2D-ultrathin MXene/DOXjade platform for iron chelation chemo-photothermal therapy.

An increased demand for iron is a hallmark of cancer cells and is thought necessary to promote high cell proliferation, tumor progression and metastasis. This makes iron metabolism an attractive therapeutic target. Unfortunately, current iron-based therapeutic strategies often lack effectiveness and can elicit off-target toxicities. We report here a dual-therapeutic prodrug, DOXjade, that allows for iron chelation chemo-photothermal cancer therapy. This prodrug takes advantage of the clinically approved iron chelator deferasirox (ExJade®) and the topoisomerase 2 inhibitor, doxorubicin (DOX). Loading DOXjade onto ultrathin 2D Ti3C2 MXene nanosheets produces a construct, Ti 3 C 2 -PVP@DOXjade, that allows the iron chelation and chemotherapeutic functions of DOXjade to be photo-activated at the tumor sites, while potentiating a robust photothermal effect with photothermal conversion efficiencies of up to 40%. Antitumor mechanistic investigations reveal that upon activation, Ti 3 C 2 -PVP@DOXjade serves to promote apoptotic cell death and downregulate the iron depletion-induced iron transferrin receptor (TfR). A tumor pH-responsive iron chelation/photothermal/chemotherapy antitumor effect was achieved both in vitro and in vivo. The results of this study highlight what may constitute a promising iron chelation-based phototherapeutic approach to cancer therapy.

Introduction

Cancer remains one of the most serious diseases affecting the global population [[1], [2], [3]]. Despite major therapeutic breakthroughs in recent decades, cancer incidence remains high. Moreover, most available treatment options fail to suppress reliably the development of chemoresistance as well as cancer reoccurrence [1]. Current treatments are also plagued by limited tumor specificity and unwanted off-target cytotoxicity, limitations that can lead to side effects such as nausea, vomiting, hair loss and increased susceptibility towards bleeding and infections [4]. To address these challenges, new advanced anticancer treatments are urgently needed.

Iron chelation therapy has gained increasing attention in recent years as a potential primary or adjuvant cancer treatment option [[5], [6], [7]]. Iron is an essential element for humans and access to iron is an absolute requirement for cellular proliferation [8]. In general, the cellular iron pool is well-balanced and is dynamically regulated by certain transmembrane proteins, such as ferroportin 1 [9]. However, owing to the rapid synthesis of DNA during cell growth, cancer cells exhibit an abnormal iron metabolism with higher demand for iron than normal cells, as reflected, for instance, in the upregulation of the iron uptake protein transferrin receptor (TfR) in various types of cancers [10,11]. As a consequence, tumor cells are more sensitive to iron deprivation. This, in turn, makes selective modulation of cellular iron metabolism a promising approach to cancer chemotherapy drug development [10]. One notable system of interest in this context is the FDA-approved iron chelating agent deferasirox (ExJade®, Scheme 1) [12,13], which has been shown to exert inhibitory effects towards cancer cells in vitro and in vivo models derived from human liver, lung and pancreatic cells. Promising preliminary success in the treatment of a patient with acute myelogenous leukemia has also been reported [[14], [15], [16]]. Unfortunately, dose-dependent toxicity and non-targeting issues constitute roadblocks in the clinical development of deferasirox for cancer-related indications.

Scheme 1

Schematic representation of DOXjade being loaded onto 2D ultrathin Ti3C2 MXene nanosheets to create constructs that allow for combined iron chelation chemo-photothermal therapy.

