Tumor microenvironment-activated theranostic nanoreactor for NIR-II Photoacoustic imaging-guided tumor-specific photothermal therapy

Abstract

Theranostic agents that can be sensitively and specifically activated by the tumor microenvironment (TME) have recently attracted considerable attention. In this study, TME-activatable 3,3′,5,5′-tetramethylbenzidine (TMB)-copper peroxide (CuO₂)@poly(lactic-co-glycolic acid) (PLGA)@red blood cell membrane (RBCM) (TCPR) nanoparticles (NPs) for second near-infrared photoacoustic imaging-guided tumor-specific photothermal therapy were developed by co-loading CuO₂ NPs and TMB into PLGA camouflaged by RBCMs. As an efficient H₂O₂ supplier, once exposed to a proton-rich TME, CuO₂ NPs can generate H₂O₂ and Cu²⁺, which are further reduced to Cu⁺ by endogenous glutathione. Subsequently, the Cu⁺-mediated Fenton-like reaction produces cytotoxic ·OH to kill the cancer cells and induce TMB-mediated photoacoustic and photothermal effects. Combined with the RBCM modification-prolonged blood circulation, TCPR NPs display excellent specificity and efficiency in suppressing tumor growth, paving the way for more accurate, safe, and efficient cancer theranostics.

Graphical Abstract

1. Introduction

Theranostic nanoplatforms, which integrate diagnostic and therapeutic functions, have paved the way for cancer management to achieve the goal of personalized medicine, owing to their unique advantages, including engineerable functionalities, early detection of tumors, visualization of the spatiotemporal biodistribution of nanoagents, and real-time assessment of therapeutic effects [1], [2], [3], [4], [5], [6]. Among various theranostic nanoplatforms, optical theranostic nanoagents have drawn increasing attention due to their minimal invasiveness, high specificity, and spatial-temporal selectivity.

Photothermal therapy (PTT), which causes cancer cell death by using incident light energy to induce hyperthermia using photothermal agents (PTAs), has substantial potential in oncotherapy. Compared to other diagnostic methods, photoacoustic imaging (PAI) has the advantages of excellent tissue penetrability and low scattering and dissipation in biological tissues; hence, PAI-based theranostic nanoplatforms have drawn increasing attention among all developed nanoplatforms. PAI/PTT theranostic nanoplatforms show great promise for tackling cancers.

Most of the reported theranostic agents lack tumor specificity, and the diagnostic signals and therapeutic effects are always in “on” mode. Intravenous (i.v.) injected nanoplatforms, which are prone to phagocytosis and retention by the reticuloendothelial system, not only lead to non-specific interference during cancer diagnosis, but also cause side effects during treatment. Therefore, it is preferable to design stimuli-responsive theranostic agents that can be exclusively activated in a specific focal area to overcome the aforementioned barriers.

Unlike normal tissues, the tumor microenvironment (TME) is generally characterized by hypoxia, acidosis, overexpressed H₂O₂, and high interstitial fluid pressure [7]. Taking advantage of these unique characteristics, TME-activatable theranostic agents (ATAs) have gained broad interest as the diagnostic and therapeutic features they induce can be “switched on” in tumors and “switched off” in normal tissues, which facilitates precise tumor detection and ablation with minimal non-specific damage. Such theranostic systems can be built by employing chemical reactions that are initiated exclusively at the tumor sites [8], [9], [10], [11], [12], [13], [14], [15]. For instance, H₂O₂-activated agents provide a feasible choice for constructing TME-activated ATAs [16], [17], [18], [19], [20], [21]. However, the H₂O₂ levels in the TME are often insufficient for ATAs to realize efficient cancer theranostics, and integrating H₂O₂-activated ATAs with self-sufficient H₂O₂ could be an effective strategy to tackle this challenge.

