively. The large Stokes shift (about 300 nm) of RuDA indicates a significant change in the geometry of the excited state and the formation of low-energy excited states. The luminescence quantum yields of Ore in CH2Cl2 and CH3OH were determined to be 3.3 and 0.6%, respectively. However, in a mixture of methanol and water (5/95, v/v), a slight redshift of the emission and a decrease in the quantum yield (0.22%) were observed, which may be due to the self-assembly of Ore.
To visualize the self-assembly of ORE, we used liquid atomic force microscopy (AFM) to visualize the morphological changes in ORE in methanol solution after adding water. When the water content was below 80%, no clear aggregation was observed (Supplementary Fig. 7). However, with a further increase in the water content to 90–95%, small nanoparticles appeared, which indicated the self-assembly of Ore. In addition, laser irradiation with a wavelength of 808 nm did not affect the absorption intensity of RuDA in aqueous solution (Fig. 2C and Supplementary Fig. 8). In contrast, the absorbance of indocyanine green (ICG as control) dropped rapidly at 779 nm, indicating excellent photostability of RuDA. In addition, the stability of RuDA-NPs in PBS (pH = 5.4, 7.4 and 9.0), 10% FBS and DMEM (high glucose) was examined by UV-visible absorption spectroscopy at various points time. As shown in Supplementary Figure 9, slight changes in RuDA-NP absorption bands were observed in PBS at pH 7.4/9.0, FBS and DMEM, indicating excellent stability of RuDA-NP. However, in an acidic medium (рН = 5.4) hydrolysis of Ore was found. We also further evaluated the stability of RuDA and RuDA-NP using high performance liquid chromatography (HPLC) methods. As shown in Supplementary Figure 10, RuDA was stable in a mixture of methanol and water (50/50, v/v) for the first hour, and hydrolysis was observed after 4 hours. However, only a wide concave-convex peak was observed for RuDA NPs. Therefore, gel permeation chromatography (GPC) was used to assess the stability of RuDA NPs in PBS (pH = 7.4). As shown in Supplementary Figure 11, after 8 hours of incubation under the tested conditions, the peak height, peak width and peak area of ​​NP RuDA did not change significantly, indicating excellent stability of NP RuDA. In addition, TEM images showed that the morphology of the RuDA-NP nanoparticles remained virtually unchanged after 24 hours in diluted PBS buffer (pH = 7.4, Supplementary Fig. 12).
Because self-assembly can confer different functional and chemical characteristics on Ore, we observed the release of 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA, indicator 1O2) in methanol-water mixtures. Ore with different water content50. As shown in Figure 2D and Supplementary Figure 13, no degradation of ABDA was observed when the water content was below 20%. With an increase in humidity to 40%, ABDA degradation occurred, as evidenced by a decrease in the intensity of ABDA fluorescence. It has also been observed that higher water content results in faster degradation, suggesting that RuDA self-assembly is necessary and beneficial for ABDA degradation. This phenomenon is very different from modern ACQ (aggregation-induced quenching) chromophores. When irradiated with a laser with a wavelength of 808 nm, the quantum yield of 1O2 RuDA in a mixture of 98% H2O/2% DMF is 16.4%, which is 82 times higher than that of ICG (ΦΔ = 0.2%)51, demonstrating a remarkable generation efficiency 1O2 RuDA in the state of aggregation.
Electron spins using 2,2,6,6-tetramethyl-4-piperidinone (TEMP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as spin traps Resonance spectroscopy (ESR) was used to identify the resulting species AFK. by RuDA. As shown in Supplementary Figure 14, it has been confirmed that 1O2 is generated at irradiation times between 0 and 4 minutes. In addition, when RuDA was incubated with DMPO under irradiation, a typical four-line EPR signal of 1:2:2:1 DMPO-OH· adduct was detected, indicating the formation of hydroxyl radicals (OH·). Overall, the above results demonstrate the ability of RuDA to stimulate ROS production through a dual type I/II photosensitization process.
To better understand the electronic properties of RuDA in monomeric and aggregated forms, the frontier molecular orbitals of RuDA in monomeric and dimeric forms were calculated using the DFT method. As shown in fig. 3A, the highest occupied molecular orbital (HOMO) of monomeric RuDA is delocalized along the ligand backbone and the lowest unoccupied molecular orbital (LUMO) is centered on the TDP acceptor unit. On the contrary, the electron density in the dimeric HOMO is concentrated on the ligand of one RuDA molecule, while the electron density in the LUMO is mainly concentrated on the acceptor unit of another RuDA molecule, which indicates that RuDA is in the dimer. Features of CT.
A The HOMO and LUMO of Ore are calculated in monomeric and dimeric forms. B Singlet and triplet energy levels of Ore in monomers and dimers. C Estimated levels of RuDA and possible ISC channels as monomeric C and dimeric D. Arrows indicate possible ISC channels.
The distribution of electrons and holes in the low-energy singlet excited states of RuDA in the monomeric and dimeric forms was analyzed using the Multiwfn 3.852.53 software, which were calculated using the TD-DFT method. As indicated on the additional label. As shown in Figures 1-2, monomeric RDA holes are mostly delocalized along the ligand backbone in these singlet excited states, while electrons are mostly located in the TDP group, demonstrating the intramolecular characteristics of CT. In addition, for these singlet excited states, there is more or less overlap between holes and electrons, suggesting that these singlet excited states make some contribution from local excitation (LE). For dimers, in addition to intramolecular CT and LE features, a certain proportion of intermolecular CT features were observed in the respective states, especially S3, S4, S7, and S8, based on intermolecular CT analysis, with CT intermolecular transitions as the main ones (Supplementary Table). 3).
