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Hypoxia-responsive core-cross-linked supramolecular nanoprodrug based on dendritic drug-drug conjugates for synergetic anticancer therapy

Journal of Nanobiotechnology volume 23, Article number: 316 (2025) Cite this article

Abstract

Background

Recently, the strategy of self-assembling dendritic drug-drug conjugates into supramolecular nanoprodrug was widely explored in biomedical applications. Herein, we construct a hypoxia-responsive core-cross-linked supramolecular nanoprodrug (CSN-IR806/CB) based on a dendritic drug-drug conjugate.

Methods

We prepared a hypoxia-responsive dendritic drug-drug conjugates IR806-(Azo-CB)4, which was combined with β-cyclodextrin-pendant poly(ethylene glycol)-block-poly(glutamic acid) block copolymer (PEG-PGlu-CD) to construct the core-cross-linked supramolecular nanoprodrug (CSN-IR806/CB) with enhanced physiological stability through the synergy of π-π stacking interaction, host-guest complexation, hydrogen bonds, and hydrophobic interaction.

Results

The near-infrared (NIR) light irradiation of the CSN-IR806/CB treated tumor cells induced IR806-mediated PDT and PTT, and aggravated hypoxia, which triggered the disassembly of CSN-IR806/CB and the subsequent release of activated CB for synergetic cancer cell killing.

Conclusions

The CSN-IR806/CB can realize a synergistic triple therapeutic effect of photothermal therapy (PTT), photodynamic therapy (PDT), and chemotherapy (CT; i.e., PTT-PDT-CT).

Background

Nanomedicine-based drug delivery systems (DDSs), which possess some advantages of simultaneous delivering multiple drugs, enhancing drug solubility, and prolonging blood circulation, can effectively deliver anticancer drugs to tumor site via the enhanced permeation and retention (EPR) effect, and then specifically release anticancer drugs in tumor site [1,2,3,4,5]. Among them, nanoprodrug has received increasing attention owing to higher drug-loading efficiency and lower potential systematic toxicity, which is established through various conjugation approaches such as polymer-drug conjugates and drug-drug conjugates [6,7,8,9]. The drug-drug conjugates are always synthesized by coupling two or more types of drug molecules through stimuli-responsive linkages, which can be disintegrated under corresponding stimulus to release activated drugs [10,11,12].

Especially, dendrimer is emerging as promising drug-drug conjugates on account of its unique dendritic structure and multivalent cooperativity, resulting from its highly branched yet precisely controllable architecture [13, 14]. However, the dendritic drug-drug conjugates still present certain obstacles for constructing nanoprodrug, such as complex synthesis process, uncontrolled drug release behaviors, and the limited selection of anticancer drugs [15]. In light of this, the introduction of macrocyclic molecules with a cavity may provide an available strategy for improving the above issues by encapsulating the dendrimer-based drug-drug conjugate via host-guest complexation [16,17,18,19]. Against this background, supramolecular dendritic drug-drug conjugates show great potential for the development of desired nanomedicine.

Inspired by the key characteristic of hypoxia in numerous solid tumors, which is caused by rapid cell proliferation and insufficient neovasculature, a series of bio-reductive prodrugs containing hypoxia-responsive linkers have been employed in hypoxic imaging and therapy [20,21,22,23,24]. Azobenzene (AZO) linker is the most widely used function group in the design of hypoxia-responsive DDSs, which can be reduced to aniline group in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and azo reductase under hypoxic tumor microenvironment [25,26,27,28]. In addition, photodynamic therapy (PDT) is generally combined with the hypoxia-responsive DDSs for that PDT can aggravate the underlying hypoxia by employing photosensitizers to facilitate the conversion of oxygen into cytotoxic reactive oxygen species (ROS) under light irradiation [29,30,31].

Herein, we rationally designed a hypoxia-responsive dendritic drug-drug conjugates IR806-(Azo-CB)4, which was synthesized by appending chlorambucil (CB) to the terminals of IR806-containing dendritic structure via azobenzene linkages with the aim of achieving hypoxia-responsive drug release under hypoxic conditions. Subsequently, IR806-(Azo-CB)4 self-assembled into core-cross-linked supramolecular nanoprodrug CSN-IR806/CB combined with β-cyclodextrin grafted PEG-block-poly(glutamic acid) block copolymer (PEG-PGlu-CD) via the synergy of π-π stacking interaction, host-guest complexation, hydrogen bonds, and hydrophobic interaction (Scheme 1). Outstandingly, the host-guest complexation between β-cyclodextrin and chlorambucil (CB) endowed CSN-IR806/CB with a cross-linked core, which greatly enhanced the stability of this supramolecular nanoprodrug. After internalization of CSN-IR806/CB into HepG2 cells, IR806 mediated photothermal therapy (PTT) and PDT under near-infrared (NIR) light irradiation. Due to the consumption of oxygen, the creating PDT-enhanced hypoxic environment accelerated the release of activated CB through the cleavage of AZO linkers. Therefore, we have successfully fabricated a hypoxia-responsive core-cross-linked supramolecular nanoprodrug CSN-IR806/CB to achieve a synergistic triple therapeutic effect of PTT, PDT, and chemotherapy (CT; i.e., PTT-PDT-CT).