A promising approach to improve the therapeutic efficacy of deferasirox would be to make it part of a combination therapy. Here, the idea, as with other so-called drug cocktails, is to exploit multiple therapeutic strategies simultaneously to increase synergistically the anticancer activity of the individual treatments without reaching their respective dose-limiting toxicities [[17], [18], [19], [20], [21]]. One modality that appears attractive for combination with deferasirox is photothermal therapy (PTT), which relies on photonic energy to generate local hyperthermia to damage cancer cells [[22], [23], [24]]. PTT takes advantage of the susceptibility of cancer cells toward heat to induce apoptosis [25,26]. Especially, because of its O2-independent mechanism of action, PTT has emerged as an important strategy for improving the sensibility of conventional cancer treatments, such as radio- and chemotherapy [[27], [28], [29], [30]]. We therefore postulated that rationally designed photothermal systems that include an iron chelation element would prove promising in terms of combating malignant tumors. Unfortunately, simple combination strategies, such as those that rely on, e.g., direct loading of an active drug form into nanocomposites, usually do not address the problem of off-target toxicities that plague traditional cancer treatments.

Considering the limitations in combination therapy, we sought to develop a “dual-therapeutic prodrug nanomedicine” approach involving iron chelation chemo-photothermal therapy as illustrated in Scheme 1. Here, the key elements are 1) a new tumoral acid microenvironment responsive dual-therapeutic conjugate (DOXjade) that incorporates in one molecular construct both the iron chelator deferasirox and the clinically approved topoisomerase 2 inhibitor, doxorubicin (DOX), and 2) ultrathin two-dimensional (2D) Ti3C2 MXene nanosheets designed to act as a photothermal generator and drug delivery carrier. The two drug components making up DOXjade are tethered through a pH-sensitive linker, namely a hydrazone bond (Scheme 1). Our design expectation was that the choice of an acid-labile hydrazone linker would enable the inherent iron chelation and chemotherapeutic functions of DOXjade to be activated preferentially at tumor sites, thereby reducing off-target toxicity effects.

Nanomaterials, especially those with good biocompatibility and drug loading efficiencies, are attracting increasing attention within the context of cancer nanomedicine [[31], [32], [33], [34], [35], [36]]. As an emerging member of 2D material family, Ti3C2 MXene nanosheets showed several advantages for nanocarrier biomedicine application including significant light absorbed ability in near infrared (NIR) light, large specific surface area and excellent air-stability and solution stability [32]. In this context, 2D ultrathin Ti3C2 MXene nanosheets, benefitting from a planar structure with a large surface area, were expected to interact strongly with DOXjade. The resulting construct, termed TiC-PVP@DOXjade, was, in turn, expected to display good photothermal efficiency [37]. In fact, as detailed below, the Ti3C2 nanosheets present in TiC-PVP@DOXjade displayed photothermal conversion efficiencies of up to 40% and provided excellent PTT performance when subject to NIR light irradiation at 808 nm within the so-called “optical-therapeutic window (650–900 nm)” [38,39]. Moreover, a sensitive tumor pH-responsive iron chelation/PTT/chemotherapy antitumor effect was seen with TiC-PVP@DOXjade.