To better understand the experimental results, we further explored the properties of RuDA excited states to explore the differences between monomers and dimers (Supplementary Tables 4–5). As shown in Figure 3B, the energy levels of the singlet and triplet excited states of the dimer are much denser than those of the monomer, which helps to reduce the energy gap between S1 and Tn. It has been reported that the ISC transitions could be realized within small energy gap (ΔES1-Tn < 0.3 eV) between S1 and Tn54. It has been reported that the ISC transitions could be realized within a small energy gap (ΔES1-Tn < 0.3 eV) between S1 and Tn54. Сообщалось, что переходы ISC могут быть реализованы в пределах небольшой энергетической щели (ΔES1-Tn <0,3 эВ) между S1 и Tn54. It has been reported that ISC transitions can be realized within a small energy gap (ΔES1-Tn <0.3 eV) between S1 and Tn54.据报道,ISC 跃迁可以在S1 和Tn54 之间的小能隙(ΔES1-Tn < 0.3 eV)内实现。据报道,ISC 跃迁可以在S1 和Tn54 之间的小能隙(ΔES1-Tn < 0.3 eV)内实现。 Сообщалось, что переход ISC может быть реализован в пределах небольшой энергетической щели (ΔES1-Tn < 0,3 эВ) между S1 и Tn54. It has been reported that the ISC transition can be realized within a small energy gap (ΔES1-Tn < 0.3 eV) between S1 and Tn54. In addition, only one orbital, occupied or unoccupied, must differ in bound singlet and triplet states to provide a non-zero SOC integral. Thus, based on the analysis of the excitation energy and the orbital transition, all possible channels of the ISC transition are shown in Figs. 3C,D. Notably, only one ISC channel is available in the monomer, while the dimeric form has four ISC channels that can enhance the ISC transition. Therefore, it is reasonable to assume that the more RuDA molecules are aggregated, the more accessible the ISC channels will be. Therefore, RuDA aggregates can form two-band electronic structures in the singlet and triplet states, reducing the energy gap between S1 and available Tn, thereby increasing the efficiency of ISC to facilitate 1O2 generation.
To further elucidate the underlying mechanism, we synthesized a reference compound of the arene-Ru(II) complex (RuET) by replacing two ethyl groups with two triphenylamine phenyl groups in RuDA (Fig. 4A, for full characterization, see ESI, Supplementary 15-21 ) From donor (diethylamine) to acceptor (TDF), RuET has the same intramolecular CT characteristics as RuDA. As expected, the absorption spectrum of RuET in DMF showed a low energy charge transfer band with strong absorption in the near infrared region in the region of 600–1100 nm (Fig. 4B). In addition, RuET aggregation was also observed with increasing water content, which was reflected in the redshift of the absorption maximum, which was further confirmed by liquid AFM imaging (Supplementary Fig. 22). The results show that RuET, like RuDA, can form intramolecular states and self-assemble into aggregated structures.
Chemical structure of RuET. B Absorption spectra of RuET in mixtures of various ratios of DMF and water. Plots C EIS Nyquist for RuDA and RuET. Photocurrent responses D of RuDA and RuET under the action of laser radiation with a wavelength of 808 nm.
The photodegradation of ABDA in the presence of RuET was evaluated by irradiation with a laser with a wavelength of 808 nm. Surprisingly, no degradation of ABDA was observed in various water fractions (Supplementary Fig. 23). A possible reason is that RuET cannot efficiently form a banded electronic structure because the ethyl chain does not promote efficient intermolecular charge transfer. Therefore, electrochemical impedance spectroscopy (EIS) and transient photocurrent measurements were performed to compare the photoelectrochemical properties of RuDA and RuET. According to the Nyquist plot (Figure 4C), RuDA shows a much smaller radius than RuET, which means that RuDA56 has faster intermolecular electron transport and better conductivity. In addition, the photocurrent density of RuDA is much higher than that of RuET (Fig. 4D), confirming the better charge transfer efficiency of RuDA57. Thus, the phenyl group of triphenylamine in Ore plays an important role in providing intermolecular charge transfer and formation of a banded electronic structure.
To increase tumor accumulation and in vivo biocompatibility, we further encapsulated RuDA with F127. The average hydrodynamic diameter of RuDA-NPs was determined to be 123.1 nm with a narrow distribution (PDI = 0.089) using the dynamic light scattering (DLS) method (Figure 5A), which promoted tumor accumulation by increasing permeability and retention. EPR) effect. The TEM images showed that Ore NPs have a uniform spherical shape with an average diameter of 86 nm. Notably, the absorption maximum of RuDA-NPs appeared at 800 nm (Supplementary Fig. 24), indicating that RuDA-NPs may retain the functions and properties of self-assembling RuDAs. The calculated ROS quantum yield for NP Ore is 15.9%, which is comparable to Ore. The photothermal properties of RuDA NPs were studied under the action of laser radiation with a wavelength of 808 nm using an infrared camera. As shown in fig. 5B,C, the control group (PBS only) experienced a slight increase in temperature, while the temperature of the RuDA-NPs solution increased rapidly with increasing temperature (ΔT) to 15.5, 26.1, and 43.0°C. High concentrations were 25, 50, and 100 µM, respectively, which indicates a strong photothermal effect of RuDA NPs. In addition, heating/cooling cycle measurements were taken to evaluate the photothermal stability of RuDA-NP and compare with ICG. The temperature of Ore NPs did not decrease after five heating/cooling cycles (Fig. 5D), which indicates the excellent photothermal stability of Ore NPs. In contrast, ICG exhibits lower photothermal stability as seen from the apparent disappearance of the photothermal temperature plateau under the same conditions. According to the previous method58, the photothermal conversion efficiency (PCE) of RuDA-NP was calculated as 24.2%, which is higher than existing photothermal materials such as gold nanorods (21.0%) and gold nanoshells (13.0%)59 . Thus, NP Ore exhibit excellent photothermal properties, which makes them promising PTT agents.
