Tumor-microenvironment-responsive poly-prodrug encapsulated semiconducting polymer nanosystem for phototherapy-boosted chemotherapy

Abstract

Phototherapy-induced hypoxia in the tumor microenvironment (TME) is responsible for diminished therapeutic efficacy. Designing an intelligent nanosystem capable of responding to hypoxia for TME-responsive drug delivery will, to some extent, improve the therapeutic efficacy and reduce side effects. Semiconducting polymers with high photothermal conversion efficiency and photostability have tremendous potential as phototheranostics. In this paper, hypoxia-activatable tirapazamine (TPZ) was conjugated onto poly(ethylene glycol) to form a pH-sensitive poly-prodrug, PEG–TPZ, that can be triggered by the low acidity of the TME to cleave the acylamide bond for controllable drug release. PEG–TPZ was then used to encapsulate a semiconducting polymer (TDPP) for NIR-II-fluorescence-imaging-guided synergistic therapy. The reactive oxygen species (ROS) generation and ultrahigh photothermal conversion efficiency (∼58.6%) of the TDPP@PEG–TPZ NPs leads to the destruction of the tumor blood vessels, thus further activating the hypoxia-induced chemotherapy of TPZ. As a result, effective tumor regression was achieved after laser irradiation.

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New concepts

Although intelligent nano-platforms with tumor microenvironment responsiveness have been reported, producing multi-responsive ones for synergistic therapy remains a challenge. Semiconducting-polymer-based phototheranostics have the benefits of non-invasiveness and excellent phototherapeutic efficacy. We designed a multi-responsive nanoplatform for phototherapy-enhanced chemotherapy based on the poly-prodrug (PEG–TPZ) encapsulated semiconducting polymer TDPP. In the first step, tirapazamine (TPZ) can be released by the hydrolysis of the acylamide bond. Next, efficient reactive oxygen species (ROS) and photothermal conversion can be triggered by laser irradiation, leading to the consumption of cellular oxygen as well as damage to the blood vessels. The aggravated hypoxia contributes to the activation of tirapazamine for ROS generation, in turn enhancing the therapeutic efficacy. The cascade reaction based on both pH- and hypoxia-responsiveness will benefit not only the rational design and synthesis of nanomaterials, but also nanomedicine-based oncology and pharmacology, etc.

Introduction

Osteosarcoma (OS) is the most common primary malignant tumor of bones in children and adolescents, and generally originates from mesenchymal tissues.¹ Currently, the pathogenesis and mechanism of OS are still unknown in the clinic,² and early diagnosis remains challenging.^3,4 OS is characterized by high malignancy, fast growth, substantial invasion, and metastasis, and can destroy adjacent normal bone tissue.⁵ Due to its late diagnosis, OS develops rapidly and metastasizes hematogenously. More than half of patients exhibit small metastatic lesions, especially lung metastasis, followed by bone, resulting in poor prognosis.^6–8 Therefore, it is urgent to improve the efficacy of both diagnostic and therapeutic techniques for osteosarcoma.

Phototherapy has attracted increasing attention because of its low toxicity, minimized side effects, and high biocompatibility. It has been applied to the clinical treatment of various malignant tumors.^9–13 Osteosarcoma tumors with few tissue vessels result in aggravated hypoxia in the tumor microenvironment.^14–16 Organic semiconducting materials (OSMs) composed of p-conjugated building blocks as the optically active components have recently emerged as a promising category of biophotonic agents. OSMs can convert light energy into cytotoxic free radicals or heat, allowing for effective cancer phototherapy, and possess standard features, including excellent optical properties, good photostability, and biologically benign composition.¹⁷ In addition, OSMs are also used as contrast agents for cell imaging, targeted tumor imaging, drug release tracking, and monitoring of physiological indicators and real-time biomarkers, as well as phototherapeutic agents for cancer treatment.^18–20 Therefore, hypoxia-activated prodrugs may be potential candidates for the treatment of osteosarcoma.