PTT, which causes cancer cell death by using light energy to induce hyperthermia via PTAs, has substantial potential in oncotherapy owing to its minimal invasiveness and short treatment period [22]. Exploring PTAs with high photothermal conversion efficiency (PCE) and strong near-infrared (NIR) absorption, especially in the second near-infrared (NIR-II) band (1000–1350 nm), is critical for improving the antitumor efficacy of PTT [23], [24], [25]. Noble metal nanoparticles (NPs), metal chalcogen NPs, polyoxometalates, semiconducting polymers, and MXene nanomaterials are the most common NIR-II-absorbing materials [26], [27], [28]. Among these, organic PTAs based on NIR-II-absorbing p-conjugated semiconducting polymers have been used for PTT and PAI of deep-seated tumors owing to their high extinction coefficient, good biocompatibility, flexible structure, and engineerable physicochemical properties. Although the PCE of PTAs has dramatically improved in recent years, the side effects induced by strong hyperthermia on adjacent normal tissues cannot be ignored. Thus, there is an urgent need to develop novel PTAs with enhanced specificity and efficiency to improve the patient therapeutic outcomes, while reducing the potential side effects.

Copper peroxide (CuO₂) NPs, with a high H₂O₂ self-production capacity, and 3,3′,5,5′-tetramethylbenzidine (TMB), with excellent hydroxyl radical (·OH)-activated photothermal efficiency in the NIR-II region, were co-loaded into biodegradable poly(lactic-co-glycolic acid) (PLGA), which was subsequently coated with the red blood cell membrane (RBCM) of mice to fabricate TMB-CuO₂@PLGA@RBCM (TCPR) NPs for in vivo TME-activated theranostics (Scheme 1).

Scheme 1.

(a, b) Schematic illustration of the synthesis of 3,3′,5,5′-tetramethylbenzidine (TMB)-copper peroxide (CuO₂)@poly(lactic-co-glycolic acid) (PLGA)@red blood cell membrane (RBCM) (TCPR) nanoparticles (NPs) and the mechanism of H₂O₂-activated specific photothermal therapy (PTT) guided by second near-infrared (NIR-II) photoacoustic imaging (PAI).

Specifically, TCPR NPs present various desirable properties: (1) With the aid of RBCMs, TCPR NPs can be efficiently accumulated in tumor locations due to the enhanced permeability and retention (EPR) effect. (2) The loaded CuO₂ in TCPR can generate H₂O₂ via the reaction between CuO₂ and protons in the mildly acidic TME to increase the concentration of H₂O₂ in situ. (3) The Fenton-like reaction mediated by Cu ions and H₂O₂ (both endogenous and supplied) can efficiently generate sufficient •OH to oxidize TMB for NIR-II PAI and PTT. The theranostic performance of the rationally designed nanosystem was investigated in vitro and in vivo.

2. Section of Experimentation

2.1. Materials

The Shanghai Xinbao Fine Chemical Factory supplied CuCl₂·2H₂O. H₂O₂, CH₂Cl₂, NaOH, and PLGA were purchased from Nanjing Chemical Reagent Co. Ltd. Poly(vinylpyrrolidone) (PVP), polyvinyl alcohol (PVA), and TMB were obtained from Adamas-beta Co. Ltd.

2.2. Characterization

A scanning electron microscope (SEM, S4800; Hitachi, Japan) was used to characterize the morphology of the samples, and a Zetasizer Nano series (Nano ZS90; Malvern Instrument Ltd.) was used to characterize the hydrodynamic size distribution and zeta potential. Similarly, an ultraviolet (UV)–visible (Vis)–NIR spectrometer (UV-3600; Shimadzu, Japan) was used to measure the absorption spectrum, whereas inductively coupled plasma mass spectrometry (NeXion 300X) was used to measure the concentration of Cu. An infrared camera (FLIR) was used to capture the thermal pictures, and photoacoustic (PA) imaging was done using LOIS-3D (TomoWave Laboratories, USA). Flow cytometry (BD FACSCalibur) was used to detect the apoptotic rate of the cells, and biochemical analysis was performed using an automatic biochemical analyzer (Chemray 800; Leidu Life Technology, China). Lastly, an X-ray photoelectron spectroscopy (XPS) analysis was performed to further confirm the valence state of Cu.

2.3. Synthesis of CuO₂, TCP, and TCPR NPs

PVP (500 mg) was dissolved in a CuCl₂·2H₂O (1 mL, 0.05 M) solution, and NaOH (10 mL, 0.01 M) and H₂O₂ (100 L) were then added to the mixture, from which the CuO₂ NPs were obtained via ultrafiltration after 30 min of stirring.