Scheme 1.
scheme 1

Schematic illustration for the fabrication of hypoxia-responsive core-cross-linked supramolecular nanoprodrug CSN-IR806/CB and the mechanism of PTT-PDT-CT

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Materials and methods

Synthesis of Boc-D2-(Azo-CB)4

The Azo-CB-COOH (456.3 mg, 1.25 mmol) and (7-Azabenzotriazol-1-yl)-N, N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) (760 mg, 2 mmol) were dissolved in 10 mL of anhydrous DMF and stirred at 0 °C for 5 h. Following this, DIPEA (348 mg, 2.7 mmol) introduced into the mixture, which was then allowed to warm up to room temperature. Solution of Boc-D2 (105.6 mg, 0.125 mmol) in 5 mL dry DMF was added gradually while stirring continued for an additional forty-eight hours. The reaction was terminated by adding water and subsequently extracted with dichloromethane (DCM). The organic phase was dried using sodium sulfate before removing the solvent under reduced pressure. Finally, the crude product underwent purification through column chromatography on silica gel yielding an orange-red solid Boc-D2-(Azo-CB)4 (128.3 mg. 46.2% yield).

Synthesis of IR806-(Azo-CB)4

Initially, trifluoroacetic acid (TFA) (1 mL) was added slowly to solution of Boc-D2-(Azo-CB)4 (130 mg, 0.058 mmol) in 1 mL DCM and stirred for 2 h. The solvent along with any unreacted TFA was then evaporated under reduced pressure, resulting in an orange-red solid of D2-(Azo-CB)4 after vacuum drying. Next, IR806 (80 mg, 0.1 mmol) and HATU (58 mg, 0.15 mmol) were dissolved in 5 mL dry DMF and stirred for 3 h at 0 ℃. DIPEA (0.5 mL, 0.003 mmol) was introduced into the mixture which was then allowed to warm up to room temperature. A solution of D2-(Azo-CB)4 (217.5 mg, 0.097 mmol) in 2 mL dry DMF was added dropwise into the front solution while stirring continued for another 24 h. The resulting solution was placed into a dialysis tube (MWCO 2000 Da), dialyzed against DMSO for twenty-four hours followed by ultrapure water for an additional twenty-four hours before being freeze-dried to yield IR806-(Azo-CB)4 (242.67 mg, 86.3% yield).

Synthesis of PEG-PBGlu

PEG-NH2 (187 mg, 0.037 mmol) was used as the initiator and added to a solution of Bn-Glu-NCA (300 mg, 1.28 mmol) in 4 mL of dry DMF while stirring continuously at room temperature for 48 h under a nitrogen atmosphere. The reaction mixture was then precipitated by adding diethyl ether (20 mL) to collect the white precipitate via centrifugation. This precipitate was dried overnight in a vacuum oven to yield PEG-PBGlu (196.6 mg, 86.4%).

Synthesis of PEG-PGlu-CD

PEG-PBGlu (100 mg, 0.074 mmol) was dissolved in 20 mL of a 1 M sodium hydroxide solution and stirred vigorously while refluxing at 100 °C overnight. The resulting reaction mixture was then placed into dialysis tubing and dialyzed against deionized water for 48 h to eliminate sodium hydroxide and dissociated protective groups, yielding PEG-PGlu (70.4 mg, 85% yield).

Subsequently, PEG-PGlu (50 mg, 0.0074 mmol), EDC (12 mg, 0.077 mmol), DMAP (5 mg, 0.04 mmol), and β-Cyclodextrin (106.3 mg, 0.094 mmol) were dissolved in 5 mL dry DMF. The mixture was then stirred for an additional twenty-four hours at room temperature. Finally, the reaction mixture was precipitated by adding diethyl ether (20 mL) to collect the white solid sediment of PEG-PGlu-CD (91.96 mg, 35.3% yield).

Fabrication of core-cross-linked supramolecular nanoprodrug CSN-IR806/CB

Typically, PEG-PGlu-CD (4 mg) and IR806-(Azo-CB)4 (2 mg) was dissolved in 1 mL of DMF. Subsequently, 5 mL of PBS was added to the solution while stirring vigorously overnight. The resulting mixture was then placed into a dialysis tube (MWCO 3500 Da) and dialyzed against PBS to yield CSN-IR806/CB.

