Appropriate introduction of nitrile groups to balance NIR-II fluorescence imaging with photothermal therapy/photoacoustic imaging

Abstract

Phototheranostics, especially the combination of second near-infrared window (NIR-II) fluorescence imaging and photothermal therapy (PTT), is a novel biotechnological approach that integrates diagnosis and therapy to enable accurate and effective tumor killing. However, it is difficult to balance the competition between generating fluorescence by radiation decay and generating heat by non-radiation decay. In this paper, we propose a nitrilation strategy to design A–D–A′–D–A photothermal agents (PTAs) to balance this inherent conflict and achieve both high fluorescence quantum yield (FLQY) and good photothermal conversion ability. Our experimental results show that the photothermal conversion efficiency (PCE) and FLQY of nanoparticles (NPs) were significantly enhanced after the induction of nitrile groups and appropriate nitrilation. Theoretical calculations demonstrate that moderate induction of nitrile groups can maximize electrostatic potential difference (ΔESP) and increase intramolecular/intermolecular D–A interactions, resulting in tighter molecular packing and further promoting an increased molar extinction coefficient. Among the three types of nanoparticles we investigated, the NIR-II emission of BTP-TCID-2CN NPs with two nitrile groups extended to 1200 nm, exhibiting the highest FLQY (3.7%), good PCE (33.5%), and excellent photoacoustic imaging capacity. In addition, both biological safety and cellular internalization ability were significantly improved after appropriate nitrilation. These characteristics enable BTP-TCID-2CN NPs to support high-resolution NIR-II FLI/PAI-guided PTT in vivo. This study provides a feasible basis for the future research and development of NIR-II organic PTAs with excellent overall performance.

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Introduction

Cancer is one of the most serious diseases endangering human health. However, traditional cancer treatments have critical disadvantages, including poor targeting, associated trauma, a long repair period, and significant side effects, that limit their efficacy.^1,2 Therefore, it is necessary to explore new effective treatment methods for cancer. Phototheranostics, a novel biotechnology that integrates therapy and diagnosis, has aroused increasing attention in the cancer field.^3–7 Photothermal therapy (PTT) is one of the main therapeutic modalities of phototheranostics,^8,9 and its mechanism can be explained through the Jablonski diagram. Photothermal agents (PTAs) absorb energy to enable their electrons to reach the excited state from the ground state, after which they release energy through non-radiation decay usually accompanied by a photoacoustic signal for photoacoustic imaging (PAI). When energy is released in the form of radiation decay, fluorescence is generated, thereby achieving fluorescence imaging (FLI).^10–12 PAI, a non-invasive and non-ionizing imaging modality, can be generated in parallel with PTT. However, its application is often limited by a low signal-to-noise ratio (SNR), low image contrast and poor spatial resolution in deep tissue due to optical attenuation.^13,14 FLI, especially in the second near-infrared window (NIR-II) (1000–1700 nm), has higher sensitivity and distinct advantages with respect to reducing autofluorescence, leading to deeper penetration and higher SNR,^15–17 and enabling real-time tumor monitoring, intraoperative visualization, and improved surgical accuracy.^18–20 Therefore, the combination of FLI and PTT/PAI into a single platform for cancer diagnosis and treatment promises to improve the efficiency of tumor detection and monitoring, allowing more accurate tumor elimination. However, due to competition between non-radiative decay and radiative decay, it is necessary to develop molecules with the ability to balance NIR-II FLI and PTT/PAI, as well as to explore strategies for their simultaneous enhancement.

Nitrile groups (–CN) with strong polarity and high N atom electronegativity play important roles in dyes,^21,22 agrochemicals,^23,24 pharmaceuticals^25,26 and electronic materials.^27,28 The introduction of –CN groups, which have strong electron-absorbing ability and stretching vibration characteristics, can not only highly polarize the adjacent electron density, enhance electrostatic interactions and promote absorption redshift, but also induce photothermal aggregation and enhance photoacoustic signals.^29–32 However, differences in the amount of –CN introduced can lead to different effects on the molecule's spatial site resistance, in turn altering the effects of FLI, PTT and PAI. Thus, the exploration of a nitrilation strategy to optimize the introduction of –CN groups in phototheranostics is a meaningful pursuit that has yet to be explored.