Synergistic therapy overcomes the disadvantages of monotherapy, enhancing therapeutic efficacy and leading to tumor regression.^21–23 Most solid tumors feature a hypoxic tumor microenvironment (TME), and PDT treatment will further consume oxygen, which in turn aggravates the hypoxic condition in the tumor, reducing the efficacy of PDT.^24–26 Great efforts have been devoted to overcoming the hypoxicity of tumors, including transporting oxygen molecules to relieve hypoxia and developing oxygen-dependent or oxygen-independent photosensitizers.^27–30 As a simple and operable strategy, combining an anti-tumor hypoxia-activated prodrug and PDT can significantly enhance the overall anti-tumor effect. Tirapazamine (TPZ), a classic hypoxia-activated drug,³¹ is a nitrogen oxide Hypoxia-activated prodrug (HAP) and can destroy the structure of biological macromolecules and selectively kill hypoxic tumor cells.³² Gratifying progress has been made in the experimental research of TPZ in the treatment of malignant tumors. However, TPZ still suffers from poor permeability into tumor tissue and fast metabolism, and thus can hardly reach the core of the tumor.^33,34 In this regard, constructing a tumor-targeted nano-drug delivery system is considered to increase the in vivo stability and tumor accumulation effectively, thus improving the anti-tumor efficacy.^35–37

In this paper, we have designed and prepared a new intelligent TPZ-functionalized poly(ethylene glycol) (PEG–TPZ) that can respond to the low acidity of the tumor microenvironment through the cleavage of an acylamide bond for controllable drug release (Scheme 1). PEG–TPZ was then used to encapsulate a new semiconducting polymer (TDPP) for near-infrared region-II (NIR-II)-imaging-guided therapy.^38–41 Semiconducting polymers with high photostability have great potential for phototherapy, and NIR-II imaging, with its diminished tissue scattering and high signal-to-noise ratio, will guarantee a low background ratio and effectively distinguish the lesion from normal tissues. It was envisaged that the ultrahigh photothermal conversion efficiency of TDPP@PEG–TPZ NPs could induce blood vessel damage via the hyperthermia effect. Therefore, tumor hypoxia was enhanced to activate the chemotherapy of TPZ.⁴² The NIR-II fluorescence imaging using TDPP@PEG–TPZ NP-guided therapy leads to complete tumor regression. This result provides a paradigm for designing intelligent nano-formulations for photothermal-therapy-boosted chemotherapy.^43–46

Scheme 1 Illustration of TDPP@PEG–TPZ nanoformulation for phototherapy-enhanced chemotherapy.

Experimental section

Materials and apparatus

DSPE–PEG–COOH (DSPE = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine) was purchased from ZZBIO Co. Ltd, DPPSn (2,5-bis(2-octyldodecyl)-3,6-bis(5-(trimethylstannyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione) was purchased from Derthon OPV Co LTD (Shenzhen, China) and other reagents were available from Sigma Aldrich. The human osteosarcoma cell line 143B and human embryonic kidney cells HEK-293 used in this study were obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (CAS). The ¹H NMR and ¹³C NMR spectra were measured in CDCl₃ solution using a Bruker DRX NMR spectrometer (400 MHz) with the solvent residual as the internal standard. UV-vis spectra were obtained using a UV-3600 spectrophotometer (Shimadzu, Japan). The size of the nanoparticles was recorded using a 90-Plus particle size analyzer (Brookhaven Instruments, USA). The morphology of the nanoparticles was determined using a transmission electron microscope (JEM-2100, JEOL). The bio-images of the tumor, heart, liver, spleen, lung and kidneys were recorded using a Nitroptics Series III 900/1700. The apoptosis of the cells was then analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

Synthesis and preparation of TDPP

For synthesis of TDPP, 2,5-dibromothieno[3,2-b]thiophene (59.2 mg, 0.2 mmol), DPPSn (286.6 mg, 0.2 mmol) and Pd (PPh3)4 (9.3 mg, 0.0080 mmol) were dissolved in anhydrous toluene (5 mL). Nitrogen purging was used to drive off any possible oxygen and water. The mixture was then sealed and stirred at 110 °C for 24 h under a nitrogen atmosphere. The reaction mixture was poured into methanol (200 mL) after cooling to ambient temperature. The crude product was then subjected to Soxhlet extraction with methanol, acetone, and hexane. Eventually, TDPP was extracted with chloroform, concentrated and precipitated with methanol again, leading to the formation of dark solids. Yield: 158 mg. PEG–TPZ was synthesized by treating DSPE–PEG–COOH (M_w 2000, 100 mg, 0.05 mmol) with TPZ (15 mg, 0.084 mmol) with EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) as the catalysts. Yield: 194 mg, 90%. The crude product was purified via dialysis in distilled water to obtain the final product after freeze-drying.

Preparation of TDPP@PEG-TPZ NPs

A mixture of PEG–TPZ (20 mg) and TDPP (2 mg) was dissolved in tetrahydrofuran. It was then dropped into distilled water (5 mL) under ultrasonic conditions. The mixture was then stored in a fume hood to drive off the tetrahydrofuran and then stored in the dark for further use. The THF was then removed under vacuum, and the TDPP@PEG–TPZ NPs were filtrated with a 220 nm filtration membrane for further application.