PLGA (10 mg) was dissolved in 2 mL of CH₂Cl₂, and a mixture of 1% PVA (500 µL) and CuO₂ NP solution (200 mg mL⁻¹, 250 µL) was added. The microemulsion bubbles were formed twice using an ultrasonic cell grinder for 15 min, and TMB (5 mg mL⁻¹, 7 mL) in dimethyl sulfoxide (DMSO) was then mixed with the microemulsion. Finally, the prepared solution was progressively added to 6 mL of 2% PVA solution under ultrasonic pulverization for 10 min. The TMB-CuO₂@PLGA (TCP) NPs were collected via centrifugation after stirring overnight at 25 °C.

While RBCM was obtained from fresh blood of mice, hypotonic phosphoric acid buffer (1:50) was added to red blood cells (RBCs) and placed in the refrigerator overnight. The RBC solution was then purified via centrifugation (9000 rpm/min) to obtain pure RBCM, while the TCP NPs were stirred with RBCM for two hours to obtain TCPR NPs using the nanoprecipitation method.

2.4. Detection of H₂O₂

KMnO₄ (10 µg mL⁻¹) solution was treated with H₂O₂, phosphate-buffered saline (PBS), and TCPR NPs to detect the production of H₂O₂. Subsequently, UV–Vis–NIR spectra ranging from 400–1100 nm was measured. The presence of H₂O₂ was detected by the appearance and increase in the absorbance of oxTMB at 650 nm, which could also react with KI to generate I³⁻, resulting in an absorbance peak at 350 nm. Finally, the H₂O₂ concentration was determined by plotting a standard curve.

2.5. Photothermal Effect

The FLIR thermal camera was used to measure the temperature curves of TCPR NPs, which were incubated for 8 h for TMB oxidization before measuring UV–Vis absorption spectra, with varying concentrations at various pH values under 1060 nm laser irradiation. Meanwhile, the temperature change associated with TCPR NP dispersion was also observed in a quartz tube under laser irradiation with various power intensities (1.0, 0.8, 0.6, 0.4, and 0.2 W cm⁻²), and their optical stability (100 mg L⁻¹) under acidic conditions (pH 5.5) was also tested. The following equation was used to calculate the PCE (η):

$$\eta =h_S\left(T_{max}-T_{sur}\right)-Q_{Dis}/I(1-{10}^{-A1060})$$ (1)

The symbol η denotes the PCE, while the cell surface area is denoted by “s” and the heat transfer coefficient is denoted by “h”. Similarly, the equilibrium temperature is T_Max, ambient temperature is T_Sur, and I is the incident laser power. When a quartz cell and water are irradiated with laser light, QDis is the baseline energy generated, and A₁₀₆₀ is the absorbance of the sample at 1060 nm. “hs” is calculated from substituting equations:

$$\tau =mc/h_S$$ (2)

The mass and capacity of pure water are “m” and “c,” respectively, and the following formula can be used to compute the time constant (τ):

$$t=-\tau ln\theta$$ (3)

$$\theta =\left(T-T_{Sur}\right)/\left(T_{Max}-T_{Sur}\right)$$ (4)

in which θ is the driving force temperature of the solution.

2.6. In vitro cytotoxicity assay

Mouse breast cancer cells (4T1) and human-immortalized keratinocytes (HaCaTs) were cultured in a medium (Roswell Park Memorial Institute-1640 for 4T1 and Dulbecco's minimal essential medium for HaCaTs purcharsed form Wuhan Servicebio technology Co. Ltd. containing 10% fetal bovine serum and 1% penicillin-streptomycin in the presence of 5% CO₂ at 37 °C). In regards to the cytotoxicity assay, a 96-well plate was inoculated with HaCaTs (2.0 × 10⁴ cells per well), and the cells were incubated with different concentrations of TCPR NPs in a 100 µL complete culture medium. After a 12-h incubation period, each well received 20 µL of 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg mL⁻¹), and the plate was kept at 37 °C for additional four hours. DMSO (200 µL) was used to dissolve the formazan crystals in living cells, and the absorbance of the purple-colored formazan/DMSO solution at 490 nm was measured using a microplate reader (MK3, Thermo Scientific, America) for the calculation of cell viability.

Finally, a 2,7-dichlorodi-hydrofluorescein diacetate probe was used to detect intracellular •OH. Dead cells emitted red fluorescence when stained with propidium iodide (PI), whereas live cells were stained with calcein acetoxymethyl ester (Calcein-AM), which emitted green fluorescence.