Herein, we propose a nitrilation strategy that employs three A–D–A′–D–A type organic molecules as PTAs carrying different amounts of nitrile groups. The ladder-type electron-deficient core (BTP) with multi-fused rings provides multiple D–A interactions, which can increase the push–pull effect, reduce the energy gap, facilitate intramolecular charge transfer, improve the light capture ability of the near-infrared (NIR) window, and endow FLI capacity.^33–35 Moreover, long alkyl chains act as shielding groups to inhibit intermolecular interactions, in addition to providing increased space to promote intramolecular motion.^36,37 The single bond connecting the donor-core BTP and acceptor allows intramolecular rotation and further promotes the generation of thermal energy, contributing to PTT.^38–40 Furthermore, 2-(5,6-dichloro-3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile and 4,5,6,7-tetrachloro-1H-indene-1,3(2H)-dione (TCID) were selected as acceptors, due to not only their strong electron-withdrawing abilities but also the ability to replenish the intramolecular rotation of –CN, further improving photothermal conversion.^41–43 In addition, these three A–D–A′–D–A type organic molecules were further encapsulated with Pluronic F127 to form nanoparticles (NPs), promoting their solubility in water and generating simultaneous bathochromic shift due to J-aggregation.^44–46 Comparing these three NPs, we found that BTP-TCID-2CN containing two nitrile groups, exhibited the highest QY of 3.7%, an appropriate photothermal conversion efficiency (PCE) of 33.5%, satisfactory PAI ability and low cytotoxicity. Therefore, BTP-TCID-2CN NPs are ideal candidates with enhanced biosafety and a good balance between NIR-II FLI and PTT/PAI. The nitrilation strategy we report herein is a useful basis for further research and development of NIR-II organic PTAs with excellent overall performance (Scheme 1).

Scheme 1 Schematic diagram of nitrile-induced molecules enabling balance between NIR-II FLI and PTT/PAI.

Results and discussion

Synthesis and characterization

We utilized alkyl-chain-grafted BTP as an electron donor and one of two electron-withdrawing end units, 2-(5,6-dichloro-3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile or 4,5,6,7-tetrachloro-1H-indene-1,3(2H)-dione (TCID), as electron acceptors to synthesize BTP-2TCID and BTP-TCID-2CN through the Knoevenagel reaction, according to the reported procedure.⁴⁷¹H NMR spectroscopy confirmed the successful synthesis of these two molecules (Fig. S1 and 2†). To comprehensively assess the impact of the number of nitrile groups on performance, we purchased BTP-4CN, a compound with four nitrile groups, and used 2-(5,6-dichloro-3-oxo-2,3-dihydro-1H-inden-1-ylidene) malononitrile as an electron acceptor. The structures of these three molecules are shown in Fig. 1A. To further explore the rationality of molecular design, we calculated HOMO–LUMO distributions and differences in electrostatic potential (ΔESP) based on density functional theory (DFT). As shown in Fig. 1D, the HOMO of these three molecules was distributed mainly on the donor core, while the LUMO was principally distributed on the receptor. With increasing numbers of nitrile groups, the electron-withdrawing ability of the receptor increases, while the LUMO becomes more widely distributed. Therefore, the HOMO–LUMO differences of BTP-2TCID, BTP-TCID-2CN and BTP-4CN showed a decreasing trend with the increase in the number of nitrile groups (2.12 eV for BTP-2TCID, 1.98 eV for BTP-TCID-2CN and 1.92 eV for BTP-4CN), where a smaller HOMO–LUMO difference is beneficial for promoting absorption and emission redshift. All absorption spectra (Fig. 1B) showed clear THF absorption peaks, with the peaks of BTP-2TCID, BTP-TCID-2CN and BTP-4CN centered at around 651 nm, 693 nm and 722 nm, respectively; these values are consistent with the HOMO–LUMO distributions and confirm that the introduction of nitrile groups promotes redshift. In addition, the fluorescence spectra of BTP-2TCID, BTP-TCID-2CN and BTP-4CN exhibited a maximum fluorescence emission at 705 nm, 798 nm and 804 nm, respectively (Fig. 1C), and also reflected a tendency towards increasing redshift as the number of nitrile groups increases. Moreover, BTP-TCID-2CN exhibited the largest Stokes shift among the three molecules, potentially increasing SNR, improving the FLI resolution ratio, and generally offering significant potential for fluorescence imaging.^48,49

Fig. 1 (A) Molecular structures of BTP-2TCID, BTP-TCID-2CN and BTP-4CN. (B) Normalized absorption and (C) fluorescence spectra of those three molecules in THF. (D) HOMO–LUMO distribution, (E) energy-level diagram.