Results and discussion

Synthesizes and Characterization of Ti3C2 nanosheets

Ti3C2 nanosheets were synthesized via a hydrofluoric acid (HF) etching method (see Methods section for full details). Bulk Ti3AlC2 powder was initially ground into a fine black powder (400 mesh) (Fig. 1a). As revealed by scanning electron microscope (SEM) images, the bulk Ti3AlC2 MAX phase consists of layered Ti3C2 and planar aluminum (Al) atomic sheets (Fig. 1b). HF etching was then used to remove the atomic Al sheets and facilitate the formation of a multilayer Ti3C2 material with a typical “accordion” morphology (Fig. 1c). After intercalation and sonication, delaminated Ti3C2 nanosheets with a thin and transparent structure were obtained, as confirmed by transmission electron microscopy (TEM) (Fig. 1d). As shown in Fig. 1e, cross-sectional TEM images of the stacked layers of Ti3C2 revealed interlayer distances of 9.2 Å, which is in good agreement with previous reports [37,40,41]. Scanning transmission electron microscopy (STEM) with energy dispersive X-ray spectroscopy (EDS) was used to detect the presence of Ti, C and F, as well as the excellent overlap of these three elements (Fig. 1f). The fluorine atoms present in the sample presumably originate from the HF etching and exist on the surface of Ti3C2 sheets as surface terminating moieties. The X-ray diffraction (XRD) patterns of the Ti3AlC2 samples prepared as above (Fig. 1g) proved to be in agreement with the Joint Committee on Powder Diffraction Standards (JCPDS PDF#52–0875). This was taken as evidence that the crystallinity of Ti3AlC2 MAX phase is maintained. In comparison to bulk Ti3AlC2, the most intense peak at 39° 2θ was absent in the case of the Ti3C2 nanosheets (Fig. 1g), which was ascribed to exfoliation [40]. Moreover, the (002) peaks at 9.6° in the original Ti3AlC2 samples were found to shift to a lower angle (7.2°) in the case of the Ti3C2 nanosheets. Broadening of this peak was also seen (Fig. 1g). These are typical features of Ti3C2. The UV–vis absorption spectrum of Ti3C2 was shown to have a noticeable and broad absorption in the visible and NIR region (600–900 nm), which is attributed to a surface plasmon resonance of Ti3C2 (Fig. 1h) [37]. The results of high-resolution XPS analyses of the Ti 2p region are shown in Fig. 1i and fittings of the Ti 2p peaks proved consistent with previous reports [42]. Binding energies of 459.3 eV and 465.1 eV were assigned to the Ti–O bonds in TiO2, while the peaks corresponding to the Ti–C bonds in Ti3C2 were found to have binding energies of 454.1 eV and 455.5 eV, respectively. The peak at a binding energy of 461.6 eV is assigned to the Ti–F bond, further substantiating the formation of Ti–F bonds as surface termination moieties. The XPS spectra of the Ti3C2 nanosheets (Fig. S1a) displayed four peaks assigned to the F 1s, O 1s, Ti 2p and C 1s orbitals, respectively, reflecting the main elements found in the Ti3C2 nanosheets. Fitting the high‐resolution XPS spectra of the O 1s orbital provided support for overlap between the O–Al, O–H and O–Ti bonds (Fig. S1b). High resolution XPS spectra of the Al 2p orbital in Ti3C2 and Ti3AlC2 are shown in Fig. S1c. Importantly, the peak corresponding to the Al 2p orbital was absent in the case of the Ti3C2 sample, which was taken as evidence for the removal of Al atoms from Ti3AlC2 during the synthesis of Ti3C2. Atomic force microscopy (AFM) was used to investigate the thickness of the Ti3C2 sheets, giving an average height of 6.07 ± 2.7 nm, which corresponds to the thickness expected for a few Ti3C2 nanosheet layers (Fig. S2) [37].

Fig. 1

Characterization of Ti3AlC2 MAX phase and Ti3C2 nanosheets. a) Digital photograph and b) SEM image of bulk Ti3AlC2. c) SEM images of multilayer Ti3C2. d) TEM images of the Ti3C2 nanosheets. e) Cross-sectional TEM image of the Ti3C2 nanosheets. f) EDS elemental mapping results of theTi3C2 nanosheets. g) The XRD patterns of theTi3C2 nanosheet (red graph) and bulk Ti3AlC2 (blue graph). h) UV–vis absorption spectrum of Ti3C2 nanosheets at different concentrations in deionized water. i) High-resolution XPS spectra of the Ti 2p orbital.