Calculation of drug loading content (DLC) and drug loading efficiency (DLE):

DLC (wt%) = mass of loaded drug/total mass of loaded drug and polymer × 100% (1)

DLE (%) = mass of loaded drug/mass of theoretical drug × 100% (2)

Singlet oxygen detection

Singlet oxygen generation was evaluated using both singlet oxygen sensor green (SOSG) and electron spin resonance (ESR) spectroscopy with 2,2,6,6-tetramethylpiperidine (TEMP) as a spin trapper. Generally, a mixture of TDPP@PEG–TPZ NPs (5 mM) and SOSG was prepared, and the fluorescence intensity of SOSG was recorded after irradiation at different time points (0, 1, 2, 3 and 4 min). The triplet peaks observed in a 1 : 1 : 1 ratio confirmed the production of ¹O₂ under the laser-irradiation of TDPP@PEG–TPZ NPs.

Measurement of TPZ release

10 mg PEG–TPZ was dissolved in water (0.5 mL). The solution was dialysed against 10 mL PBS (pH 7.4 and 5.5), respectively. The UV-vis spectra of the PBS solutions were measured and compared with the standard curve of the absorbance of TPZ to calculate the amount of TPZ. The release percentage was then calculated according to the following equation:

Percentage = amount of released TPZ/amount of total TPZ × 100% (3)

Cell culture

The 143B cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific), and HEK-293 cells were cultured in Dulbecco's minimum essential medium (Gibco; Thermo Fisher Scientific) with 10% FBS. All the complete culture media contained 100 units per mL penicillin and 100 mg mL⁻¹ streptomycin. All cells were cultured at 37 °C in a humidified incubator with 5% CO₂.

Cellular uptake and MTT assay

143B cells were seeded in a 6-well dish for 24 h and incubated with TDPP@PEG–TPZ NPs (10 μg mL⁻¹) in a 5% CO₂ incubator, after which the cells were collected and the fluorescence intensity of cells at different time points was detected using flow cytometry.

The 143B cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cells were seeded into 96-well plates with 1 × 10⁴ cells per well in 100 μL culture medium. The cells were incubated for 24 h and then exposed to the drug at various concentrations of TDPP@PEG–TPZ NPs or TPZ for the indicated time points, and the control cells were treated with 0.5% DMSO. The TPZ treatment group was divided into normoxia and hypoxia culture conditions, respectively. Cells in the illumination groups (TDPP@DSPE–PEG or TDPP@PEG–TPZ NPs) were irradiated with a laser (808 nm, 0.5 W cm⁻²) for 5 min. After incubation, the cells were incubated for a further 12 h. Subsequently, 10 μL MTT solution (5 mg mL⁻¹ in PBS) was added and incubated for another 4 h. The supernatants were then removed and 100 μL per well DMSO was added to dissolve formazan crystals. The absorbance was recorded at 492 nm using a Thermo Mk3 microplate reader. A cell growth inhibition curve was generated by plotting cell growth inhibition against drug concentration, and the half-maximal inhibitory concentration (IC₅₀) was determined using GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA).

Annexin V-FITC/propidium iodide (PI) staining

The apoptosis was analyzed using Annexin V-FITC/propidium iodide (PI) dual-staining. 143B osteosarcoma cells were harvested after treatment with TDPP@DSPE–PEG, TDPP@PEG–TPZ NPs or TPZ and then stained with Annexin V-FITC/PI Cell Apoptosis Detection Kit (KeyGen Biotech, Nanjing, China) according to the manufacturer's protocol. The apoptosis rates of the cells were then analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

Fluorescence imaging of intracellular reactive oxygen species

Intracellular ROS levels were indicated by a green fluorescent probe, 6-carboxy-2′,7′-dichlorofluoroscein diacetate (DCF-DA, Sigma-Aldrich). 143B cells were seeded into a confocal dish, and incubated with media containing TDPP@PEG–TPZ NPs (10 μg mL⁻¹) for 24 h; after incubation, the cells were stained with 2 mL DCFH-DA (10 μM) at 37 °C for 20 min. The cells were then washed three times with PBS and irradiated with a laser (808 nm, 0.5 W cm⁻²) for 10 min. The fluorescence images were observed using an Olympus IX 70 inverted microscope excited at 633 nm and collected from 640 to 700 nm for DCF detection.