Analysis of DLS and TEM images of RuDA NPs (inset). B Thermal images of various concentrations of RuDA NPs exposed to laser radiation at a wavelength of 808 nm (0.5 W cm-2). C Photothermal conversion curves of various concentrations of ore NPs, which are quantitative data. B. D Temperature increase of ORE NP and ICG over 5 heating-cooling cycles.
Photocytotoxicity of RuDA NPs against MDA-MB-231 human breast cancer cells was evaluated in vitro. As shown in fig. 6A, B, RuDA-NPs and RuDA exhibited negligible cytotoxicity in the absence of irradiation, implying lower dark toxicity of RuDA-NPs and RuDA. However, after exposure to laser radiation at a wavelength of 808 nm, RuDA and RuDA NPs showed strong photocytotoxicity against MDA-MB-231 cancer cells with IC50 values ​​(half-maximum inhibitory concentration) of 5.4 and 9.4 μM, respectively, demonstrating that RuDA-NP and RuDA have potential for cancer phototherapy. In addition, the photocytotoxicity of RuDA-NP and RuDA was further investigated in the presence of vitamin C (Vc), an ROS scavenger, to elucidate the role of ROS in light-induced cytotoxicity. Obviously, cell viability increased after the addition of Vc, and the IC50 values ​​of RuDA and RuDA NPs were 25.7 and 40.0 μM, respectively, which proves the important role of ROS in the photocytotoxicity of RuDA and RuDA NPs. Light-induced cytotoxicity of RuDA-NPs and RuDA in MDA-MB-231 cancer cells by live/dead cell staining using calcein AM (green fluorescence for live cells) and propidium iodide (PI, red fluorescence for dead cells). confirmed by cells) as fluorescent probes. As shown in Figure 6C, cells treated with RuDA-NP or RuDA remained viable without irradiation, as evidenced by intense green fluorescence. On the contrary, under laser irradiation, only red fluorescence was observed, which confirms the effective photocytotoxicity of RuDA or RuDA NPs. It is noteworthy that green fluorescence appeared upon addition of Vc, which indicates a violation of the photocytotoxicity of RuDA and RuDA NPs. These results are consistent with in vitro photocytotoxicity assays.
Dose-dependent viability of A RuDA- and B RuDA-NP cells in MDA-MB-231 cells in the presence or absence of Vc (0.5 mM), respectively. Error bars, mean ± standard deviation (n = 3). Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. Непарные двусторонние t-критерии *p <0,05, **p <0,01 и ***p <0,001. Unpaired two-tailed t-tests *p<0.05, **p<0.01, and ***p<0.001.未配对的双边t 检验*p < 0.05、**p < 0.01 和***p < 0.001。未配对的双边t 检验*p < 0.05、**p < 0.01 和***p < 0.001。 Непарные двусторонние t-тесты *p <0,05, **p <0,01 и ***p <0,001. Unpaired two-tailed t-tests *p<0.05, **p<0.01, and ***p<0.001. C Live/dead cell staining analysis using calcein AM and propidium iodide as fluorescent probes. Scale bar: 30 µm. Representative images of three biological repeats from each group are shown. D Confocal fluorescence images of ROS production in MDA-MB-231 cells under different treatment conditions. Green DCF fluorescence indicates the presence of ROS. Irradiate with a laser with a wavelength of 808 nm with a power of 0.5 W/cm2 for 10 minutes (300 J/cm2). Scale bar: 30 µm. Representative images of three biological repeats from each group are shown. E Flow cytometry RuDA-NPs (50 µM) or RuDA (50 µM) treatment analysis with or without 808 nm laser (0.5 W cm-2) in the presence and absence of Vc (0.5 mM) for 10 min . Representative images of three biological repeats from each group are shown. F Nrf-2, HSP70 and HO-1 of MDA-MB-231 cells treated with RuDA-NPs (50 µM) with or without 808 nm laser irradiation (0.5 W cm-2, 10 min, 300 J cm-2) , cells express 2). Representative images of two biological repeats from each group are shown.
Intracellular ROS production in MDA-MB-231 cells was examined using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method. As shown in fig. 6D, cells treated with RuDA-NPs or RuDA exhibited distinct green fluorescence when irradiated with the 808 nm laser, indicating that RuDA-NPs and RuDA have an efficient ability to generate ROS. On the contrary, in the absence of light or in the presence of Vc, only a weak fluorescent signal of the cells was observed, which indicated a slight formation of ROS. Intracellular ROS levels in RuDA-NP cells and RuDA-treated MDA-MB-231 cells were further determined by flow cytometry. As shown in Supplementary Figure 25, the mean fluorescence intensity (MFI) generated by RuDA-NPs and RuDA under 808 nm laser irradiation was significantly increased by about 5.1 and 4.8 times, respectively, compared to the control group, confirming their excellent formation AFK. capacity. However, intracellular ROS levels in RuDA-NP or MDA-MB-231 cells treated with RuDA were only comparable to controls without laser irradiation or in the presence of Vc, similar to the results of confocal fluorescence analysis.