Fabrication and optical properties of nanoparticles

To improve the biocompatibility and biostability of hydrophobic molecules for further biological applications, we used nanoprecipitation to assemble them with Pluronic F127 (Fig. 2A). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed that BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs possess spherical morphologies with average sizes of approximately 171.8 nm, 178.3 nm and 112.3 nm, respectively (Fig. 2B and S5†). The encapsulation efficiency of BTP-4CN NPs was calculated to be 1.09%, lower than those of BTP-2TCID NPs (2.86%) and BTP-TCID-2CN NPs (2.92%). To confirm that these three molecules were effectively encapsulated, we measured their zeta potentials. While the initial zeta potential of Pluronic F127 was −9.4 mV, significant changes after nanoprecipitation with BTP-2TCID, BTP-TCID-2CN and BTP-4CN generated values of −28.5 mV, −12.5 mV and −15.1 mV, respectively, further confirming effective NPs formation (Fig. 2C). Moreover, the changes in particle size for BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs after nine days of storage were negligible, and their absorption values remained similarly unchanged under 30 min of laser irradiation at 808 nm. These results suggest remarkable dimensional stability and photostability (Fig. 2D and E) and lay a strong foundation for subsequent biological experiments.

Fig. 2 (A) Schematic illustration of NPs preparation. (B) Average hydrodynamic size of the three NPs. (C) Zeta potentials of F127, BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs in deionized water. (D) Dimensional stability after nine days of storage in deionized water. (E) Absorbance changes under 30 min of 808 nm laser irradiation (1.0 W cm⁻²) in deionized water. (F) Normalized absorption spectra of the three NPs in deionized water. (G) Normalized fluorescence spectra of the three NPs in deionized water. (H) NIR-II FLQY values of various NPs. (I) NIR-II fluorescence images of different NPs (20 μg mL⁻¹) under 808 nm laser irradiation.

Next, we investigated the optical properties of NPs in water. Bathochromic shifts are generated in absorption and fluorescence spectra after NPs preparation, which was related to J-aggregation. The optical properties were further investigated in different mixtures of THF and water to verified J-aggregation in molecules, we found that with the increase of the proportion of water, the absorption of the three molecules showed different degrees of redshift and the absorption range widened (Fig. S6†), which proved the existence of J-aggregation.^44,50,51 The maximum absorption peaks of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs were all redshifted and broadened (BTP-2TCID NPs to extend 950 nm, while BTP-TCID-2CN NPs and BTP-4CN NPs even extended to 1000 nm) (Fig. 2F). As shown in Fig. 2G, the fluorescence emission peaks of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs were at 814 nm, 914 nm and 928 nm, respectively, and all persisted until 1150 nm, reaching the NIR-II window. In addition, the FLQY values of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs were estimated by the sphere integration method as 0.56%, 3.77% and 0.86%, respectively (Fig. 2H). Notably, BTP-TCID-2CN NPs with two nitrile groups exhibited the highest FLQY value by far. However, FLQY decreased upon the introduction of additional nitrile groups, indicating that more nitrile groups are not necessarily better, and only appropriate nitrile groups can significantly improve FLQY. This phenomenon can be further explained through ΔESP. The ESP distributions of the three molecules are shown in Fig. S3.† Electrostatic potential in the red region is negative, corresponding to electron-withdrawing groups, while the positive blue region corresponds to electron-donating groups. We found that the ΔEPS values of BTP-2TCID, BTP-TCID-2CN and BTP-4CN were 67.13 kcal mol⁻¹, 69.02 kcal mol⁻¹ and 68.94 kcal mol⁻¹, respectively, and the ΔEPS of BTP-TCID-2CN was larger than those of the other two. A larger ΔEPS is indicative of stronger intermolecular electrostatic interactions, which may offset the intermolecular π–π interactions to some extent, thereby increasing FLQY.³⁰ The high FLQY of BTP-TCID-2CN NPs is shown visually in Fig. 2I. At the same concentration, the fluorescence images generated with BTP-TCID-2CN NPs were much brighter and clearer, demonstrating the superior FLI ability of BTP-TCID-2CN NPs. These results indicate that appropriate nitrilation can promote redshift and improve FLQY.