Detection of 1O2 generation

ABDA (0.05 mg/mL) was introduced into 20 mL of PBS containing CSN-IR806/CB. To trigger the release of 1O2 using NIR, this solution was exposed to NIR irradiation at 808 nm with an intensity of 1.0 W/cm² for 5 min. Due to ABDA’s ability to effectively capture ROS through a rapid reaction with its anthracene moiety, the initial UV-Vis absorption of ABDA would gradually decrease in the presence of 1O2.

In vitro drug release

A solution of CSN-IR806/CB (1 mg/mL) was mixed with varying concentrations of Na2S2O4 at 1.6, 3.2, 6.4, 12.8, or 25.6 mM. After stirring the mixture for 2 h, it was placed in a dialysis bag (MWCO: 500 Da) and immersed in methanol (15 mL) while stirring for 24 h. The methanol solution outside the dialysis bag was then evaporated and analyzed using HPLC.

In vitro cytotoxicity

HepG2, HeLa, and MCF-7 cells were seeded in a 96-well plate at a density of 1 × 104 cells per well (200 µL) and incubated in DMEM for 12 h. After this period, the medium was replaced with fresh DMEM containing varying concentrations of CSN-IR806/CB and incubated for an additional 6 h under either hypoxic or normoxic conditions. For the irradiation groups, the cells received NIR exposure (808 nm, 1.0 W/cm², for 5 min), followed by another incubation period of 18 h. The cytotoxicity was then assessed using MTT assays. In addition, live/dead staining was performed on HepG2 cells using fluorescein diacetate and propidium iodide before imaging them with a fluorescent microscope.

Cell internalization

HepG2 cells were plated in a 12-well plate at a density of 1.0 × 105 cells per well and incubated in DMEM for 12 h. Following this, the medium was replaced with fresh DMEM containing either CSN-IR806/CB or free IR806 at a concentration of 4 µg/mL. After incubation for either 1–4 h under hypoxic or normoxic conditions, the cells were stained with DAPI for 20 min and then observed using a confocal laser scanning microscope (CLSM).

Intracellular ROS generation

HepG2 cells were plated in a 12-well plate at a density of 1.0 × 105 cells per well and incubated in DMEM for 12 h. Subsequently, the medium was replaced with fresh DMEM containing CSN-IR806/CB at an IR806 concentration of 4 µg/mL. After a 4 h incubation period, the irradiation groups received NIR exposure (808 nm, 1.0 W/cm², for 5 minutes). Following this, the cells were stained with 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 min and examined using a fluorescence microscope.

In vivo biodistribution

The experimental protocol was approved by the Nantong University Institutional Animal Care and Use Committee. Nude mice with HepG2 tumors (approximately 80 mm³) were randomly assigned to three groups (n = 3) and treated intravenously with either free IR806, free CB, or CSN-IR806/CB, using a dose of 5 mg/kg for CB or 4.2 mg/kg for IR806. At specified time points, in vivo tumor accumulation and biodistribution were assessed using a Kodak multimode imaging system. Major organs and tumors were collected at 12 h and 24 h post-injection for ex vivo distribution imaging. The amount of IR806 was extracted and quantified through fluorescence spectroscopy.

In vivo antitumor activity

Nude mice bearing HepG2 tumors (60–70 mm³) were randomly assigned to five groups (n = 4) and treated intravenously with PBS, free CB, free IR806 + L, CSN-IR806/CB, or CSN-IR806/CB + L at CB equivalent dose of 5 mg/kg and IR806 dose of 4.2 mg/kg on day 0 and day 5. Mice in the IR806 + L and CSN-IR806/CB + L groups underwent NIR irradiation (808 nm, 1.0 W/cm² for 5 min) 12 h after injection. Tumor size and body weight were monitored at designated time points. The tumor volume (V) was calculated according to equation: V = 0.5 × Length× Width2, and the tumor inhibitory rates (TIR) was calculated by equation: TIR (%) = 100 × (mean V of PBS group - mean V of others)/(mean V of PBS group).

Statistical analysis

All data were provided as mean ± standard deviation (SD). The statistical significance of different groups was given by using Student’s t-test.

Results and discussion

Synthesis and characterization of core-cross-linked supramolecular nanoprodrug CSN-IR806/CB

The hypoxia-responsive dendritic drug-drug conjugates IR806-(Azo-CB)4 was successfully synthesized by appending the chlorambucil (CB) to the IR806-initial-modified dendrimer terminals via hypoxia-cleavable azobenzene linkages with the aim of achieving controllable drug release, and the PEG-PGlu-CD was prepared by grafting β-cyclodextrin onto poly(ethylene glycol)-block-poly(glutamic acid) via biodegradable ester bonds (Additional file 1: Fig. S1). The relevant nuclear magnetic resonance spectroscopy illustrated in Additional file 1: Fig. S2-S5, was confirm the structure of IR806-(Azo-CB)4 and PEG-PGlu-CD. Additionally, the appearance of characteristic absorption peaks of Azo-CB and IR806 in UV-vis spectrophotometry and fluorescence spectra were both further approved the successful synthesize of IR806-(Azo-CB)4 (Fig. 1A-C).