It has been shown that mitochondria are the main target of Ru(II)-arene complexes60. Therefore, the subcellular localization of RuDA and RuDA-NPs was investigated. As shown in Supplementary Figure 26, RuDA and RuDA-NP show similar cellular distribution profiles with the highest accumulation in mitochondria (62.5 ± 4.3 and 60.4 ± 3.6 ng/mg protein, respectively). However, only a small amount of Ru was found in the nuclear fractions of Ore and NP Ore (3.5 and 2.1%, respectively). The remaining cell fraction contained residual ruthenium: 31.7% (30.6 ± 3.4 ng/mg protein) for RuDA and 42.9% (47.2 ± 4.5 ng/mg protein) for RuDA-NPs. In general, Ore and NP Ore are mainly accumulated in mitochondria. To assess mitochondrial dysfunction, we used JC-1 and MitoSOX Red staining to assess mitochondrial membrane potential and superoxide production capacity, respectively. As shown in Supplementary Fig. 27, intense green (JC-1) and red (MitoSOX Red) fluorescence was observed in cells treated with both RuDA and RuDA-NPs under 808 nm laser irradiation, indicating that both RuDA and RuDA-NPs highly fluorescent It can effectively induce mitochondrial membrane depolarization and superoxide production. In addition, the mechanism of cell death was determined using flow cytometry based analysis of annexin V-FITC/propidium iodide (PI). As shown in Figure 6E, when irradiated with 808 nm laser, RuDA and RuDA-NP induced a significantly increased early apoptosis rate (lower right quadrant) in MDA-MB-231 cells compared to PBS or PBS plus laser. processed cells. However, when Vc was added, the apoptosis rate of RuDA and RuDA-NP decreased significantly from 50.9% and 52.0% to 15.8% and 17.8%, respectively, which confirms the important role of ROS in the photocytotoxicity of RuDA and RuDA-NP. . In addition, slight necrotic cells were observed in all groups tested (upper left quadrant), suggesting that apoptosis may be the predominant form of cell death induced by RuDA and RuDA-NPs.
Since oxidative stress damage is a major determinant of apoptosis, the nuclear factor associated with erythroid 2, factor 2 (Nrf2) 62, a key regulator of the antioxidant system, was investigated in RuDA-NPs-treated MDA-MB-231. Mechanism of action of RuDA NPs induced by irradiation. At the same time, expression of the downstream protein heme oxygenase 1 (HO-1) was also detected. As shown in Figure 6F and Supplementary Figure 29, RuDA-NP-mediated phototherapy increased Nrf2 and HO-1 expression levels compared to the PBS group, indicating that RuDA-NPs may stimulate oxidative stress signaling pathways. In addition, to study the photothermal effect of RuDA-NPs63, the expression of the heat shock protein Hsp70 was also evaluated. It is clear that cells treated with RuDA-NPs + 808 nm laser irradiation showed increased expression of Hsp70 compared to the other two groups, reflecting a cellular response to hyperthermia.
The remarkable in vitro results prompted us to investigate the in vivo performance of RuDA-NP in nude mice with MDA-MB-231 tumors. The tissue distribution of RuDA NPs was studied by determining the content of ruthenium in the liver, heart, spleen, kidneys, lungs, and tumors. As shown in fig. 7A, the maximum content of Ore NPs in normal organs appeared at the first observation time (4 h), while the maximum content was determined in tumor tissues 8 hours after injection, possibly due to Ore NPs. EPR effect of LF. According to the distribution results, the optimal duration of treatment with NP ore was taken 8 hours after administration. To illustrate the process of accumulation of RuDA-NPs in tumor sites, the photoacoustic (PA) properties of RuDA-NPs were monitored by recording the PA signals of RuDA-NPs at different times after injection. First, the PA signal of RuDA-NP in vivo was assessed by recording PA images of a tumor site after intratumoral injection of RuDA-NP. As shown in Supplementary Figure 30, RuDA-NPs showed a strong PA signal, and there was a positive correlation between RuDA-NP concentration and PA signal intensity (Supplementary Figure 30A). Then, in vivo PA images of tumor sites were recorded after intravenous injection of RuDA and RuDA-NP at different time points after injection. As shown in Figure 7B, the PA signal of RuDA-NPs from the tumor site gradually increased with time and reached a plateau at 8 hours post-injection, consistent with tissue distribution results determined by ICP-MS analysis. With respect to RuDA (Supplementary Fig. 30B), the maximum PA signal intensity appeared 4 hours after injection, indicating a rapid rate of entry of RuDA into the tumor. In addition, the excretory behavior of RuDA and RuDA-NPs was investigated by determining the amount of ruthenium in urine and faeces using ICP-MS. The main route of elimination for RuDA (Supplementary Fig. 31) and RuDA-NPs (Fig. 7C) is via the faeces, and effective clearance of RuDA and RuDA-NPs was observed during the 8-day study period, which means that RuDA and RuDA-NPs may efficiently eliminated from the body without long-term toxicity.
A. Ex vivo distribution of RuDA-NP in mouse tissues was determined by the Ru content (percentage of administered dose of Ru (ID) per gram of tissue) at different times after injection. Data are mean ± standard deviation (n = 3). Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. Непарные двусторонние t-критерии *p <0,05, **p <0,01 и ***p <0,001. Unpaired two-tailed t-tests *p<0.05, **p<0.01, and ***p<0.001.未配对的双边t 检验*p < 0.05、**p < 0.01 和***p < 0.001。未配对的双边t 检验*p < 0.05、**p < 0.01 和***p < 0.001。 Непарные двусторонние t-тесты *p <0,05, **p <0,01 и ***p <0,001. Unpaired two-tailed t-tests *p<0.05, **p<0.01, and ***p<0.001. B PA images of in vivo tumor sites at 808 nm excitation after intravenous administration of RuDA-NPs (10 µmol kg-1) at different time points. After intravenous administration of RuDA NPs (10 µmol kg-1), C Ru was excreted from mice with urine and faeces at different time intervals. Data are mean ± standard deviation (n = 3).