Surface functionalization and the photothermal performance of Ti3C2 nanosheets

For 2D nanomaterials to be used in drug delivery and phototherapeutic applications, solution stability and biocompatibility are required. Ti3C2 synthesized via acid etching affords a negatively charged surface due to the presence of hydroxyl and fluorine units anchored to the surface [43,44]. This negative surface charge affords acid etched Ti3C2 nanosheets with good stability in aqueous media. Unfortunately, in the presence of positively charged ions, such as those present as counter cations in phosphate buffered saline (PBS) and Dulbecco's modified eagle medium (DMEM), aggregation or precipitation of Ti3C2 occurs as shown in Fig. 2a. To address this issue, we subjected our etched Ti3C2 to surface functionalization with polyvinylpyrrolidone (PVP) (Methods section). The resulting material, Ti3C2-PVP, as well as Ti3C2 and PVP, were characterized by Fourier transform infrared spectroscopy (FTIR) (Fig. S3a). PVP displayed infrared absorption peaks ascribable to the expected C–O, C–N, –CH2 functionalities. This observation was taken as evidence that PVP had been anchored successfully onto the Ti3C2 nanosheets. Dynamic laser scattering (DLS) analyses revealed that the Ti3C2-PVP nanosheets were larger than the unfunctionalized Ti3C2 nanosheets (Fig. S3b), as expected for a functionalized system. The aqueous stability of the Ti3C2-PVP nanosheets was evaluated in PBS and DMEM solution (Fig. 2b). No discernible aggregation was observed when Ti3C2-PVP was subjected to centrifugation at 3000 rpm for 5 min. In contrast, under the same conditions, unfunctionalized Ti3C2 nanosheets aggregated. We thus suggest that Ti3C2-PVP is appropriate for use in biological studies.

Fig. 2

Photothermal performance and stability studies of Ti3C2 nanosheets in different media. a) Photographs of aqueous solutions of Ti3C2 and Ti3C2-PVP nanosheets dispersed in different solvents (water, PBS and DMEM). b) Photographs of Ti3C2 and Ti3C2-PVP nanosheets after centrifugation at 3000 rpm. Photothermal performance of different concentrations of Ti3C2-PVP under laser irradiation intensities of c) 1 W cm−2 and d) 1.5 W cm−2. e) Photothermal performance of Ti3C2-PVP (100 μg mL−1) under different laser irradiation intensities. f) Temperature change curves for Ti3C2-PVP (100 μg mL−1) dispersed in deionized water after five laser on/off cycles under different laser irradiation intensities. g) Photothermal effect of Ti3C2-PVP under 808 nm photo-irradiation showing one heating and cooling cycle.

The photothermal performance of Ti3C2-PVP was then evaluated. In these studies, different concentrations of Ti3C2-PVP were subject to 808 nm laser irradiation at 1 W cm−2 (Fig. 2c) and 1.5 W cm−2 (Fig. 2d), respectively. Ti3C2-PVP displayed excellent photothermal properties. For instance, with irradiation at 1.5 W cm−2 for 360 s, a 100 μg mL−1 solution in Ti3C2-PVP reached a temperature of 63 °C (Fig. 2d). A concentration dependence was seen for these photoinduced temperature changes (Fig. 2e). To test the photostability of Ti3C2-PVP, Ti3C2-PVP solutions were subject to NIR laser radiation (laser on) followed by natural cooling back to room temperature (laser off) for five on/off cycles. As inferred from the associated temperature change profiles, no discernible degradation was observed. Similar results were seen at different power settings (Fig. 2f). On this basis we conclude that Ti3C2-PVP would prove stable under conditions of photo-activation in vivo.

To determine the photothermal conversion efficiency (η) of Ti3C2-PVP, the temperature change was recorded under NIR laser radiation for 10 min (laser on) followed by natural cooling to room temperature (laser off) as seen in Fig. 2g. The time constant for heat transfer from the system was determined to be 310 s by plotting the linear time data from the cooling period versus the negative natural logarithm of the driving force temperature, as shown in Fig. S4. Using conventional methods [32,37], the η value for Ti3C2-PVP was determined to be 40%, highlighting the potential of Ti3C2-PVP as a possible PTT agent for cancer treatment.