Performance of nanoparticles

To further investigate the impact of the nitrile group's number on the photochemical properties of A–D–A′–D–A typed molecules, we first characterized the PTT performance of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs in a concentration- and power density-dependent manner under 808 nm laser irradiation and demonstrated a positive dependence between them (Fig. S7A–F†). After 10 min irradiation, the temperatures of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs rose by 19.6 °C, 25.8 °C and 37.9 °C, respectively (Fig. 3A), and photothermal images visually reveal a huge difference in temperature rise between the three NPs and deionized water (Fig. 3C). In particular, BTP-4CN NPs exhibited the highest temperature rise after 10 minutes of 808 nm laser irradiation (1.0 W cm⁻²), associated with its greater absorption at 808 nm. We then measured the PCEs of these three NPs to qualitatively compare their heating abilities. According to experimental calculations, the PCEs of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs are 10.23%, 33.5% and 43.3%, respectively (Fig. S7G–I†). These findings demonstrate that an increase in the number of nitrile groups is beneficial for promoting intramolecular rotation and further improving PCE. Good photothermal stability is important to the application value of PTAs with high PCE. Therefore, we investigated this quality through four successive cycles of 808 nm laser irradiation (1.0 W cm⁻², 10 min) with free cooling. We found that all three NPs rose to similar temperatures as before, verifying their stable temperature-increasing capacity and subsequent experimental and application value (Fig. 3B).

Fig. 3 (A) Temperature rise of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs under 808 nm laser irradiation (1.0 W cm⁻²) for 10 min. (B) Temperature elevation of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs (20 μg mL⁻¹) under four irradiation/cooling cycles (808 nm laser, 1.0 W cm⁻², 10 min). (C) Thermal images of deionized water, BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs (20 μg mL⁻¹) under 808 nm laser irradiation (1.0 W cm⁻²). (D) PA intensities of different NPs (20 μg mL⁻¹) in the tissue simulation experiment. (E) NIR-II fluorescence images of different concentrations of BTP-TCID-2CN NPs (0, 5, 10, 20, 40, and 60 μg mL⁻¹). (F) Corresponding fluorescence intensities of (E). (G) ROS generation of three NPs and ICG assessed using DPBF as a probe under 808 nm laser irradiation (1.0 W cm⁻²) for 20 s. (H) ¹O₂ quantum yield values for the three NPs.

PTAs with excellent PTT performance can usually release photoacoustic signals during PTT, thereby enabling PAI capabilities. To this end, we validated and studied the effective generation of photoacoustic signals by these three NPs accompanying PTT. As shown in Fig. 3D, the photoacoustic intensity of BTP-4CN NPs was a litter higher than that of BTP-TCID-2CN NPs, and the photoacoustic intensities of BTP-4CN NPs and BTP-TCID-2CN NPs were much higher than that of BTP-2TCID NPs without a nitrile group. These findings indicate that, as additional nitrile groups were introduced, the PA intensity strengthened, in accordance with the PCE results. However, in agreement with the FLQY values, only BTP-TCID-2CN NPs exhibited outstanding FLI ability. For BTP-TCID-2CN NPs, the fluorescence intensity and definition of NIR-II fluorescence images increased as the concentration increased (Fig. 3E and F), further demonstrating that appropriate nitrile groups can endow excellent performance in PTT and NIR-II FLI.

In order to comprehensively explore the energy dissipation patterns of these three NPs, we characterized their photodynamic properties. Under 808 nm laser irradiation, solutions of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs all displayed concentration-dependent and power density-dependent decreases in absorption when DPBF was added as a ROS indicator (Fig. S8–S10†), indicating ROS generation. Comparing the rates of ROS generation for BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs following 808 nm laser irradiation (1.0 W cm⁻²) for 20 s, we found that, although the ROS production ability of BTP-TCID-2CN NPs with two nitrile groups was slightly increased, it was not much different than that of the nitrile-group-lacking BTP-2TCID NPs. However, the ROS production of BTP-4CN NPs, with four nitrile groups, was significantly increased to a much higher value (Fig. 3G), implying that increasing the number of nitrile groups can promote ROS generation, with the most significant enhancing effect seen with the introduction of four nitrile groups. To further verify ROS species under 808 nm laser irradiation, electron spin resonance (ESR) spectrometry was performed. Only the NPs solution containing TEMP showed an obvious EPR signal, confirming that only ¹O₂ was produced (Fig. S11†). However, the ¹O₂ quantum yields of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs were very low, only 0.38%, 0.42% and 0.45%, respectively (Fig. 3H and S12†). These findings indicate that increasing the number of nitrile groups can enhance ¹O₂ generation, but this improvement effect is limited and virtually negligible. This weak ¹O₂ generation capacity also suggests that the treatment of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs occurs mainly through PTT. Upon irradiation of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs, their energies are dissipated primarily in the forms of radiation and non-radiation decay, and the appropriate introduction of nitrile groups can balance these two forms of energy dissipation and achieve the equilibrium necessary to establish a theragnostic nanoplatform with excellent FLI and PTT properties.