In vitro microcapillary formation assay

Matrigel Matrix was thawed at 4 °C for 24 h to form a gel, added to angiogenesis slides (IBIDI GMBH, 10 μL per well) and incubated at 37 °C. Human umbilical vein endothelial cells (HUVECs) were seeded on the matrix-coated well (2 × 10⁴ cells per well) to generate a complete network. The medium was then replaced with fresh medium containing 0 or 10 μg mL⁻¹ of TDPP@PEG–TPZ NPs and incubated for a further 24 h. Micro-tube images were then captured using a TS100 microscope (Nikon, Japan).

Live and dead viability assay

The effect of TDPP@DSPE–PEG NPs or TDPP@PEG–TPZ on 143B cell viability was assessed using a living/dead cell double-staining kit (Sigma-Aldrich (Shanghai) Trading Co. Ltd.) following the manufacturer's protocol. The kit contains calcein-AM (Calcein acetoxymethyl) and PI (Propidium iodide), which fluorescently stain viable and dead cells, respectively. The slides were photographed under an Olympus FV1000 confocal microscope.

In vivo NIR-II and photothermal imaging

The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Tech University. The animal study was performed according to the rules of Nanjing Tech University.

Generally, 200 μL TDPP@PEG–TPZ NPs (200 μg mL⁻¹) in saline was intravenously administered to nude mice. In vivo fluorescence images were recorded using a Niroptics Series III 900/1700 at different time points after intravenous injection. Infrared imaging was conducted using an FLIR thermal camera under irradiation with an 808 nm laser for 24 h starting 8 min after injection. Mice injected with saline were evaluated as a control.

Antitumor effects in nude mice

Thirty male BALB/c nude mice (3–4 weeks, 10–12 g) were purchased from the Comparative Medicine Centre of Yangzhou University and maintained in a specific-pathogen-free animal facility (21 ± 2 °C and 45 ± 10% humidity) on a 12 h light–dark cycle with food and water supplied freely during the entire experiment.

To establish the OS model, 143B cells (5 × 10⁶) suspended in 100 μL PBS were subcutaneously inoculated in the right flank of each mouse. When the tumor size reached around 100 mm³, the nude mice were randomly assigned to six groups (n = 5). The mice were intravenously injected with different materials. The dosing of each group was as follows: (i) saline + laser (100 μL), (ii) free TPZ (20 μg mL⁻¹, 100 μL), (iii) TDPP@DSPE–PEG NPs without irradiation (100 μg mL⁻¹, 100 μL), (ix) TDPP@DSPE–PEG NPs with irradiation (100 μg mL⁻¹, 100 μL), (v) TDPP@PEG–TPZ without irradiation (100 μg mL⁻¹, 100 μL), (vi) TDPP@PEG–TPZ with irradiation (100 μg mL⁻¹, 100 μL). All groups received tail vein injections every 2 days. The tumors of the control and illumination groups were irradiated using a laser (808 nm, 0.5 W cm⁻²). Tumor volume (V) was measured every two days and calculated according to the equation:

V = length × width²/2.

Histology examination and TUNEL assay

All nude mice were sacrificed at the end point of the experiment, and their blood and major organ tissues (including tumors, heart, liver, spleen, lung, and kidney) were collected. Biochemical analysis of the blood was performed to evaluate drug toxicity. All tissues were fixed in 10% buffered formalin for 24 h at 4 °C and embedded in paraffin. 5 μm thick sections of the tissues were sliced and stained with hematoxylin and eosin (H&E) using a standard procedure for histopathological observation. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to analyze the apoptosis induction in the tumor tissues. The slides were photographed under a confocal microscope (Nikon, Chiyodaku, Tokyo, Japan).

Statistical analysis

All in vitro data are shown as mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses were performed using one-way ANOVA analysis of variance with Dunnett's test. All comparisons were made relative to untreated controls, and the significance of the difference is indicated as *P < 0.05 and **P < 0.01.