In a 6-well plate, 4T1 cells (5 × 10⁶ cells per well) were seeded and incubated overnight. Different substances (PBS, TPR, TPR + NIR, TCPR, and TCPR + NIR) were added to each well at a concentration of 100 µg mL⁻¹. The laser irradiation group received 10 min of 1060 nm laser irradiation (1 W cm⁻²) after a 4-h incubation, and the cells were cultured for another 8 h before conducting western blotting analysis.

2.7. NIR-II PA imaging

TCPR NP aqueous suspensions (25, 50, 75, and 100 µg mL⁻¹) were simultaneously injected into PA glass tubes, and the PA intensity of the samples under 1060 nm laser irradiation was subsequently determined.

To detect the in vivo PA signals at the tumor sites, 4T1 tumor-bearing mice were i.v. injected with 200 µL of TCPR NPs (500 µg mL⁻¹), and placed into the LOIS-3D machine to gather PA signals.

2.8. Animal models

The Yangzhou University Comparative Medicine Centre provided female mice (BALB/c, 5–6 weeks old), and 4T1 cells were inoculated subcutaneously in the right rear to build a local tumor model. All animal experiments were monitored and authorized by the School of Pharmaceutical Science, Nanjing Tech University. Female Balb/c mice were ordered from the Comparative Medicine Centre of Yangzhou University's animal ethics (permit number:SCXK (SU) 2017-0007).

2.9. In vivo assay

The 4T1 tumor-bearing mice were randomly distributed into five groups (n = 4) and i.v. injected with 100 µL of the following formulations when the tumor volume reached 100–120 mm³: (1) control (PBS), (2) CuO₂ NPs, (3) TPR NPs + NIR, (4) TCPR NPs, and (5) TCPR NPs + NIR. The concentration of the NPs was 4 mg/kg. After administration for 8 h, 10 min of laser irradiation (0.5 W cm⁻², 1060 nm) was applied to the 4T1 tumor-bearing mice in groups (3) and (5), and tumor volumes and body weights were measured every two days.

2.10. Intratumoral dihydroethidium (DHE) staining and copper ion detection

While frozen sections of fresh tumors were stored at –20 °C, DHE could enter the cells and be oxidized by intracellular reactive oxygen species (ROS) to emit red fluorescence. The brighter the red fluorescence, the higher were the cellular ROS levels.

Tumor tissue sections were treated with ethanolic ruby acid and sodium acetate solutions to detect Cu ions, while excess rubric acid combined to form dark green-black rubric Cu salt precipitates.

2.11. In vivo histochemical analysis

The mice were sacrificed at the end of the treatment, and their spleen, liver, heart, kidney, lung, and tumor tissues were fixed with 4% paraformaldehyde (Wuhan Servicebio technology Co. Ltd.). Ki-67 and hematoxylin and eosin (H&E) were used to stain the tissues before examination under an optical microscope.

3. Results and discussion

The smart TME-triggered theranostic system is expected to remarkably improve the efficiency of tumor management and resultant patient outcomes. Hence, TME-responsive PTT is extremely beneficial to patients. As illustrated in Scheme 1, CuO₂ NPs (∼12 nm) were first synthesized according to a previous study [29], and the crystal structure was confirmed by the XRD spectrum (Fig. S1). TCP NPs were fabricated by co-loading TMB and CuO₂ NPs into a water/oil/water (w/o/w) double emulsion formed by PLGA to realize NIR-II PAI-guided tumor-specific PTT. RBCM was employed to modify TCP NPs (TCPR NPs) via self-assembly to achieve long-term blood circulation.