Drug (DOXjade) loading onto the Ti3C2 nanosheets

The dual-therapeutic agent, DOXjade, was synthesized via the acid mediated (trifluoroacetic acid, TFA) condensation between a hydrazide functionalized deferasirox derivative (ExHydra) and DOX (see Methods for full synthesis) - Scheme S1. Ti3C2-PVP and DOXjade were then mixed in PBS. DOXjade was added at different feeding ratios, set at 1.0, 1.5 2.0, 2.5 and 3.0, respectively, and stirred overnight under an inert atmosphere. As shown in Fig. S3b, after anchoring DOXjade to the surface of Ti3C2-PVP, the resulting TiC-PVP@DOXjade constructs were larger than Ti3C2-PVP, as evidenced by DLS. The loading of DOXjade onto Ti3C2-PVP (to give TiC-PVP@DOXjade) resulted in a change in the zeta potential from negative to positive (Fig. S3c). This finding provides further support for the suggestion that DOXjade was successfully loaded onto the Ti3C2-PVP nanosheets.

To determine the drug loading capacity of Ti3C2-PVP, we first recorded a standard concentration-absorbance curve for DOXjade at 440 nm (Fig. S5a). Presumably as a consequence of their planar structure and large surface area [32], the present 2D ultrathin MXene Ti3C2-PVP nanosheets exhibited a loading potency for DOXjade of 210% (Fig. S5b). This relatively high drug loading efficiency was viewed as being advantageous since it was expected to allow DOXjade to be delivered efficiently to tumor sites via, e.g., a classic EPR (enhanced permeability and retention) effect, while minimizing potentially non-specific accumulation within normal tissues.

pH-triggered drug release and cytotoxicity of DOXjade

In accord with our design expectations, DOXjade was found to hydrolyze into its respective components (DOX and ExHydra) under acidic conditions (Fig. 3a), as inferred from LC-MC analyses (Figs. S17–S19). This cleavage was found to occur in a pH-dependent manner. As shown in Fig. 3b, in acetate buffer (pH = 5.3), the maximum intensity of the fluorescence emission feature ascribed to DOX at 600 nm increased in a time-dependent manner (0–72 h) to a maximum of 6.9-fold. In contrast, in neutral medium (pH = 7.4), the intensity of the 600 nm emission feature increased by only 1.4-fold under otherwise identical conditions (Fig. 3c and d). These results provide support for a key hypothesis underlying the present study, namely that DOXjade would be able to act as a prodrug capable of releasing its two constituents, ExHydra and DOX in acidic tumor microenvironments. ExHydra possesses the same chelating motif as deferasirox and it was found to display near-identical UV–vis spectral changes when exposed to Fe (III) (Fig. 3e). Thus, the mode of action of ExHydra was assumed to be the same as deferasirox [45].

Fig. 3

Hydrolysis of DOXjade seen at low pH and results of cytotoxicity assays carried out with DOX, ExHydra and DOXjade. (a) Scission of DOXjade (hydrazone bond cleavage) results in a fluorescence ‘turn-on’ response and concurrent release of both drugs (ExHydra and DOX). (b) Fluorescence spectra of DOXjade (10 μM) recorded as a function of time (0–72 h) in acetate buffer (pH = 5.3), and (c) PBS buffer (pH = 7.4). λex = 500 nm. Slit widths: ex = 10 nm and em = 10 nm. (d) Relative fluorescence intensity changes of aqueous samples of DOXjade at λem = 600 nm as a function of time (0–72 h); data are derived from (b) and (c). (e) Absorbance profiles of ExHydra (30 μM), FeCl3 (30 μM), alone and in combination in acetate buffer at pH = 5. The interaction between ExHydra and Fe3+ is inferred based on analogy to deferasirox and is supported by the observation of a broad band at around 510 nm. (f) Viability of human colorectal cancer cells HCT116 seen upon incubation with different concentrations of ExHydra, DOX and DOXjade, as determined from CCK-8 assays. (g) Dose dependent cytotoxicity of ExHydra, DOX and DOXjade towards HMEC-1 cells. Mean ± SD (n = 3). **p < 0.01 vs. control.