Results and discussion

Synthesis and characterization of TDPP@PEG–TPZ NPs

TDPP was prepared using the Stille reaction by treating 2,5-dibromothiophene (DPPSn) in toluene with Pd(PPh₃)₄ as the catalyst (Scheme 1). PEG–TPZ was synthesized by the condensation of DSPE–PEG–COOH and tirapazamine. First, the absorbance and fluorescence spectra were recorded to investigate the successful encapsulation of TDPP@PEG–TPZ. The ¹H NMR spectrum and GPC of TDPP are shown in Fig. S3 and S4 (ESI†), and the ¹H NMR spectrum of PEG–TPZ is presented in Fig. S5 (ESI†). As shown in Fig. 1a, TDPP in THF shows absorption intensity maxima of 368 and 644 nm, while the NPs present two broad absorption peaks at 378 and 674 nm. In the emission spectra, TDPP@PEG–TPZ shows emission intensity maxima at 711 and 817 nm (Fig. 1b), while the NPs show maxima at 712 and 820 nm. The red-shift in the UV-vis and fluorescence spectra was caused by the aggregation of these NPs in water. The near-infrared absorbance of the NPs indicates the deep penetration depth of the photosensitizer. Additionally, the transmission electron microscopy (TEM) (Fig. 1c) image is consistent with dynamic light scattering (DLS) results of TDPP@PEG–TPZ with a mean size of 65 nm (Fig. 1d). The drug loading content (DLC) and drug loading efficiency (DLE) are 16.7% and 50.0%, respectively. DSPE–PEG encapsulated TDPP NPs (TDPP@DSPE–PEG) was chosen as the control group. Similarly, TEM and DLS indicate the spherical morphology with an average diameter of 92 nm (Fig. S6, ESI†). The stability of TDPP@PEG–TPZ under physiological condition was investigated by measuring the DLS in PBS, FBS and DMEM at different time points (0, 24 and 48 h). The results suggests the high stability of TDPP@PEG–TPZ because the diameter remained almost unchanged (Fig. S7–S9, ESI†).

Fig. 1 (a) Absorbance spectra of TPZ, PEG–TPZ and TDPP@PEG–TPZ NPs in water. (b) NIR-II fluorescence spectra of TDPP@PEG–TPZ NPs. (c) TEM of TDPP@PEG–TPZ NPs. (d) DLS of TDPP@PEG–PTZ NPs.

ROS generation, photothermal conversion efficiency and pH triggered TPZ release

Ideal photosensitizers should possess high ROS generation ability and photothermal conversion efficiency. Singlet oxygen sensor green (SOSG) was used to detect the ROS generation of TDPP@PEG–TPZ NPs with or without laser irradiation (Fig. 2a). The fluorescence intensity of SOSG was enhanced four-fold in the presence of TDPP@PEG–TPZ, while those for SOSG only or TDPP@PEG–TPZ without irradiation were almost negligible, indicating the strong ROS generation ability of these NPs with laser irradiation. In addition, the triplet peaks observed in a 1 : 1 : 1 ratio confirmed the production of ¹O₂ under laser irradiation of TDPP@PEG–TPZ NPs (Fig. 2b). The photothermal conversion efficiency was investigated by recording the heating and cooling curve of TDPP@PEG–TPZ with or without irradiation (Fig. 2c). A considerable temperature elevation (ΔT) of 41.8 °C was observed when TDPP@PEG–TPZ NPs were irradiated with a laser (808 nm, 0.5 W cm⁻²), indicating an extremely high photothermal conversion efficiency of 58.6%, according to the linear fitting of time versus −ln θ curve (Fig. S10, ESI†).

Fig. 2 (a) Relative fluorescence intensity of SOSG only and of TDPP@PEG–TPZ with or without laser irradiation. (b) ESR spectra of TEMP in the presence of TDPP@PEG–TPZ with irradiation. (c) Heating and cooling curves of water versus TDPP@PEG–TPZ NPs with laser irradiation. (d) TPZ release by the cleavage of the acylamide bond under different pH conditions.

The pH-controlled TPZ release was investigated under either neutral or acidic conditions, since the acylamide bond in the prodrug can be hydrolyzed in acidic conditions (Fig. 2d). Under neutral conditions (pH 7.4), the TPZ release within 36 h was rather low (<18%), while at pH 6.5, efficient TPZ release (>80%) was observed within 36 h because of the cleavage of the acylamide bond, indicating the potential for pH-controlled TPZ release in the tumor microenvironment. This phenomenon is attributed to the fact that the two oxygen atoms of TPZ with their strong electron withdrawing ability can enhance the acidity of the acylamide bond and promote its hydrolysis even at pH 6.5.

In vitro cellular uptake, cell viability, and ROS generation

A confocal live cell imaging system and flow cytometry were used to study the cellular uptake behavior of TDPP@PEG–TPZ NPs. Strong and homogenous fluorescence intensity in the cytoplasm (red fluorescence) was observed following 24 h incubation, indicating the ability of TDPP@PEG–TPZ NPs for endosome escape as well as the suitability of such NPs for cell imaging and therapy (Fig. 3a). 2′,7′-dichlorofluorescein diacetate (DCF-DA), a commercial probe, was used to investigate the ROS generation of TDPP@PEG–TPZ NPs in 143B cells. Strong green fluorescence was observed upon excitation at 488 nm (Fig. 3a), indicating the efficient singlet oxygen generation of TDPP@PEG–TPZ NPs under irradiation in vitro. Moreover, flow cytometry was used to detect the fluorescence intensity after incubation with TDPP@PEG–TPZ NPs at 1, 2, 4, and 8 h, respectively. The fluorescence enhancement also suggests the efficient cellular uptake of TDPP@PEG–TPZ NPs by 143B cells (Fig. S11, ESI†). To evaluate the inhibition of angiogenesis by TDPP@PEG–TPZ, we validated it via microcapillary formation assay using HUVECs (Fig. S12, ESI†).