The heating capacity of RuDA-NP in vivo was studied in nude mice with MDA-MB-231 and RuDA tumors for comparison. As shown in fig. 8A and supplementary Fig. 32, the control (saline) group showed less temperature change (ΔT ≈ 3 °C) after 10 minutes of continuous exposure. However, the temperature of RuDA-NPs and RuDA increased rapidly with maximum temperatures of 55.2 and 49.9 °C, respectively, providing sufficient hyperthermia for in vivo cancer therapy. The observed increase in high temperature for RuDA NPs (ΔT ≈ 24°C) compared to RuDA (ΔT ≈ 19°C) may be due to its better permeability and accumulation in tumor tissues due to the EPR effect.
Infrared thermal images of mice with MDA-MB-231 tumors irradiated with 808 nm laser at different times 8 hours after injection. Representative images of four biological repeats from each group are shown. B Relative tumor volume and C Average tumor mass of different groups of mice during treatment. D Curves of body weights of different groups of mice. Irradiate with a laser with a wavelength of 808 nm with a power of 0.5 W/cm2 for 10 minutes (300 J/cm2). Error bars, mean ± standard deviation (n = 3). Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. Unpaired, two-sided t tests *p < 0.05, **p < 0.01, and ***p < 0.001. Непарные двусторонние t-критерии *p <0,05, **p <0,01 и ***p <0,001. Unpaired two-tailed t-tests *p<0.05, **p<0.01, and ***p<0.001.未配对的双边t 检验*p < 0.05、**p < 0.01 和***p < 0.001。未配对的双边t 检验*p < 0.05、**p < 0.01 和***p < 0.001。 Непарные двусторонние t-тесты *p <0,05, **p <0,01 и ***p <0,001. Unpaired two-tailed t-tests *p<0.05, **p<0.01, and ***p<0.001. E H&E staining images of major organs and tumors from different treatment groups, including Saline, Saline + Laser, RuDA, RuDA + Laser, RuDA-NPs, and RuDA-NPs + Laser groups. E H&E staining images of major organs and tumors from different treatment groups, including Saline, Saline + Laser, RuDA, RuDA + Laser, RuDA-NPs, and RuDA-NPs + Laser groups. Изображения окрашивания E H&E основных органов и опухолей из разных групп лечения, включая группы физиологического раствора, физиологического раствора + лазера, RuDA, RuDA + Laser, RuDA-NPs и RuDA-NPs + Laser. E H&E staining images of major organs and tumors from different treatment groups, including saline, saline + laser, RuDA, RuDA + Laser, RuDA-NPs, and RuDA-NPs + Laser groups.来自不同治疗组的主要器官和肿瘤的E H&E 染色图像,包括盐水、盐水+ 激光、RuDA、RuDA + 激光、RuDA-NPs 和RuDA-NPs + 激光组。来自不同治疗组的主要器官和肿瘤的E H&E Окрашивание E H&E основных органов и опухолей из различных групп лечения, включая физиологический раствор, физиологический раствор + лазер, RuDA, RuDA + лазер, RuDA-NPs и RuDA-NPs + лазер. E H&E staining of major organs and tumors from various treatment groups including saline, saline + laser, RuDA, RuDA + laser, RuDA-NPs, and RuDA-NPs + laser. Scale bar: 60 µm.
The effect of phototherapy in vivo with RuDA and RuDA NPs was evaluated in which naked mice with MDA-MB-231 tumors were intravenously injected with RuDA or RuDA NPs at a single dose of 10.0 µmol kg-1 via the tail vein, and then 8 hours after injection. laser irradiation with a wavelength of 808 nm. As shown in Figure 8B, tumor volumes were significantly increased in the saline and laser groups, indicating that saline or laser 808 irradiation had little effect on tumor growth. As in the saline group, rapid tumor growth was also observed in mice treated with RuDA-NPs or RuDA in the absence of laser irradiation, demonstrating their low dark toxicity. In contrast, after laser irradiation, both RuDA-NP and RuDA treatment induced significant tumor regression with tumor volume reductions of 95.2% and 84.3%, respectively, compared to the saline treated group, indicating excellent synergistic PDT. , mediated by the RuDA/CHTV effect. – NP or Ore. Compared with RuDA, RuDA NPs showed a better phototherapeutic effect, which was mainly due to the EPR effect of RuDA NPs. Tumor growth inhibition results were further assessed by tumor weight excised on day 15 of treatment (Fig. 8C and Supplementary Fig. 33). The mean tumor mass in RuDA-NP treated mice and RuDA treated mice was 0.08 and 0.27 g, respectively, which was much lighter than in the control group (1.43 g).