Fig. 1
figure 2

(A) The UV-vis absorption spectra of Azo-CB, free IR806, IR806-(Azo-CB)4, PEG-PGlu-CD, and CSN-IR806/CB. (B) Fluorescence spectra of Azo-CB, IR806-(Azo-CB)4, and CSN-IR806/CB at the excitation wavelength of 225 nm. (C) Fluorescence spectra of free IR806, IR806-(Azo-CB)4, and CSN-IR806/CB at the excitation wavelength of 440 nm. (D) DLS data, (E) TEM image and (F) SEM image of CSN-IR806/CB

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Sequentially, IR806-(Azo-CB)4 and PEG-PGlu-CD were self-assembled into core-cross-linked supramolecular nanoprodrug CSN-IR806/CB with ultrahigh stability via intra- and intermolecular noncovalent interactions, e.g. π-π stacking interaction, host-guest complexation, hydrogen bonds, and hydrophobic interaction [32, 33]. The PEG component functioned as the outer hydrophilic shell to enhance stability, inhibit non-specific adsorption, and prolong the blood circulation time [34]. While loading dendritic drug-drug conjugates IR806-(Azo-CB)4, the formation of crosslinked core tremendously enhanced the stability of CSN-IR806/CB and avoided the premature leakage of drugs during the blood circulation. The coexistence of Azo-CB and IR806 in supramolecular nanoprodrug was confirmed by the characteristic absorption peaks at 273, 417, and 805 nm in UV-vis absorption spectra of CSN-IR806/CB (Fig. 1A). The drug loading efficiency of IR806 in CSN-IR806/CB was determined to be 10.6% according to the UV-vis absorbance, and then the drug loading efficiency of CB was calculated to be 12.7% according to the chemical structure of IR806-(Azo-CB)4. Additionally, fluorescence spectra of TAPP and Azo-CB were detected inside CSN-IR806/CB. As shown in Fig. 1B, C, significant fluorescence of Azo-CB and IR806 were observed in CSN-IR806/CB upon excitation at 225 nm and 440 nm, respectively.

Subsequently, the size distribution and morphology of CSN-IR806/CB were investigated using dynamic light scattering (DLS), scanning electron microscope (SEM), and transmission electron microscopy (TEM) analyses. As shown in Fig. 1D-F, the hydrodynamic diameter (Dh) of CSN-IR806/CB was 205.3 ± 0.96 nm with a PDI of 0.145 ± 0.03, while the TEM and SEM image both exhibited uniform spherical morphology of CSN-IR806/CB with average diameter of 128.6 ± 6.3 nm and 135.8 ± 5.5 nm, respectively. Outstandingly, the core-shell structure of CSN-IR806/CB was clearly observed in the enlarged TEM image, which showed an outer PEG layer of about 10 nm and a cross-linked core with a diameter of approximate 92 nm (Fig. 1E). Furthermore, the biostability of SNP was assessed by measuring Dh in phosphate buffered saline (PBS) solution at 37 °C with 5%, 10%, or 15% fetal bovine serum (FBS) (Fig. 2A). The absence of significant changes in Dh within 24 h indicated the excellent biostability of CSN-IR806/CB under simulated physiological environments [35].

Fig. 2
figure 3

(A) Hydrodynamic diameter distribution of CSN-IR806/CB incubated in different media for 24 h. (B) Photothermal heating effects and (C) infrared thermal images of PBS, free IR806, and CSN-IR806/CB solutions under 808 nm NIR irradiation (1.0 W/cm2, 5 min). Photothermal heating effects of CSN-IR806/CB solution (D) under various power intensities or (E) with different concentrations. (F) Photothermal cycle curves of CSN-IR806/CB (40 µg/mL IR806 equiv.) under NIR irradiation (808 nm, 1.0 W/cm2, 5 min) for four cycles

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Photothermal and photodynamic properties