Fig. 3 (a) Cellular uptake of TDPP@PEG–TPZ NPs and ROS generation with DCF-DA as a probe using confocal imaging. (b) MTT assay of TDPP@DSPE–PEG NPs with/without irradiation using 143B cells. (c) MTT assay of TPZ under normoxic and hypoxic conditions. (d) MTT assay of TDPP@PEG–TPZ with/without irradiation. (e) MTT assay of TDPP@PEG–TPZ on normal cells, including HEK-293 and HUVEC. (f) Flow cytometry of 143B cells using Annexin V/PI co-staining to determine the apoptosis of the groups treated with TDPP@DSPE–PEG (laser−/laser+), TDPP@PEG–TPZ (laser−/laser+) and TPZ.

To validate the phototherapeutic effects of TDPP@PEG–TPZ NPs, we evaluated the cytotoxicity of TDPP@PEG–TPZ with/without irradiation. Additionally, the cell viability of TPZ in normoxic and hypoxic conditions was also investigated. After treatment of 143B cells with NPs for 24 h, the dark toxicity was negligible, while the phototoxicity was considerably high, with an IC₅₀ of 13.84 μg mL⁻¹ (Fig. 3b). The IC₅₀ values of TPZ under normoxic and hypoxic conditions are 112.5 and 15.33 μg mL⁻¹, respectively (Fig. 3c). The superior cytotoxicity of TDPP@PEG–TPZ was confirmed using the MTT assay, in which the oxygen consumption in 143B cells by PDT resulted in a decrease in the intracellular oxygen level, thus activating the chemotherapeutic effect of TPZ (Fig. 3d). We then investigated the cytotoxicity of TDPP@PEG–TPZ NPs on normal cells, including HEK-293 (human embryonic kidney 293 cells) and HUVEC. Similarly, the TDPP@PEG–TPZ NPs show very low dark toxicity in normal cells (Fig. 3e).

An annexin V-FITC/PI co-staining assay was further used to confirm the pro-apoptotic effect of TDPP@PEG–TPZ NPs or TPZ. The results showed that the apoptosis rates of 143B cells in the TDPP@DSPE–PEG(Laser−) (3.99%) group were similar to that of the control group (2.32%), suggesting low dark toxicity. In comparison, significant apoptosis of the TDPP@DSPE–PEG (Laser+) group (44.3%) demonstrated that TDPP@DSPE–PEG NPs could induce typical apoptosis in 143B cells (Fig. 3g). In addition, the apoptosis of those treated with TPZ under hypoxia was obvious (33.8%), and similar to that of those treated with TDPP@PEG–TPZ NPs (33.8%). Among the six groups, TDPP@PEG–TPZ NPs (Laser+) treatment induced the most efficient cell apoptosis (55.5%), indicating the synergistic effect of this combination (Fig. 3f).

In addition, calcein-AM/PI co-staining was performed to further assess the in vitro therapy effects. No apparent red fluorescence was detected in the control or TDPP@PEG–TPZ(Laser−) groups, while a small amount of red fluorescence could be observed in the TPZ and TDPP@PEG–TPZ(Laser−) groups. The effects of TDPP@PEG–TPZ NPs on HUVEC microcapillary formation were determined as previously described. The results indicated that TDPP@PEG–TPZ NPs can effectively inhibit angiogenesis with laser irradiation, compared with the control group (Fig. S12, ESI†). After laser irradiation, the TDPP@PEG–TPZ(Laser+) group showed the greatest cell death, indicating its excellent synergistic antitumor effect. Additionally, calcein-AM/PI co-staining was performed to directly test the ROS generation and apoptosis (Fig. S13, ESI†). The results were consistent with those of flow cytometry. Overall, TDPP@PEG–TPZ achieved a synergistic effect of phototherapy and hypoxia-activated chemotherapy.