As shown in Fig. 1a, the TCP NPs exhibited a regular homogeneous spherical shape, with an average diameter of 124 nm. The roughness of the surface of the TCPR NPs increased after surface modification with RBCM (Fig. 1b). In turn, the low zeta potential of TCPR NPs compared to that of TCP NPs further confirmed effective surface modification (Fig. S2). The hydrodynamic size distribution of TCPR NPs in water was ∼125 nm (Fig. 1c), which is consistent with the SEM results. The atomic composition was confirmed by XPS (Fig. 1d and S3). The Cu 2p_1/2 and Cu 2p_3/2 peaks of the XPS Cu spectrum in Fig. 1e deconvoluted into their peak at 932.3 eV from Cu⁺ and at 952.5 eV from Cu²⁺ [30]. KMnO₄ was chosen as the detection probe to investigate the H₂O₂ generation ability of the developed TCPR NPs, whose characteristic UV–Vis–NIR absorption peaks would dissolve due to H₂O₂ oxidation. As shown in Fig. 1f, five distinct KMnO₄ peaks were identified in the control group, but when KMnO₄ was treated with H₂O₂ or TCPR NPs, the characteristic absorption disappeared, indicating the H₂O₂ generating capability of TCPR NPs. The existence of Cu⁺/Cu²⁺ redox couples together with the H₂O₂ generating capability of TCPR NPs verified the H₂O₂ self-supply property of TCPR NPs in an acidic environment (CuO₂ + 2H⁺ → Cu²⁺ + H₂O₂). Under acidic conditions, the Fenton-like reaction mediated by hydrolyzed Cu ions and endogenous/generated H₂O₂ produces •OH, further oxidizing TMB into oxTMB (Fig. S4). The absorption spectra of TCPR NPs were analyzed at pH 7.4 (to mimic normal tissue) and pH 5.5 (to mimic tumor tissue) to assess the selectivity of TCPR NPs for acidity. The characteristic oxTMB absorption spectrum showed that the release rate was faster at pH 5.5, within 10 h of the test, with the release amount corresponding to pH 5.5, which was nearly three times that at pH 7.4, confirming the specificity of TCPR in acidic environments (Fig. 1g). Furthermore, the release of TMB at pH 5.5 reached a maximum at 10 h, which was nearly 3-fold the release at pH condition of 7.4 (Fig. 1h). In addition, KI was used as the probe to detect self-supplied H₂O₂ at pH 5.5, and pH 6.5. At pH 5.5, the generation rate of H₂O₂ was significantly higher than that at pH 7.4, as shown in Fig. 1i. These results revealed that the recently obtained TCPR NPs possess the capability of H₂O₂ self-supplementary and specific release of TMB in an acidic environment, enabling self-enhanced and TME-activated PTT.

Fig. 1.

(a,b) Scanning electron microscope (SEM) images of 3,3′,5,5′-tetramethylbenzidine (TMB)-copper peroxide (CuO₂)@poly(lactic-co-glycolic acid) (PLGA) (TCP) and TCP@red blood cell membrane (RBCM) (TCPR) nanoparticles (NPs). (c) Hydrodynamic size distribution of TCPR NP solution (polydispersity index: 0.258). (d) X-ray photoelectron spectroscopy (XPS) survey spectrum. (e) Cu 2p XPS spectra at high resolution. Ultraviolet (UV)–visible (Vis)–near-infrared (NIR) absorption spectra of (f) KMnO₄ incubated with PBS (Control), TCPR NPs, and H₂O₂ for detecting the generation of H₂O₂, (g) TCPR NPs at pH 5.5 and 7.4. (h) Absorbance of the released TMB at different time points under pH 5.5 and 7.4. (i) Concentration changes of H₂O₂ with time changes generated by TCPR NPs at pH 6.5 and 5.5 (***p < 0.001, **p < 0.01).

Compared with the NIR-I biowindow (750–1000 nm), the NIR-II biowindow (1000–1350 nm) has very recently attracted more attention due to its higher maximum allowable exposure (MPE) to laser (e.g., 0.33 W cm⁻² for 808 nm light, 1 W cm⁻² for 1060 nm light), as well as its deeper tissue penetration depth. We further evaluated the photothermal performance of TCPR NPs under different conditions. As shown in Fig. 2a–c, TCPR NPs showed concentration-, pH-, and laser power density-dependent photothermal efficiency. Impressively, benefiting from the effective generation of H₂O₂ under acidic conditions (pH 5.5), the temperature of the TCPR NPs solution (100 µg mL⁻¹) increased by over 25 °C, which was significantly higher than that in the neutral pH condition. This again confirmed the selective oxidation of TMB by introducing CuO₂ NP and ensured tumor-specific PTT with minimal side effects to adjacent normal tissues (Fig. 2a and b). Under 1060 nm laser irradiation, the TCPR NPs presented an excellent PCE efficiency (ƞ = 54%, Fig. S5) and outstanding photothermal stability (Fig. 2d) as a photothermal agent.

Fig. 2.