In addition, the body weight of mice was recorded every three days to study the dark toxicity of RuDA-NPs or RuDA in vivo. As shown in Figure 8D, no significant differences in body weight were observed for all treatment groups. Furthermore, the hematoxylin and eosin (H&E) staining of the major organs (heart, liver, spleen, lung, and kidney) from different treatment groups were undertaken. Furthermore, the hematoxylin and eosin (H&E) staining of the major organs (heart, liver, spleen, lung, and kidney) from different treatment groups were performed. Кроме того, было проведено окрашивание гематоксилином и эозином (H&E) основных органов (сердца, печени, селезенки, легких и почек) из разных групп лечения. In addition, hematoxylin and eosin (H&E) staining of major organs (heart, liver, spleen, lungs, and kidneys) from different treatment groups was performed.此外,对不同治疗组的主要器官(心脏、肝脏、脾脏、肺和肾脏)进行苏木精和伊红(H&E) 染色。 (H&E) Кроме того, проводили окрашивание гематоксилином и эозином (H&E) основных органов (сердца, печени, селезенки, легких и почек) в различных группах лечения. In addition, hematoxylin and eosin (H&E) staining of major organs (heart, liver, spleen, lung, and kidney) was performed in different treatment groups. As shown in Fig. 8E, the H&E staining images of five major organs from the RuDA-NPs and RuDA groups exhibit no obvious abnormalities or organ damages. 8E, the H&E staining images of five major organs from the RuDA-NPs and RuDA groups exhibit no obvious abnormalities or organ damages. As shown in fig. 8E, изображения окрашивания H&E пяти основных органов из групп RuDA-NPs и RuDA не демонстрируют явных аномалий или повреждений органов. 8E, H&E staining images of five major organs from the RuDA-NPs and RuDA groups show no obvious organ abnormalities or lesions.如图8E 所示,来自RuDA-NPs 和RuDA 组的五个主要器官的H&E 染色图像没有显示出明显的异常或器官损伤。如图8E 所示,来自RuDA-NPs 和RuDA 组的五个主要器官的H&E Как показано на рисунке 8E, изображения окрашивания H&E пяти основных органов из групп RuDA-NPs и RuDA не показали явных аномалий или повреждения органов. As shown in Figure 8E, H&E staining images of the five major organs from the RuDA-NPs and RuDA groups showed no obvious abnormalities or organ damage. These results showed that neither RuDA-NP nor RuDA showed signs of toxicity in vivo. Moreover, H&E staining images of tumors showed that both the RuDA + Laser and RuDA-NPs + Laser groups could cause severe cancer cell destruction, demonstrating the excellent in vivo phototherapeutic efficacy of RuDA and RuDA-NPs. Moreover, H&E staining images of tumors showed that both the RuDA + Laser and RuDA-NPs + Laser groups could cause severe cancer cell destruction, demonstrating the excellent in vivo phototherapeutic efficacy of RuDA and RuDA-NPs. In addition, hematoxylin-eosin stained tumor images showed that both RuDA+Laser and RuDA-NPs+Laser groups can induce severe destruction of cancer cells, demonstrating the superior phototherapeutic efficacy of RuDA and RuDA-NPs in vivo.此外,肿瘤的H&E 染色图像显示,RuDA + Laser 和RuDA-NPs + Laser 组均可导致严重的癌细胞破坏,证明了RuDA 和RuDA-NPs 的优异的体内光疗功效。此外 , 肿瘤 的 & e 染色 显示 , ruda + laser 和 ruda-nps + laser 组均 导致 的 癌细胞 破坏 , 证明 了 ruda 和 ruda-nps 的 的 体内 光疗。。。。。。。。。。。。。。。。 In addition, hematoxylin and eosin stained tumor images showed that both RuDA+Laser and RuDA-NPs+Laser groups resulted in severe destruction of cancer cells, demonstrating superior phototherapeutic efficacy of RuDA and RuDA-NPs in vivo.
In conclusion, the Ru(II)-arene (RuDA) organometallic complex with DA-type ligands was designed to facilitate the ISC process using the aggregation method. Synthesized RuDA can self-assemble through non-covalent interactions to form RuDA-derived supramolecular systems, thereby facilitating 1O2 formation and efficient photothermal conversion for light-induced cancer therapy. It is noteworthy that monomeric RuDA did not generate 1O2 under laser irradiation at 808 nm, but could generate a large amount of 1O2 in the aggregated state, demonstrating the rationality and efficiency of our design. Subsequent studies have shown that the supramolecular assembly endows RuDA with improved photophysical and photochemical properties, such as redshift absorption and photobleaching resistance, which are highly desirable for PDT and PTT processing. Both in vitro and in vivo experiments have shown that RuDA NPs with good biocompatibility and good accumulation in the tumor exhibit excellent light-induced anticancer activity upon laser irradiation at a wavelength of 808 nm. Thus, RuDA NPs as effective bimodal supramolecular PDT/PTW reagents will enrich the set of photosensitizers activated at wavelengths above 800 nm. The conceptual design of the supramolecular system provides an efficient route for NIR-activated photosensitizers with excellent photosensitizing effects.
All chemicals and solvents were obtained from commercial suppliers and used without further purification. RuCl3 was purchased from Boren Precious Metals Co., Ltd. (Kunming, China). [(η6-p-cym)Ru(fendio)Cl]Cl (fendio = 1,10-phenanthroline-5,6-dione) and 4,7-bis[4-(N,N-diphenylamino)phenyl]-5 ,6-Diamino-2,1,3-benzothiadiazole was synthesized according to previous studies64,65. NMR spectra were recorded on a Bruker Avance III-HD 600 MHz spectrometer at Southeastern University Analytical Test Center using d6-DMSO or CDCl3 as solvent. Chemical shifts δ are given in ppm. with respect to tetramethylsilane, and the interaction constants J are given in absolute values ​​in hertz. High resolution mass spectrometry (HRMS) was performed on an Agilent 6224 ESI/TOF MS instrument. Elemental analysis of C, H, and N was performed on a Vario MICROCHNOS elemental analyzer (Elementar). UV-visible spectra were measured on a Shimadzu UV3600 spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-6000 spectrofluorimeter. EPR spectra were recorded on a Bruker EMXmicro-6/1 instrument. The morphology and structure of the prepared samples were studied on FEI Tecnai G20 (TEM) and Bruker Icon (AFM) instruments operating at a voltage of 200 kV. Dynamic light scattering (DLS) was performed on a Nanobrook Omni analyzer (Brookhaven). Photoelectrochemical properties were measured on an electrochemical setup (CHI-660, China). Photoacoustic images were obtained using the FUJIFILM VisualSonics Vevo® LAZR system. Confocal images were obtained using an Olympus FV3000 confocal microscope. FACS analysis was performed on a BD Calibur flow cytometer. High performance liquid chromatography (HPLC) experiments were performed on a Waters Alliance e2695 system using a 2489 UV/Vis detector. Gel Permeation Chromatography (GPC) tests were recorded on a Thermo ULTIMATE 3000 instrument using an ERC RefratoMax520 refractive index detector.