In vivo NIR-II-fluorescence-imaging-guided phototherapy-boosted chemotherapy

NIR-II fluorescence imaging, which has a high signal-to-noise ratio and low tissue scattering was used to investigate the uptake of TDPP@PEG–TPZ NPs in vivo. At different time intervals after the intravenous injection of TDPP@PEG–TPZ NPs (200 μg mL⁻¹, 200 μL), fluorescence images were recorded (Fig. 4a). The fluorescence signals of the tumors increased rapidly and remained high from 1 to 48 h after injection, suggesting that TDPP@PEG–TPZ NPs could effectively accumulate at the tumor sites due to the enhanced permeability and retention (EPR) effects. At 24 h post-injection, the fluorescence intensity reached its maximum level, suggesting that 24 h may be a suitable time point for phototherapy after administration. Furthermore, the fluorescence intensity of tumor was still high 48 h after injection, indicating that TDPP@PEG–TPZ NPs can be used as a long-term fluorescence imaging agent. Finally, the mice were sacrificed, and the bio-distribution showed that the NPs remained in the tumor, as indicated by the fluorescence intensity (Fig. 4b).

Fig. 4 (a) In vivo NIR-II fluorescence imaging of 143B tumor-bearing mice at different time points, and bio-distribution of TDPP@PEG–TPZ NPs in the main tissues (tumor, heart, liver, spleen, lung, and kidney) after i.v. injection. (b) Relative fluorescence intensity of the tumor, heart, liver, lung, spleen and kidneys with the intensity of the tumor as the control. (c) In vivo photothermal imaging of the tumor after injection with saline or TDPP@PEG–TPZ NPs under laser irradiation. (d) Tumor temperature change curve for saline or TDPP@PEG–TPZ NPs under laser irradiation.

The high photothermal conversion efficiency of TDPP@PEG–TPZ NPs promises high photothermal therapeutic efficacy in vivo. 24 h after the injection of TDPP@PEG–TPZ NPs, photothermal imaging pictures were recorded at different time intervals (0, 2, 4, 6, 8 min) (Fig. 4c and d). Fig. 4c showed that the temperature of the tumor site increased by only 4.8 °C in the saline group, which was almost negligible. In contrast, the tumor surface temperature of the mouse that was injected with TDPP@PEG–TPZ NPs increased to 58.6 °C (ΔT = 21.8 °C) under the same conditions; this temperature is high enough to kill the tumor cells.

Then, 30 nude mice bearing osteosarcoma tumors were divided into groups to assess the PDT/PTT effects of TDPP@PEG–TPZ NPs and the synergistic therapeutic efficacy combined with chemotherapy of TPZ in this study. Due to the high photothermal conversion efficiency in water, photothermal imaging was conducted to show the temperature elevation to assess the photothermal therapy efficacy of TDPP@PEG–TPZ NPs in vivo. The in vivo fluorescence imaging results showed that 8 h post-injection is the most appropriate time point for phototherapy. Therefore, the mouse was irradiated with the laser for 0, 2, 4, 6, and 8 min at 8 h post-injection, and the infrared imaging was collected. Fig. 4c shows that the temperature of the tumor site increased by only 4.8 °C in the saline group, which was almost negligible. In contrast, the tumor surface temperature of the mouse injected with TDPP@PEG–TPZ NPs increased to 58.6 °C (ΔT = 21.8 °C) under the same conditions; this temperature is high enough to kill the tumor cells.

To further investigate the synergistic therapeutic efficacy of TDPP@PEG–TPZ in vivo, 30 nude mice bearing osteosarcoma tumors were divided into 6 groups, and their tumor volume and body weight were recorded every two days (Fig. 5a and b). The tumor volume in the TDPP@DSPE–PEG only group was similar to that of the control group, indicating the low dark toxicity of the TDPP@DSPE–PEG NPs themselves. In contrast, significant tumor suppression was observed for TDPP@DSPE–PEG and TDPP@PEG–TPZ with the help of the laser. The therapeutic efficacy of the TDPP@PEG–TPZ NPs is superior to that of all the other groups, suggesting the effectiveness of the synergistic therapy. The nude mice of all six groups were then sacrificed. Photographs of the tumors are shown in Fig. S14 (ESI†). The results showed that after laser irradiation, the photothermal and photodynamic effects were activated. A large amount of oxygen in the tumor was consumed, resulting in aggravated hypoxia and stimulating the chemotherapeutic effect of TPZ, thus achieving the effect of multiple combination therapy.

Fig. 5 (a) Relative tumor volume. (b) Body weight change of the mice (**P < 0.01). (c) H&E, TUNEL, HIF-1α and Ki67 staining pictures of the tumors from the control, TDPP@DSPE–PEG (laser−/laser+), TPZ and TDPP@PEG–TPZ(laser−/laser+) NP groups.