(a,b) Quantitative temperature curve of TCPR NPs with different concentrations at pH 7.4 and 5.5 (1060 nm, 1 W cm⁻², 10 min). (c) Temperature variations of TCPR NPs (100 µg mL⁻¹) irradiated by 1060 nm laser at different power densities. (d) Photothermal stability of TCPR NPs exposed to laser irradiation (1060 nm, 1 W cm⁻²) for 5 on/off cycles. (e) Photoacoustic (PA) images of TCPR NP suspensions at various concentrations (25–100 µg mL⁻¹). (f) Quantitative PA intensities corresponding to the PA signals in (e).

As expected, TCPR NPs at pH 5.5 showed desirable PAI performance and a concentration-dependent increase in PA signal (Fig. 2e and f), which revealed that TCPR NPs exhibit a TME-responsive photothermal effect and satisfactory PAI performance for directing and assessing the treatment process. Inspired by their superior physicochemical properties, the anticancer effects of the TCPR NPs were further tested at the cellular level. By incubating 4T1 cells with fluorescein isothiocyanate (FITC)-labeled TCPR NPs for different time intervals, we found that TCPR NPs were effectively endocytosed within 4 h and were distributed in both the nucleus and cytoplasm (Fig. 3a). TCPR NPs at concentration of 0-250 µg mL⁻¹ exhibited no apparent toxicity to HaCaTs (Fig. 3b), whereas they were dose-dependently cytotoxic to 4T1 tumor cells, presumably because cancer cells have a relatively higher H₂O₂ level than normal cells. 4T1 cells were treated with nanoparticles of different formulations, with or without laser irradiation, to further investigate the anticancer mechanism of TCPR NPs. The relative survival rate of cancer cells treated with TPR nanoparticles (without CuO₂) did not decrease significantly during laser irradiation, as seen in Fig. 3c. Interestingly, when 4T1 cells were cultured with the TCPR NPs for 6 h, a large number (∼40%) of cancer cells were killed, which could be due to the Fenton-like reaction of Cu ions with the self-supplied H₂O₂ and endogenous H₂O₂., which is demonstrated by the significantly increased ROS levels in the TCPR and TCPR + NIR groups (Fig. 3d).

Fig. 3.

(a) Cellular uptake and localization of fluorescein isothiocyanate (FITC)-labeled TCPR NPs (green fluorescence) at a concentration of 100 µg mL⁻¹ at different time points. The cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). (b) Cell viability of human-immortalized keratinocytes (HaCaTs) incubated with TCPR NPs at various concentrations. (c) Viability of 4T1 cells cultured with NPs of TPR or TCPR at different concentrations with and without laser irradiation (10 min, 1 W cm⁻², 1060 nm). (d) Reactive oxygen species (ROS) staining for intracellular radical detection (incubated with NPs [50 µg mL⁻¹] for 8 h). (e) Live/dead staining of 4T1 cells after treatment (50 µg mL⁻¹) under laser irradiation (10 min, 1 W cm⁻², 1060 nm). (f) Annexin-V-FITC/propidium iodide (PI) flow cytometry assay. (g) Western blotting assay of caspase-3, B-cell lymphoma (Bcl)-2, and Bcl-2-associated X (Bax) protein expression. (h) Schematic illustration of cell apoptosis caused by TCPR NP-induced hypothermia (***p < 0.001).