Considering the NIR photothermal responsiveness of IR806-based materials, the photothermal properties of CSN-IR806/CB was subsequently examined by monitoring temperature fluctuations in water solutions under 808 nm NIR irradiation. As depicted in Fig. 2B, C, the maximum temperature increment (ΔT) of CSN-IR806/CB solutions (40 µg/mL IR806 equiv.) rapidly increased to 21.6 °C under NIR irradiation (808 nm, 1.0 W/cm2, 5 min), which was obviously higher than that of the PBS control solution (3.8 °C) and free IR806 (14.6 °C) under the same NIR irradiation condition. In comparison to ΔT of free IR806, the ΔT increases of CSN-IR806/CB can be attributed to the enhanced π-π stacking interactions between IR806-(Azo-CB)4 molecules within the supramolecular nanoprodrug CSN-IR806/CB, which can promote the energy relaxation [35]. Meanwhile, the photothermal effect of CSN-IR806/CB exhibited a dependence on both concentration and laser power intensity (Fig. 2D, E). Notably, PTT performance of CSN-IR806/CB remained stable after four cycles of repeated NIR irradiation (808 nm, 1.0 W/cm2, 5 min), indicating excellent photostability (Fig. 2F). Based on the linear relationship between the cooling period of 808 nm laser off and ln (θ), the PTT conversion efficiency (η) of CSN-IR806/CB was calculated to be 33.4% (Additional file 1: Fig. S6) [36]. Consequently, the supramolecular nanoprodrug CSN-IR806/CB exhibit remarkable photothermal effect and photostability, making it highly suitable for in vitro and in vivo PTT.

Fig. 3
figure 4

(A) Absorption spectra of ABDA after NIR irradiation by 1O2 generated from CSN-IR806/CB. (B) ESR analysis of 1O2 production at the predetermined time points. (C) HPLC analysis of CB release triggered by different concentrations of Na2S2O4. (D) Size changes of CSN-IR806/CB incubated with Na2S2O4 of 25.6 mM. TEM images of CSN-IR806/CB incubated with Na2S2O4 of 6.4 mM for (E) 0.5 h, (F) 2 h, and (G) 4 h

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Sequentially, the 1O2 generation ability of CSN-IR806/CB under NIR irradiation was evaluated by using 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA) as probe, which could specifically react with 1O2 and then induce the decrease in its characteristic absorbance peaks [37]. Upon NIR irradiation (808 nm, 1.0 W/cm2, 5 min) of the CSN-IR806/CB solution containing ABDA, the corresponding absorption intensities of ABDA at 397 nm decreased rapidly, indicating the efficient production of 1O2 (Fig. 3A). By comparison, CSN-IR806/CB had the similar 1O2 generation efficacy as free IR806, suggesting that the 1O2 generation ability of photosensitizer IR806 was not affected by the conjugation with CB and loading in supramolecular nanoprodrug (Additional file 1: Fig. S7). Furthermore, the 1O2 generation capability of CSN-IR806/CB under 808 nm NIR irradiation was estimated by electron spin resonance (ESR) spectroscopy using 2, 2, 6, 6-tetramethylpiperide (TEMP) as capture agent. As shown in Fig. 3B, we observed that the intensity of typical 1:1:1 ESR peaks gradually increased within 10 min under NIR irradiation ascribed to the characteristic triplet signal of TEMP/1O2 adduct (TEMPO), which was in accordance with the results of UV-vis absorbance [38]. Overall, these results validated the outstanding photodynamic property of CSN-IR806/CB.

Hypoxia-responsive drug release and property of CSN-IR806/CB

As for the reduction of an azobenzene linker to two aniline derivatives is tremendously influenced by the degree of hypoxia during this process, we monitored the hypoxia-responsive drug release by using high-performance liquid chromatography (HPLC) in the presence of sodium dithionite (Na2S2O4) with different concentrations. Here, the Na2S2O4 is generally applied to mimic different hypoxic condition for 2 h [39]. As illustrated in Fig. 3C, following the increase of Na2S2O4 concentration, the peaks representing CB at 11.8 min gradually heightened. Simultaneously, the color of the CSN-IR806/CB solution was changed from green to yellow after the addition of Na2S2O4 (Additional file 1: Fig. S8). These results indicated that the activated CB could be effectively released from CSN-IR806/CB under hypoxic condition. In addition, DLS and TEM were used to measure the corresponding size and morphological changes of CSN-IR806/CB incubated with Na2S2O4 (25.6 mM) for 0.5 h, 1 h, and 2 h (Fig. 3D-G). The hydrodynamic diameter of CSN-IR806/CB distinctly became smaller and its initial spherical shape gradually disintegrated with time on account of the hypoxia-responsive disassembly.