Anti-tumor ability in vitro

Considering the excellent FLI performance of BTP-TCID-2CN NPs and the superior PCE of BTP-4CN NPs, we chose U87 cells to further assess these inhibitory effects at the cellular level. Low dark-toxicity and high phototoxicity are prerequisites for the use of PTAs in biological applications. To this end, we first assessed the toxicity of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs using an MTT assay. Different concentrations of BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs were incubated with U87 cells for 24 h with or without 808 nm laser irradiation (1.0 W cm⁻², 5 min). As shown in Fig. 4A, the cell viability remained over 80% for all NPs without laser irradiation but was apparently decreased under irradiation, demonstrating the low dark-toxicity and high phototoxicity of these NPs. Interestingly, although BTP-2TCID NPs and BTP-TCID-2CN NPs were less phototoxic than BTP-4CN NPs, the viability of cells treated with BTP-2TCID NPs or BTP-TCID-2CN NPs without laser irradiation was much higher than that of those treated with BTP-4CN NPs. The cell viability for BTP-2TCID NPs and BTP-TCID-2CN NPs in the dark was still above 70% at a concentration of 2 μg mL⁻¹, while that of BTP-4CN NPs was about 75% at a concentration of 0.5 μg mL⁻¹. These results indicate that increasing the number of nitrile groups can improve phototoxicity, but reducing the number of nitrile groups can reduce dark-toxicity and improve biosafety (Fig. S13†); indeed, BTP-TCID-2CN NPs with appropriate nitrile groups exhibit both high phototoxicity and low dark toxicity. The cell-killing ability of these NPs was visually assessed in live/dead cell-staining images, with green and red fluorescence representing the living and dead cells, respectively; these results were consistent with those of the MTT assay (Fig. 4B). To verify that heat is the main factor killing these cells, we used DCFH-DA to evaluate intracellular ROS generation. Comparing the irradiated groups for BTP-2TCID NPs, BTP-TCID-2CN NPs and BTP-4CN NPs, we found that a green fluorescence was brightest for BTP-4CN NPs and weakest for BTP-2TCID NPs without nitrile groups; these results intuitively demonstrate that increasing nitrile groups can promote the generation of ¹O₂. However, the green fluorescence of all three NPs was very weak, consistent with their ¹O₂ quantum yields (Fig. S14†). These results corroborate negligible ROS generation in these cells and prove that cells are primarily killed by PTT.

Fig. 4 (A) Dark-toxicity and phototoxicity of different NPs. (B) Live/dead assay of U87 cells treated with different NPs (0.5 μg mL⁻¹) with or without 808 nm irradiation (1.0 W cm⁻²). Scale bars: 50 μm. (C) CLSM images of various NPs. Scale bars: 20 μm.

For PTAs to achieve efficient phototherapy and imaging, they must be effectively internalized; therefore, we measured intracellular uptake of the NPs using confocal laser scanning microscopy (CLSM). After 4 h incubation, BTP-2TCID NPs exhibited stronger red color, while that of BTP-4CN NPs was inapparent, indicating that fewer nitrile groups may improve cellular uptake (Fig. 4C). We further used flow cytometry to determine the optimum time point for cellular uptake and enable precise treatment. We found that the cellular uptake of BTP-4CN NPs and BTP-TCID-2CN NPs increased over time, and the cellular uptake of BTP-2TCID NPs decreased after increasing for 6 h; these results indicate that the optimal uptake period of BTP-2TCID NPs is 6 h, while the uptake rates of BTP-4CN NPs and BTP-TCID-2CN NPs were significantly higher after 6 h (Fig. S15†).