To a certain extent, the laser promoted ROS production, and the relative cell viability in the TCPR NPs + NIR group decreased remarkably, indicating the highly efficient tumor-killing effect of this TCPR NP-based treatment, which could be attributed to the oxidation of TMB. Calcein-AM and PI were used to stain live and dead cells, respectively, to further confirm the treatment performance of TCPR NPs (Fig. 3e). Although the TPR, TPR + NIR, and TCPR groups exhibited limited lethality to 4T1 cells and most of them showed green fluorescence, significant red fluorescence was observed in the TCPR group, which indicated an effective anticancer effect triggered by the prominent synergistic effect of enhanced PTT. Flow cytometric analysis was used to quantify cell apoptosis and necrosis. As shown in Fig. 3f, the annexin V-FITC/PI double staining assay was used to examine apoptotic and necrotic cells. While almost no apoptotic or necrotic cells were observed when 4T1 cells were treated with TPR and TPR+NIR groups, 27.33% of apoptotic/necrotic cells were induced when treated with TCPR NPs alone, and 55.87% of apoptotic/necrotic cells in the TCPR group were observed. With the assistance of TME responsiveness and laser irradiation, TCPR NPs demonstrated excellent cytotoxicity in tumor cells and a favorable synergistic therapeutic effect. Additionally, as the key regulators of cell apoptosis and necrosis, the expression levels of the B-cell lymphoma (Bcl)-2 family (Bcl-2-associated X protein (Bax) and Bcl-2) and caspase family proteins (Caspase-3) in 4T1 cells after different treatments (PBS, TPR, TPR + NIR, TCPR, and TCPR + NIR) were analyzed by western blotting (Fig. 3g). Bcl-2 was downregulated, Bax was upregulated, and its value gradually decreased after the promotion of cell apoptosis, consistent with the upregulation of Caspase-3. As discussed above, the TCPR NPs self-supplied with H₂O₂ in cancer cells could activate TMB oxidization with Cu ions, which would induce a potent photothermal effect and kill cancer cells by activating the apoptotic/necrotic cascade (Fig. 3h).

Bioimaging provides information on lesions and monitors the spatiotemporal distribution of nanomedicines, thus enabling more precise phototherapy and reducing the potential damage to normal tissues. Here, we investigated the in vivo photothermal performance of TCPR NPs using the absorption of TCPR in the NIR-II range, in which T was injected subcutaneously into normal tissue and intratumorally (Fig. 4a and b). Notably, while the temperature in normal tissue only increased to 39.9 °C, the tumor temperature increased to 48.4 °C after 10 min of laser irradiation (1060 nm, 1 W cm⁻²), indicating that TCPR NPs could be triggered explicitly by the TME while maintaining stealth in normal tissues. As the TME-triggered imaging ability of the TCPR NPs could be used to guide in vivo therapy, PAI was further used to assess the in vivo performance of TCPR NPs after i.v. injection. As shown in Fig. 4d and e, the PA signal of the tumor site appeared 2 h post-injection and gradually increased over time. The maximum tumor accumulation of the NPs occurred at 6 h, after which the PA signal intensity was gradually attenuated. Copper red acid staining was conducted to confirm the presence of TCPR NPs in the tumor. In turn, the copper salt deposition was dark green and black, and the nuclei were red. In the comparison between the control and TPR groups, copper salt deposition was observed in the TCPR group (Fig. 4c), which confirmed the apparent tumor uptake of the TCPR nanoparticles.

Fig. 4.

(a) Photothermal pictures of the treatment with TCPR NPs (50 µg mL⁻¹, 200 µL), which were irradiated with 1060 nm laser (1 W cm⁻²) at the tumor sites for 10 min. (b) Corresponding temperature change in (a). (c) Copper red acid staining of tumor tissues. (d) PA images of mice at various time points after the intravenous injection of TCPR NPs. (e) Corresponding PA signal intensities in (d) (***p < 0.001).

Mice were randomly distributed into five groups and injected with different formulations through the tail vein (CuO₂, TPR + NIR, TCPR, TCPR + NIR, and control, n = 4) to assess the in vivo antitumor performance of TCPR NPs-based PTT. The temperature of the tumor injected with the TCPR NPs increased to 47.2 °C 6 h later, with 10 min of laser irradiation, while the control group and TPR + NIR group only increased to approximately 36 °C, which confirmed the photothermal effect (Fig. 5a and b).

Fig. 5.

(a) Photothermal pictures of mice treated under 1060 nm laser irradiation (1 W cm⁻²) for different time points after being injected with TPR or TCPR NPs (200 µg mL⁻¹, 50 µL) and phosphate-buffered saline (PBS.) (b) Temperature changes in mice tumor sites corresponding to (a). (c) Variation in relative tumor volume after different treatments. (d–f) Dihydroethidium (DHE; red fluorescent signal indicates ROS concentration), hematoxylin and eosin (H&E), and Ki-67 staining of tumors with various treatments (***p < 0.001).