[(η6-p-cym)Ru(fendio)Cl]Cl (fendio = 1,10-phenanthroline-5,6-dione)64 (481.0 mg, 1.0 mmol), 4,7-bis[4 -(N,N-diphenylamino)phenyl]-5,6-diamino-2,1,3-benzothiadiazole 65 (652.0 mg, 1.0 mmol) and glacial acetic acid (30 mL) were stirred at reflux refrigerator for 12 hours. The solvent was then removed in vacuo using a rotary evaporator. The resulting residue was purified by flash column chromatography (silica gel, CH2Cl2:MeOH=20:1) to obtain RuDA as a green powder (yield: 877.5 mg, 80%). anus. Calculated for C64H48Cl2N8RuS: C 67.84, H 4.27, N 9.89. Found: C 67.92, H 4.26, N 9.82. 1H NMR (600 MHz, d6-DMSO) δ 10.04 (s, 2H), 8.98 (s, 2H), 8.15 (s, 2H), 7.79 (s, 4H), 7.44 (s, 8H), 7.21 (d, J = 31.2 Hz, 16H), 6.47 (s, 2H), 6.24 (s, 2H), 2.69 (s, 1H), 2 .25 (s, 3H), 0.99 (s, 6H). 13c nmr (150 MHZ, D6-DMSO), δ (PPM) 158.03, 152.81, 149.31, 147.98, 147.16, 139.98, 136.21, 135.57, 134.68, 130.34, 130.02, 128.68, 128.01, 125.51, 124.45, 120.81, 103.49, 103.49, 103. , 86.52, 84.75, 63.29, 30.90, 22.29, 18.83. ESI-MS: m/z [M-Cl]+ = 1097.25.
Synthesis of 4,7-bis[4-(N,N-diethylamino)phenyl-5,6-diamino-2,1,3-benzothiadiazole (L2): L2 was synthesized in two steps. Pd(PPh3)4 (46 mg, 0.040 mmol) was added to N,N-diethyl-4-(tributylstannyl)aniline (1.05 g, 2.4 mmol) and 4,7-dibromo-5,6-dinitro solution - 2,1,3-benzothiadiazole (0.38 g, 1.0 mmol) in dry toluene (100 ml). The mixture was stirred at 100°C for 24 hours. After removing the toluene in vacuo, the resulting solid was washed with petroleum ether. Then a mixture of this compound (234.0 mg, 0.45 mmol) and iron powder (0.30 g, 5.4 mmol) in acetic acid (20 ml) was stirred at 80° C. for 4 hours. The reaction mixture was poured into water and the resulting brown solid was collected by filtration. The product was purified twice by vacuum sublimation to give a green solid (126.2 mg, 57% yield). anus. Calculated for C26H32N6S: C 67.79, H 7.00, N 18.24. Found: C 67.84, H 6.95, H 18.16. 1H NMR (600 MHz, CDCl3), δ (ppm) 7.42 (d, 4H), 6.84 (d, 4H), 4.09 (s, 4H), 3.42 (d, 8H ), 1.22 (s, 12H). 13С NMR (150 MHz, CDCl3), δ (ppm) 151.77, 147.39, 138.07, 131.20, 121.09, 113.84, 111.90, 44.34, 12.77. ESI-MS: m/z [M+H]+ = 461.24.
Compounds were prepared and purified following procedures similar to RuDA. anus. Calculated for C48H48Cl2N8RuS: C 61.27, H 5.14, N 11.91. Found: C, 61.32, H, 5.12, N, 11.81,1H NMR (600 MHz, d6-DMSO), δ (ppm) 10.19 (s, 2H), 9.28 (s, 2H), 8.09 (s, 2H), 7.95 (s, 4H), 6.93 (s, 4H), 6.48 (d, 2H), 6.34 (s, 2H) , 3.54 (t, 8H), 2.80 (m, 1H), 2.33 (s, 3H), 1.31 (t, 12H), 1.07 (s, 6H). 13c nmr (151 mhz, CDCL3), δ (PPM) 158.20, 153.36, 148.82, 148.14, 138.59, 136.79, 135.75, 134.71, 130.44, 128.87, 128.35, 121.70, 111.84, 110.76, 105.07, 104.23, 87.0, 84.4. , 38.06, 31.22, 29.69, 22.29, 19.19, 14.98, 12.93. ESI-MS: m/z [M-Cl]+ = 905.24.
RuDA was dissolved in MeOH/H2O (5/95, v/v) at a concentration of 10 μM. The absorption spectrum of RuDA was measured every 5 minutes on a Shimadzu UV-3600 spectrophotometer under irradiation with laser light with a wavelength of 808 nm (0.5 W/cm2). The ICG spectra were recorded under the same conditions as the standard.
The EPR spectra were recorded on a Bruker EMXmicro-6/1 spectrometer with a microwave power of 20 mW, a scanning range of 100 G, and a field modulation of 1 G. 2,2,6,6-tetramethyl-4-piperidone (TEMP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were used as spin traps. Electron spin resonance spectra were recorded for mixed solutions of RuDA (50 µM) and TEMF (20 mM) or DMPO (20 mM) under the action of laser radiation with a wavelength of 808 nm (0.5 W/cm2).