Imaging and anti-tumor therapy ability in vivo

Although BTP-TCID-2CN NPs have slightly inferior biosafety and intracellular internalization ability compared to BTP-2TCID NPs, the superior phototoxicity and potential NIR-II FLI and PAI capabilities of BTP-TCID-2CN NPs are more suitable for tumor treatment. Thus, we chose BTP-TCID-2CN NPs and further explored its NIR-II FLI, PAI, PTI and anti-tumor abilities in vivo. The evaluation of different imaging abilities was achieved via subcutaneous injection into U87 tumor-bearing mice, with the images monitored at different time points. The intensity of NIR-II fluorescence at the tumor site decreased as time elapsed, indicating that BTP-TCID-2CN NPs can be metabolized effectively (Fig. 5A and B). NIR-II fluorescence intensity in different organs was also detected after 72 h injection; importantly, the NIR-II fluorescence intensity of BTP-TCID-2CN NPs was far higher in the tumor than in other organs and rarely accumulated in living organs (Fig. 5I). The temperature of tumor site was reflected through the thermal images. The temperature of the tumor site rose to 53.4 °C under 808 nm irradiation for 2 min, similar to the value associated with BTP-4CN NPs and indicating excellent photothermal conversion capability (Fig. 5C–D and Fig. S16†). Due to the fact that PA signals are usually accompanied by PTT generation, we further validated the PAI ability of BTP-TCID-2CN NPs in vivo. Obvious PA signals could be seen at the tumor site, and the tumor contour was outlined clearly (Fig. 5E). In addition, the intensity of PA signals showed a time-dependent increase in tissue simulation experiments (Fig. 5F), enabling accurate delineation of tumor size and long-term tumor monitoring. As a whole, these results demonstrate that BTP-TCID-2CN NPs have superior photothermal conversion capacity and NIR-II FLI and PAI capabilities.

Fig. 5 (A) NIR-II fluorescence images and (B) corresponding NIR-II fluorescence intensities of U87 tumor-bearing mice injected with BTP-TCID-2CN NPs (20 μg mL⁻¹) at the tumor site and recorded at different points of time. (C) Infrared thermal images and (D) corresponding temperature changes of U87 tumor-bearing mice injected with BTP-TCID-2CN NPs (20 μg mL⁻¹) and PBS irradiated by 808 nm laser (1.0 W cm⁻²), recorded every two minutes. (E) Relative tumor volume and (F) body weight of mice throughout the therapeutic process. (G) NIR-II fluorescence intensities of different organs 72 h after injection.

We further investigated the actual therapeutic effect of BTP-TCID-2CN NPs in vivo. After 24 h post injection of BTP-TCID-2CN NPs, the tumor was treated with 808 nm laser irradiation (1.0 W cm⁻²) for 3 min. As shown in Fig. 5G, the volumes of tumors treated with BTP-TCID-2CN NPs + laser became obviously smaller, confirming the anti-tumor effects in vivo. All mice across the four treatment conditions maintained stable body weight, indicating the minimal side-effects of this therapy (Fig. 5H). In addition, cell morphology, apoptosis, and proliferation were analyzed in tumor tissues via hematoxylin and eosin (H&E) staining, terminal-deoxynucleotidyl transferase-mediated nick end labelling (TUNEL), and Ki67 staining, respectively. The BTP-TCID-2CN NPs + laser group showed increased apoptosis and decreased proliferation at the tumor site, indicating effective killing of tumor cells (Fig. S17†). Meanwhile, the BTP-TCID-2CN NPs + laser group exhibited insignificant pathological damage to major organs, as assessed by H&E staining, and the results of blood biochemistry and hematological analyses were within standard ranges, indicating excellent bio-compatibility (Fig. S18 and 19†).

Conclusions

In summary, we successfully utilized a nitrilation strategy to balance NIR-II FLI and PTT/PAI. Nitrilated molecules (BTP-4CN NPs and BTP-TCID-2CN NPs) showed greater photothermal effects (PCE of 43.3% and 33.5%, respectively) and PAI abilities than a nitrile-group-lacking molecule (BTP-2TCID NPs). Furthermore, BTP-TCID-2CN NPs showed the highest NIR-II QY (3.7%) and the largest ESP difference, enabling high-resolution NIR-II FLI in the organism. In vitro experiments indicated that molecules with fewer nitrile groups possessed better biosafety and cellular internalization abilities, while BTP-TCID-2CN NPs with appropriate nitrilated groups exhibited superior overall performance with respect to tumor-inhibiting efficiency and NIR-II FLI and PAI capabilities. This work demonstrates that nitrilation is an effective strategy to balance NIR-II FLI and PTT/PAI, broadening the prospects of exploiting high-performance NIR-II PTAs.

Author contributions

The manuscript was written with the contributions of all authors. All authors have given approval to the final version of the manuscript.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animal Center of Hangzhou Normal University and approved by Institutional Animal Care and Use Committee.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant number 21971049), “Ten-thousand Talents Plan” of Zhejiang Province (grant number 2019R52040) and the Zhejiang Provincial Natural Science Foundation of China (grant numbers LZ23B040001 and LY23E030011).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3py00461a

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