Under laser irradiation, the reaction between endogenous H₂O₂ and TPR NPs suppressed tumor growth, even in the absence of CuO₂ NPs. The growth of the tumors treated with CuO₂ and TCPR nanoparticles exhibited some inhibition, which might be due to the chemodynamic effect induced by the hydrolyzed Cu ions with endogenous and/or self-generated H₂O₂. On the other hand, the TCPR-NIR group showed significantly suppressed tumor growth (∼100% reduction in tumor volume compared to the control) after 10 min of laser irradiation, demonstrating the excellent therapeutic efficacy of TCPR NPs due to the self-supplied H₂O₂ and activated PTT (Fig. 5c and S6). Intratumoral ROS levels were determined by DHE staining (Fig. 5d). Compared to the other groups, TCPR NP treatment resulted in higher ROS levels in tumors, which were further elevated by laser irradiation-induced hyperthermia. H&E staining demonstrated apoptosis and necrosis of the tumor cells caused by PTT (Fig. 5e). The most severe damage was observed in the TCPR and TCPR + NIR groups. Furthermore, the Ki-67 (brown staining indicates cell proliferation) immunohistochemistry assay inhibited tumor cell proliferation (Fig. 5f), which showed the high antitumor efficiency of TME-responsive therapy based on TCPR NPs.

The long-term biosafety of TCPR NPs was further assessed via complete blood panel and blood biochemistry analyses, with all experimental groups maintaining normal levels of critical functional markers, such as alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine, in the blood. The levels of aminotransferase in the tested groups did not change, which demonstrated that TCPR NPs were kidney- and liver-compatible (Fig. 6a–d). The evaluated fourteen common indexes for routine blood analysis were within the normal ranges, indicating the negligible blood toxicity of TCPR NPs for 17 days (Fig. 6e). Furthermore, the weights of the mice also showed no abnormal changes (Fig. 6f), indicating negligible systematic toxicity of the treatment. Moreover, histological examination of the major organs of mice injected with PBS (as control) and TCPR NPs was performed to assess the potential toxicity (Fig. 6g), with no pathogenic abnormalities or inflammation observed in both groups. Collectively, these findings demonstrate the in vivo biocompatibility of TCPR NPs, paving the way for their future use as theranostic agents.

Fig. 6.

(a–d) Alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), and aspartate aminotransferase (AST) levels in mice after receiving TCPR NPs via an intravenous (i.v.) injection. (e) Analysis of the complete blood panel of mice after i.v. injection of TCPR NPs, including white blood cells (WBCs), hematocrit (HCT), red blood cell distribution width (RDW), platelet distribution width (PDW), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), platelets (PLTs), red blood cells (RBCs), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), lymphocyte (Lymph%), monocyte (Mon%), and granulocyte (Gran%). (f) The body weights of mice fluctuate during different treatments. (g) H&E staining of major organs in the control and TCPR + NIR groups.

4. Conclusion

In conclusion, an activatable theranostic nanoreactor was developed for TME-triggered NIR-II PTT under PAI guidance. The obtained TCPR NPs, with their rational design, showed impressive on–off switch-ability for cancer-specific therapy and outstanding pH-responsive activities to generate H₂O₂ and Cu ions, which further resulted in the generation of •OH via a Fenton-like reaction and oxidized TMB, turning on the photothermal effect via the generation of oxTMB. TCPR NPs, which also benefited from the camouflage of RBCM, achieved long-term blood circulation and successfully accumulated in tumor tissues via the EPR effect. Moreover, theranostic TCPR NRs could be specifically activated at the tumor site to achieve high-efficiency PTT under 1060 nm laser irradiation without harming the normal tissues. Therefore, use of activatable TCPR NRs has proven to be a feasible approach for tumor-specific imaging and treatment with minimal side effects.

Declaration of competing interest

The authors declare that they have no conflicts of interest in this work.

Biographies

Nan Yang is assiduously studying for a doctoral degree in the Institute of Advanced Materials, Nanjing Tech University (Nanjing Tech), Nanjing 211800, China, supervised by Prof. Xiaochen Dong. Her current research focuses on the development of functional nanomaterials with good biocompatibility and innovative strategies for designing tumor microenvironment-responsive.

Xiaochen Dong(BRID: 09638.00.66037) obtained his Ph.D. degree from Zhejiang University. Then he joined the School of Materials Science and Engineering at Nanyang Technological University as a postdoctoral. In 2012, he joined the Institute of Advanced Materials, Nanjing Tech University as a Full Professor. He has published more than 200 papers, including Adv. Mater., ACS Nano, etc. The current research involves biophotonics and bioelectronics, flexible electronics.