DFT and TD-DFT calculations for RuDA were carried out at PBE1PBE/6–31 G*//LanL2DZ levels in aqueous solution using the Gaussian program 1666,67,68. The HOMO-LUMO, hole and electron distributions of the low-energy singlet excited state RuDA were plotted using the GaussView program (version 5.0).
We first tried to measure the generation efficiency of 1O2 RuDA using conventional UV-visible spectroscopy with ICG (ΦΔ = 0.002) as a standard, but the photodegradation of ICG strongly affected the results. Thus, the quantum yield of 1O2 RuDA was measured by detecting a change in the intensity of ABDA fluorescence at about 428 nm when irradiated with a laser with a wavelength of 808 nm (0.5 W/cm2). Experiments were performed on RuDA and RuDA NPs (20 μM) in water/DMF (98/2, v/v) containing ABDA (50 μM). The quantum yield of 1O2 was calculated using the following formula: ΦΔ (PS) = ΦΔ (ICG) × (rFS/APS)/(rICG/AICG). rPS and rICG are the reaction rates of ABDA with 1O2 obtained from the photosensitizer and ICG, respectively. APS and AICG are the absorbance of the photosensitizer and ICG at 808 nm, respectively.
AFM measurements were carried out in liquid conditions using the scan mode on a Bruker Dimension Icon AFM system. Using an open structure with liquid cells, the cells were washed twice with ethanol and dried with a stream of nitrogen. Insert the dried cells into the optical head of the microscope. Immediately place a drop of the sample into the pool of liquid and place it on the cantilever using a sterile disposable plastic syringe and a sterile needle. Another drop is placed directly on the sample, and when the optical head is lowered, the two drops merge, forming a meniscus between the sample and the liquid reservoir. AFM measurements were carried out using a SCANASYST-FLUID V-shaped nitride cantilever (Bruker, hardness k = 0.7 N m-1, f0 = 120–180 kHz).
HPLC chromatograms were obtained on a Waters e2695 system equipped with a phoenix C18 column (250×4.6 mm, 5 µm) using a 2489 UV/Vis detector. The wavelength of the detector is 650 nm. Mobile phases A and B were water and methanol, respectively, and the mobile phase flow rate was 1.0 ml·min-1. The gradient (solvent B) was as follows: 100% from 0 to 4 minutes, 100% to 50% from 5 to 30 minutes, and reset to 100% from 31 to 40 minutes. Ore was dissolved in a mixed solution of methanol and water (50/50, by volume) at a concentration of 50 μM. The injection volume was 20 μl.
GPC assays were recorded on a Thermo ULTIMATE 3000 instrument equipped with two PL aquagel-OH MIXED-H columns (2×300×7.5 mm, 8 µm) and an ERC RefratoMax520 refractive index detector. The GPC column was eluted with water at a flow rate of 1 ml/min at 30°C. Ore NPs were dissolved in PBS solution (pH = 7.4, 50 μM), injection volume was 20 μL.
Photocurrents were measured on an electrochemical setup (CHI-660B, China). The optoelectronic responses when the laser was turned on and off (808 nm, 0.5 W/cm2) were measured at a voltage of 0.5 V in a black box, respectively. A standard three-electrode cell was used with an L-shaped glassy carbon electrode (GCE) as a working electrode, a standard calomel electrode (SCE) as a reference electrode, and a platinum disk as a counter electrode. A 0.1 M Na2SO4 solution was used as an electrolyte.
The human breast cancer cell line MDA-MB-231 was purchased from KeyGEN Biotec Co., LTD (Nanjing, China, catalog number: KG033). Cells were grown in monolayers in Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose) supplemented with a solution of 10% fetal bovine serum (FBS), penicillin (100 μg/ml) and streptomycin (100 μg/ml). All cells were cultured at 37°C in a humid atmosphere containing 5% CO2.
The MTT assay was used to determine the cytotoxicity of RuDA and RuDA-NPs in the presence and absence of light irradiation, with or without Vc (0.5 mM). MDA-MB-231 cancer cells were grown in 96-well plates at a cell density of approximately 1 x 105 cells/ml/well and incubated for 12 hours at 37.0°C in an atmosphere of 5% CO2 and 95% air. RuDA and RuDA NPs dissolved in water were added to the cells. After 12 hours of incubation, the cells were exposed to 0.5 W cm -2 laser radiation at a wavelength of 808 nm for 10 minutes (300 J cm -2) and then incubated in the dark for 24 hours. The cells were then incubated with MTT (5 mg/ml) for another 5 hours. Finally, change the medium to DMSO (200 µl) to dissolve the resulting purple formazan crystals. OD values ​​were measured using a microplate reader with a wavelength of 570/630 nm. The IC50 value for each sample was calculated using the SPSS software from dose-response curves obtained from at least three independent experiments.
MDA-MB-231 cells were treated with RuDA and RuDA-NP at a concentration of 50 μM. After 12 hours of incubation, the cells were irradiated with a laser with a wavelength of 808 nm and a power of 0.5 W/cm2 for 10 min (300 J/cm2). In the vitamin C (Vc) group, cells were treated with 0.5 mM Vc prior to laser irradiation. Cells were then incubated in the dark for an additional 24 hours, then stained with calcein AM and propidium iodide (20 μg/ml, 5 μl) for 30 minutes, then washed with PBS (10 μl, pH 7.4). images of stained cells.


Post time: Sep-23-2022