Cell proliferation assays were conducted to evaluate the cytotoxicity of DOXjade, DOX and ExHydra in cancer cell lines and human normal cells. The results of a cell counting kit-8 (CCK-8) cell viability assay revealed that DOX and DOXjade inhibited cancer cells growth more effectively than ExHydra alone. In the human colorectal cancer cell lines (SW480, HCT116, DLD1) and the hepatocellular carcinoma cell lines (HepG2, Huh-7), tumor cell growth was inhibited by DOXjade in a dose and time dependent manner (Fig. 3f and S6). However, in normal human mammary epithelial HMEC-1 cells, DOXjade displayed a markedly lower cytotoxicity as compared to free DOX and ExHydra (Fig. 3g), leading us to suggest that our dual-therapeutic prodrug design would display reduced off-target toxicity when tested in vivo.

Iron chelation chemo-photothermal therapy in vitro

We then evaluated the iron chelation chemo-photothermal efficiency of TiC-PVP@DOXjade in HCT116 cells using the CCK-8 assay. Ti3C2-PVP and DOXjade were included as controls. The 2D ultrathin Ti3C2-PVP nanosheets exhibited satisfactory biocompatibility and potent PTT efficiency in cells. For instance, after incubating Ti3C2-PVP in cells for 6 h, negligible cytotoxicity was noted even at concentrations up to 200 μg mL−1 (Fig. 4a and S7). In contrast, an IC50 (half maximal inhibitory concentration) of 52.01 μg mL−1 was recorded upon subjecting Ti3C2-PVP to 808 nm laser irradiation for 10 min (1 W cm−2), (Fig. 4c). This finding, which is consistent with a previous report [46], provided us with support for the notion that Ti3C2-PVP would serve as an effective PTT platform for use in conjunction with DOXjade. Studies of TiC-PVP@DOXjade were thus carried out under conditions identical to those used in the case of Ti3C2-PVP (808 nm, 1 W cm−2, 10 min). This yielded an IC50 value of 37.05 μg mL−1 (Ti3C2-PVP: 30.02 μg mL−1; DOXjade: 7.03 μg mL−1) (Fig. 4b), representing a 5.2- and 1.7-fold improvement relative to DOXjade and PTT (Ti3C2-PVP + NIR) treatments, respectively (Fig. 4c). These improvements are ascribed to a DOXjade dual-therapeutic effect (iron chelation and chemotherapy) that complements that provided by Ti3C2-PVP-mediated PTT. Live/dead cell staining using a calcein AM/7-AAD assay kit, in which the live cells produce a green fluorescence while dead cells give rise to a red fluorescence [47,48], provided support for the inference that efficient tumor cells damage is engendered as the result of the putative combination treatment (Fig. 4d). For instance, FACS analysis revealed Annexin V-FITC/7-AAD (7-aminoactinomycin D) staining for the majority of the HCT116 cells subject to photo-irradiation (Fig. 4e) reflecting an increase in cellular apoptosis.

Fig. 4

In vitro photothermal effects produced upon subjecting Ti3C2-PVP to photo-irradiation. (a) Relative cell viabilities of HCT116 cells treated with different concentrations of Ti3C2-PVP and exposed to laser irradiation at 808 nm for 10 min. (b) Relative cell viabilities of HCT116 cells treated with Ti3C2-PVP, TiC-PVP@DOXjade with or without exposure to laser irradiation (808 nm). (c) IC50 values deduced from studies of HCT116 cells treated with DOXjade, Ti3C2-PVP, TiC-PVP@DOXjade with or without NIR exposure. Note the concentration differences: #175.14 μg mL−1TiC-PVP@DOXjade (Ti3C2-PVP: 141.88 μg mL−1; DOXjade: 33.25 μg mL−1); &37.05 μg mL−1TiC-PVP@DOXjade (Ti3C2-PVP: 30.02 μg mL−1; DOXjade: 7.03 μg mL−1). (d) Fluorescent images of live/dead HCT116 cells stained using calcein AM (green, live cells) and propidium iodide (red, dead cells) after addition of Ti3C2-PVP (40.51 μg mL−1), TiC-PVP@DOXjade 50 μg mL−1 (Ti3C2-PVP: 40.51 μg mL−1; DOXjade: 9.49 μg mL−1) with or without laser irradiation (1 W cm−2). (Scale bars: 250 μm) (e) Representative annexin V-FITC/7-AAD scatter plots for HCT116 cells treated as in (d). Mean ± SD (n = 3). *p < 0.05, **p < 0.01 vs. control.