In vitro cytotoxicity

The in vitro anticancer effect of CSN-IR806/CB against HepG2, HeLa, and MCF-7 cells was estimated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Under normoxia without NIR irradiation, the cell viability decreased as the CB and IR806 concentration increased in CSN-IR806/CB and reached approximately 51.7% (HepG2), 48.8% (HeLa), and 53.2% (MCF-7) at a CB concentration and IR806 of 8 µg/mL and 6.68 µg/mL, respectively, indicating an inherent hypoxic environment within these cancer cells (Fig. 4A, S9A, and S10A). Whereas, the CSN-IR806/CB could effectively inhibit cell proliferation and enhance cell apoptosis with a half-maximal inhibitory concentration (IC50) of 1.9 µg/mL (HepG2), 1.76 µg/mL (HeLa), and 2.03 µg/mL (MCF-7) after culturing in hypoxic condition without NIR irradiation, demonstrating that the hypoxia-triggered CB release from CSN-IR806/CB resulted in higher cytotoxicity. Otherwise, the CSN-IR806/CB exhibited the minimum IC50 of 0.71 µg/mL (HepG2), 0.69 µg/mL (HeLa), and 0.72 µg/mL (MCF-7), and the strongest cytotoxicity under hypoxia with NIR irradiation owing to the synergistic triple therapeutic effect of PTT-PDT-CT (Fig. 4B, S9B, and S10B). Despite the partial limitation of PDT efficacy under hypoxia, the decrease in oxygen levels induced a stronger chemotherapeutic effect by increasing CB release, which ultimately offset the weakening of PDT and resulted in minimum cell viability combined with the unaffected PTT effect.

To further validate the cytotoxicity of CSN-IR806/CB, calcein acetoxymethyl ester (calcein-AM) and propidium iodide (PI) were utilized for staining adherent HepG2 cells with green fluorescence indicating live cells and red fluorescence indicating dead cells. As shown in Fig. 4C, CSN-IR806/CB caused increased cell death (red color) when incubated under hypoxic conditions compared to normoxic conditions without NIR irradiation. Upon 808 nm laser irradiation, much more cell death was displayed under hypoxia compared to that under normoxia, demonstrating the NIR irradiation could aggravate hypoxia to boost CD release and realize the synergistic triple therapeutic effect of PTT-PDT-CT.

Fig. 4
figure 5

In vitro cell cytotoxicity of CSN-IR806/CB against HepG2 cells after incubation for 48 h under normoxia or hypoxia (A) in the dark or (B) under NIR irradiation. (C) Live/dead assay of AM/PI co-stained HepG2 cells after various treatments. (D) Fluorescence images of ROS level after different treatments using DCFH-DA as ROS fluorescence probe. (E) CLSM images of HepG2 cells after incubation with free IR806 or CSN-IR806/CB under hypoxia for 1–4 h. (F) Flow cytometric curves of IR806, and (G) the corresponding mean fluorescence intensity after incubation with free IR806 or CSN-IR806/CB under hypoxia for 1–4 h

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In vitro ROS generation, cellular uptake, and ICD

To assess the intracellular ROS levels, HepG2 cells were incubated with CSN-IR806/CB under normoxic/hypoxic conditions with or without NIR irradiation, and the intracellular ROS levels were visualized by using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence probe (Fig. 4D). Both PBS group and CSN-IR806/CB group showed negligible green fluorescence of ROS without NIR irradiation whether under normoxia or hypoxia. Under NIR irradiation, the CSN-IR806/CB-treated cells exhibited a significantly strong green fluorescence, suggesting a high concentration of light-induced ROS generation within the cells. Furthermore, it was observed that the fluorescence in CSN-IR806/CB-treated cells under hypoxic conditions was comparatively weaker than that under normoxic conditions on account of the partial limitation on the efficacy of PDT under hypoxia.

To detect the efficient cellular uptake of CSN-IR806/CB, the internalized behavior of CSN-IR806/CB in HepG2 cells under hypoxia was analyzed by using IR806 as the fluorescent indicator through confocal laser scanning microscopy (CLSM) and flow cytometry. The nuclei displayed blue fluorescence after staining with DAPI, whereas the CSN-IR806/CB containing IR806 emitted red fluorescence. As shown in Fig. 4E, there was a gradual increase in the fluorescence intensity of both CSN-IR806/CB and free IR806-treated HepG2 cells over time. Moreover, the fluorescent intensity of CSN-IR806/CB group was significantly higher than that of free IR806 group, contributed to the more effective cellular uptake of CSN-IR806/CB via endocytosis mechanism compared to passive diffusion [40]. Based on the data of flow cytometry, the mean fluorescent intensity of CSN-IR806/CB group was 4.1 times that of free IR806 group at 4 h (Fig. 4F, G), which was consistent with the quantitative analysis by CLSM.

We next investigated whether CSN-IR806/CB could trigger tumor immunogenic cell death (ICD) effects against HepG2 cells by analyzing several typical biomarkers [41, 42]. The three typical characteristics of ICD were the secretion of adenosine triphosphate (ATP) into the extracellular environment, an increased release of high-mobility group box 1 (HMGB1), and a notable surface exposure of calreticulin (CRT) [43]. As shown in Additional file 1: Fig. S11, the CSN-IR806/CB-treated cells under hypoxia with NIR irradiation exhibited the heaviest release of ATP, CRT, and HMGB1. Therefore, the above results demonstrate that CSN-IR806/CB could effectively induce ICD in cancer cells under hypoxia with NIR irradiation.