In addition, the weight of the mice increased throughout the entire treatment process, and there was no significant difference compared with the control group, indicating that TDPP@PEG–TPZ has good biosafety and no apparent toxic or side effects. After the treatment, all the mice were sacrificed and the tumors were collected, along with the normal organs, including the heart, liver, spleen, lung, and kidneys. According to the hematoxylin and eosin (H&E) stained images of the tumors, the nucleus was damaged in the TPZ, TDPP@DSPE–PEG + Laser, and TDPP@PEG–TPZ with/without laser groups (Fig. S15, ESI†). In contrast, those in the control groups remained undamaged. Further TUNEL results indicate that the amount of positive cells increased significantly in the tumor tissues subjected to TDPP@DSPE–PEG(Laser+) and TDPP@PEG–TPZ(Laser+) treatment compared with those of the control group (Fig. 5c). The results indicated that the TDPP@DSPE–PEG NPs, as well as TDPP@PEG–TPZ, exerted anti-tumor activity without any toxicity in vivo. The enhanced therapeutic efficacy may be attributed to the fact that photodynamic therapy of TDPP@PEG–TPZ NPs consumes the intracellular oxygen and induces tumor hypoxia, which was also confirmed by the HIF-1α staining results (Fig. 5c). The damage to the blood vessels may also be responsible for the aggravated hypoxia, and this could be verified by the Ki67 staining results (Fig. 5c). The results also confirmed the biocompatibility of TDPP@PEG–TPZ NPs. Further, hematological parameters, including white blood cells, lymphocytes, monocytes, eosinophils, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet, and basophils were investigated (Fig. S16, ESI†). The results demonstrated that TDPP@PEG–TPZ NPs did not exert any adverse effects, because the parameters in the treatment group were similar to those in the control group. The optical density of immunohistochemistry was measured using Image J software. As shown in Fig. S17 (ESI†), compared with the control group, the HIF-1α in the TDPP@DSPE–PEG (Laser+) and TDPP@PEG–TPZ (Laser+) groups was significantly improved (**P < 0.01), while the Ki67 in the TDPP@DSPE–PEG (Laser+) and TDPP@PEG–TPZ (Laser+) groups was significantly decreased (**P < 0.01). These results exemplified the excellent bio-compatibility of TDPP@PEG–TPZ NPs.

Tumor hypoxia leads to advanced but dysfunctional vascularization and acquisition of the epithelial-to-mesenchymal transition phenotype, resulting in cell mobility and metastasis. This process is usually slow and mainly occurs in advanced tumors. In this paper, we established a subcutaneous tumor model. The entire treatment cycle is three weeks. Usually, osteosarcoma is prone to lung metastasis, but after the end of the experiment, no apparent metastasis phenomenon was found. During the treatment, we discovered that hypoxia activated the therapeutic effects of TPZ, overcoming some of the deficiencies caused by hypoxia.

Conclusions

In summary, we designed and synthesized an intelligent TME-responsive nanosystem, TDPP@PEG–TPZ, for phototherapy-boosted chemotherapy. The intelligent nano-platform leads to vascular damage via both ROS generation and the hyperthermia effect. Oxygen consumption during the PDT process also increases the tumor hypoxia, thus activating the chemotherapy of TPZ. As expected, the NIR-II fluorescence imaging TDPP@PEG–TPZ NP guided therapy leads to tumor regression with the help of laser irradiation, providing a paradigm to design intelligent nanoformulations for cancer theranostics and pre-clinical applications.

Author contributions

J. Zhu, Z. Wu, Q. Zhang, J. Zou, and X. Chen conceived the study. J. Zhu, J. Zou and X. Chen wrote and revised the manuscript. J. Zhu and J. Zou synthesized and characterized the materials. J. Zhu, Y. Zhang, Z. Li and X. Bao performed the in vivo NIR-II imaging and therapy. Y. Zhou and B. Ma did the in vitro cell culture and cellular uptake. Y. Xie and P. Yan performed the flow cytometry and analysed the data.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Nanjing International Science and Technology Cooperation Program (No. 201911015), National University of Singapore Startup Fund (NUHSRO/2020/133/Startup/08), NUS School of Medicine Nanomedicine Translational Research Programme (NUHSRO/2021/034/TRP/09/Nanomedicine), the National Research Foundation, Singapore, and National Medical Research Council, Singapore under its NMRC Centre Grant Programme (CG21APR1005) and Open Fund Young Individual Research Grant of Singapore (OFYIRG, MOH-001127-01)

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3mh00242j

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