Antitumor mechanistic analyses

Encouraged by the antitumor efficiency observed in vitro, we further studied the working mechanism of TiC-PVP@DOXjade. As mentioned in the introduction, cancer cells are known to have abnormal iron metabolism and are typically characterized by a relatively high dependence on iron (Fig. 5a). Certain forms of iron within the iron pool promote the growth of cancer cells by increasing reactive oxygen species, inducing DNA damage, regulating epigenetics and the transcription of oncogenic genes [10,49]. The “transferrin receptor” (TfR) plays an important role in iron uptake and iron metabolism. As shown in Fig. 5b–e, treatment of TiC-PVP@DOXjade with 808 nm laser irradiation downregulated TfR expression compared with control groups consisting of ExHydra, DOX, DOXjade, and Ti3C2-PVP + 808 nm laser irradiation (Fig. 5b–e).

Fig. 5

Antitumor mechanistic studies of TiC-PVP@DOXjade-based treatment. (a) Scheme showing key events. 1: DOXjade is released from Ti3C2-PVP; 2: Hydrolysis of DOXjade into DOX and ExHydra; 3: Iron chelation by ExHydra; 4: Inhibition of TfR expression by DOX. Tf: Transferrin Receptor. (b) Western blot analysis of the expression of the iron metabolism related protein TfR and the Bcl-2 and Bax proteins involved in apoptosis in HCT-116 cells after drug administration and laser irradiation. In this and other applicable panels, i: Control; ii: ExHydra; iii: DOX; iv: DOXjade; v: Ti3C2-PVP; vi: Ti3C2-PVP + NIR; vii: TiC-PVP@DOXjade; viii: TiC-PVP@DOXjade + NIR; quantitation of TfR (c) Bcl-2 (d), and Bax (e) protein levels. Morphology (f) and diameter (g) analyses of 3D tumor spheroid cultures of HCT-116 cells after being incubated for 72 h with ExHydra, DOX, or DOXjade. Scale bar 400 μm. (h) Results of spheroid viability expressed as a percentage with the value for the negative control cells arbitrarily set at 100%. NIR laser = 808 nm, 1.0 W cm−2, 10 min. Mean ± SD (n = 3). *p < 0.05, **p < 0.01 vs. control.

Several additional proteins that have a role in apoptosis, such as anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and the pro-apoptotic protein Bcl-2-associated X protein (Bax), were analyzed by Western blot assays. It was found that TiC-PVP@DOXjade irradiated with 808 nm laser is more effective in inhibiting Bcl-2 expression than all other treatment groups (Fig. 5b and d). The pro-apoptotic protein Bax was also upregulated in the TiC-PVP@DOXjade group relative to the other groups (Fig. 5b and e). On the basis of these findings, we conclude that iron chelation plays an important role in mediating the combined chemo- and photothermal therapeutic effect seen in the case of TiC-PVP@DOXjade.

Conventional two-dimensional (2D) cell cultures are characterized by rapid and uncontrolled growth patterns, which are considered less accurate than three-dimensional (3D) tumor spheroids in mimicking in vivo tumor environment [50]. As a consequence, 3D culture modals are being used increasingly as a bridge between 2D monolayer experiments and animal studies [51]. The cytotoxicity of TiC-PVP@DOXjade was thus further evaluated using 3D tumor spheroids. As can be seen from an inspection of Fig. 5f–h, statistically significant growth inhibition was seen in 3D tumor spheroids. Based on these data, we suggest that the combined treatment permitted by TiC-PVP@DOXjade leads to an antitumor effect in accord with the mechanism given in Fig. 5a and that iron chelation can enhance the effects of conventional chemotherapy and PTT.