In vivo pharmacokinetics, biodistribution, and photomediated imaging

Generally, nanomedicine have the potential to improve the effectiveness of antitumor treatments by prolonging the circulation of therapeutic agents in the bloodstream, leading to increased accumulation at tumor sites. To assess whether CSN-IR806/CB possess this characteristic, we conducted a study on the blood circulation profiles of free IR806 and CSN-IR806/CB. As illustrated in Fig. 5A, free IR806 was rapidly cleared from the bloodstream following intravenous administration. In contrast, CSN-IR806/CB presented significantly prolonged blood circulation time, indicating their potential as a candidate for an effective nanomedicine.

Fig. 5
figure 6

(A) Pharmacokinetics profiles of free IR806 and CSN-IR806/CB. (B) In vivo biodistribution of nude mice after intravenous injection of free IR806 and CSN-IR806/CB at different times. (C) Ex vivo imaging and (D) biodistributions of CSN-IR806/CB group in tumor tissue and major organs at 12 h and 24 h. (E) Infrared thermal images and (F) time-dependent temperature increase of HepG2 tumor-bearing mice under NIR irradiation at selected time intervals

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Sequentially, the noninvasive near-infrared optical imaging technique was utilized to investigate the biodistribution of free IR806 and CSN-IR806/CB (Fig. 5B) at predetermined time points. The intra-tumoral fluorescence was scarcely observed in the mice intravenously administered with free IR806 throughout the entire experimental period, indicating its rapid clearance in the blood circulation. Nevertheless, the mice intravenously injected with CSN-IR806/CB exhibited a progressive increase in intra-tumoral fluorescence over time, and reached the maximum at 12 h, demonstrating that CSN-IR806/CB significantly boosted the IR806 accumulation in tumors. Ex vivo imaging and semi-quantitative assay of major organs (heart, liver, spleen, lung, and kidney) and the tumor was also performed at 12 and 24 h post-injection of CSN-IR806/CB (Fig. 5C, D). At 0.5 h post-injection, CSN-IR806/CB exhibited a higher IR806 fluorescence at the tumor site and liver compared to other organs. At 24 h post-injection, the IR806 fluorescence was obviously weakened at liver induced by the metabolic and detoxification functions of the liver, and still stronger in the tumor region than in other normal organs, confirming the exceptional tumor enrichment capacity of CSN-IR806/CB [44]. Overall, CSN-IR806/CB has the superior ability to passively target tumor tissue and remain localized at the tumor site.

Based on the results of biodistribution, the tumor regions were subjected to NIR irradiation (808 nm, 1.0 W/cm2, 5 min) at 12 h after injection, and the hyperthermia study was performed by a thermal imaging camera (Fig. 5E, F). The control group with PBS injection showed negligible temperature increase with 2.2 °C. The CSN-IR806/CB-injected mice exhibited a significantly higher temperature increase (14.5 °C) compared to the IR806-injected ones (5.3 °C), suggesting enhanced accumulation of CSN-IR806/CB at the tumor site. The temperature of 46.1 °C on the tumor surfaces in CSN-IR806/CB group exhibited a mild photothermia effect, which had been demonstrated to effectively treat cancer without causing damage to the skin [45].

Fig. 6
figure 7

(A) Schematic illustration experiment design for CSN-IR806/CB + L group. (B) Time-dependent HepG2 tumor growth curves. (C) TIR for different groups. (D) Body weight changes of mice on time. (E) Photographs of tumors at the end of treatment, and H&E, TUNEL, PCNA, and HIF-1α staining images of tumor tissues at the end of experiment. *P < 0.1, and ***P < 0.001

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In vivo antitumor efficacy and biosafety

The in vivo anticancer efficacy of CSN-IR806/CB combined with NIR irradiation was evaluated by using HepG2 tumor-bearing Balb/c mice as tumor model. PBS, free CB, free IR806, and CSN-IR806/CB were administrated via tail vein injection on day 1 and day 5 (Fig. 6A). As for the light groups, the NIR irradiation (808 nm, 1.0 W/cm2, 5 min) in tumor regions 12 h post-injection was performed, and then the changes in tumor volume and body weight were monitored every two days. Free CB and free IR806 + L groups both exhibited limited tumor growth inhibition attributed to the rapid clearance of small molecule drugs (Fig. 6B). Without irradiation, CSN-IR806/CB exhibited a moderate therapeutic efficacy, achieving tumor inhibitory rate (TIR) of 59.2% due to the release of some drugs in the hypoxic conditions of the tumor tissues (Fig. 6C). In sharp contrast, CSN-IR806/CB with NIR irradiation significantly reduced tumor volume with the highest TIR of 85.4% due to the synergistic triple therapeutic effect of PTT-PDT-CT. Furthermore, in vivo biosafety of CSN-IR806/CB was further evaluated. None of the treated groups exhibited significant body weight loss or showed any apparent pathological damage in major organ during the treatment (Fig. 6D, and Additional file 1: Fig. S12), indicating that there was no apparent impairment of organ function throughout the therapy. The above results approved that the CSN-IR806/CB did not elicit any discernible adverse effects in vivo. The H&E staining, TUNEL, and PCNA assays was further used to analyze antitumor effect. (Fig. 6E). The largest area of tumor necrosis, the most cellular apoptosis, and the least cellular proliferation was observed in CSN-IR806/CB + L group, confirmed the most significant tumor-killing ability of PTT-PDT-CT synergistic triple therapies.

Fig. 7
figure 8

(A) Complete blood panel analysis of mice including WBC, RBC, HCT, HGB, MCH, MCHC, MCV, and PLT. (B) Blood biochemistry analysis of mice including ALT, AST, TP, ALP, BUN, ALB, LDH, and UA

Full size image

Finally, blood samples were obtained from the PBS, CSN-IR806/CB, and CSN-IR806/CB + L groups to assess potential in vivo toxicity through a complete blood count and biochemical analysis. As illustrated in Fig. 7A, parameters such as white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and platelets (PLT) were evaluated for the complete blood count. Concurrently, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), alkaline phosphatase (ALP), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), and uric acid levels (UA) were analyzed for biochemical assessment as shown in Fig. 7B. The results indicated that all parameters of the CSN-IR806/CB group both with and without irradiation showed minimal changes compared to the control group. These findings suggest that CSN-IR806/CB may serve as an effective and safe agent for cancer therapy under 808 nm NIR.

Conclusion

In summary, we have successfully prepared a hypoxia-responsive supramolecular nanoprodrug (CSN-IR806/CB) with a cross-linked core, which consist of a dendritic drug-drug conjugates IR806-(Azo-CB)4 and β-cyclodextrin grafted PEG-D-poly(glutamic acid) block copolymer (PEG-PGlu-CD) via the synergy of π-π stacking interaction, host-guest complexation, hydrogen bonds, and hydrophobic interaction. CSN-IR806/CB exhibited superior physiological stability, effectively preventing the toxicity of free drugs to normal tissues. Both the in vitro and in vivo anti-tumor experimental results that the treatment of IR806-(Azo-CB)4 with NIR light irradiation presented significantly strong cytotoxicity to HepG2 tumor cells and the TIR of mice reached 85.4% on day 22 resulted from the synergistic triple therapeutic effect of PTT-PDT-CT. Therefore, the IR806-(Azo-CB)4 provides a charming nanomedicine-based DDSs for combination therapy.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

This work was financially supported by the Science and Technology Project of Nantong City (MS2023040), the Natural Science Foundation of Jiangsu Province (BK20240950), Innovation Program of Shanghai Municipal Education Commission (20230548), Shanghai Key Laboratory of Cell Engineering (14DZ2272300). The Investigator-initiated Trial Program of Shanghai Pudong New Area Health Commission (the Medical and Industrial Integration Program, 2025-PWYC-04), and Project of Shanghai Municipal Health Commission (202240347).

Funding

Science and Technology Project of Nantong City, MS2023040, Yue Ding; Natural Science Foundation of Jiangsu Province, BK20240950, Yue Ding; Innovation Program of Shanghai Municipal Education Commission, 20230548, Jin Ding; Shanghai Key Laboratory of Cell Engineering, 14DZ2272300. The Investigator-initiated Trial Program of Shanghai Pudong New Area Health Commission (the Medical and Industrial Integration Program), 2025-PWYC-04, and Project of Shanghai Municipal Health Commission, 202240347, Beifang Ning.

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Authors and Affiliations

  1. School of Chemistry and Chemical Engineering, Nantong University, Nantong, 226019, P. R. China

    Yue Ding, Yu Xie, Liangshun Zheng & Tingting Chen

  2. Clinical Cancer Institute, Center for Translational Medicine, Naval Medical University, Shanghai, China

    Chang Du & Jin Ding

  3. Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China

    Mingguang Lin & Beifang Ning

  4. Department of Gastroenterology, Shanghai Pudong New Area Gongli Hospital, Shanghai, 200135, China

    Yihai Shi

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Contributions

YD, YX, and CD performed the experiments and wrote the draft manuscript. LZ, ML, TC, and YS analyzed the data and drew the Schemes and Figures. JD polished the English. YD, JD, and BN got the funding and designed the project. All authors read and approved the final manuscript.

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Ding, Y., Xie, Y., Zheng, L. et al. Hypoxia-responsive core-cross-linked supramolecular nanoprodrug based on dendritic drug-drug conjugates for synergetic anticancer therapy. J Nanobiotechnol 23, 316 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03394-y

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