In vivo antitumor efficacy of TiC-PVP@DOXjade

We next sought to assess the putative benefits of iron chelation coupled with chemo-photothermal therapy in vivo using a HCT116-based subcutaneous tumor mouse model. Prior to testing the ability of TiC-PVP@DOXjade to inhibit tumor growth in vivo, hematological studies of Ti3C2-PVP were carried out to confirm its presumed biocompatibility. Previously, the biocompatibility of Ti3C2 modified with soybean phospholipids had been demonstrated [46]. We further tested the biocompatibility of Ti3C2-PVP in terms renal and liver function. Here, BALB/c mice were sacrificed after being subject to intravenous injection with Ti3C2-PVP on days 1, 7, and 14. Blood samples were collected and isolated by centrifugation in blood coagulation tubes to obtain the plasma. Evaluation of renal function was accomplished by quantifying urea and uric acid levels, while liver function was assessed by monitoring the total protein levels and albumin/globulin ratio in serum. Compared to the control group injected with saline, no significant renal toxicity or impairment of liver function was seen for Ti3C2-PVP (Fig. S9). These data provide support for the conclusion that Ti3C2-PVP is biocompatible.

Next, efficacy studies were carried out using HCT116 tumor-bearing nude mice. The mice were randomly divided into six groups (n = 5 per group), including (i) control group (saline), (ii) ExHydra group, (iii) DOX group, (iv) DOXjade group, (v) Ti3C2-PVP group with 808 nm laser irradiation and (vi) TiC-PVP@DOXjade group with 808 nm laser irradiation. Six hours after intravenous administration of Ti3C2-PVP or TiC-PVP@DOXjade to the tumor-bearing mice, the tumor site was directly irradiated with a NIR laser (808 nm, 1.0 W cm−2, 10 min). In accord with our design expectation, a significant increase in tumor temperature was observed for the Ti3C2-PVP group (36.5–54.4 °C) (Fig. 6a and b). Monitoring the body weights of the animals in this cohort revealed no obvious changes or any observable adverse events (Fig. 6f). The Ti3C2-PVP group combined with 808 nm laser irradiation (1.0 W cm−2, 10 min) achieved modest tumor growth inhibition; however, tumor reoccurrence was observed during the two-week study period (Fig. 6c and d). In contrast, TiC-PVP@DOXjade combined with 808 nm laser irradiation achieved significant tumor eradication with no evidence of tumor regrowth over the course of the study (Fig. 6c and d).

Fig. 6

In vivo NIR imaging and antitumor effect of TiC-PVP@DOXjade. (a) Thermal images of mice bearing tumors after injection with Ti3C2-PVP or TiC-PVP@DOXjade, followed by exposure to 808 nm laser irradiation (1.0 W cm−2, 10 min). (b) Changes in tumor temperature seen in mice bearing HCT116 tumors upon laser irradiation. (c) Morphology of tumors removed from mice sacrificed at the end point of the study. Groups in this and the other studies in this figure are as follows: i: Control; ii: ExHydra; iii: DOX; iv: DOXjade; v: Ti3C2-PVP + NIR; vi: TiC-PVP@DOXjade + NIR, where NIR denotes laser irradiation (808 nm, 1.0 W cm−2, 10 min). (d) Inhibition of HCT116 tumor growth seen upon different treatments. (e) Weight of tumors excised from BALB/c nude mice involved in this study. (f) Body weight of BALB/c nude mice recorded every two days for two weeks after intravenous injection of the indicated agents. (g) Tumor growth inhibition as analyzed by Ki67 staining of tumor tissues. Scale bar: 200 μm. Mean ± SD (n = 5). **p < 0.01, ***p < 0.001 vs. control.

To assess the tumor growth inhibition effects, immunohistochemical detection of the proliferation-associated antigen Ki67 was carried out. Ki67, a labile nuclear protein, has been reported as a valuable prognostic marker in colorectal cancer [52]. As shown in Fig. 6g, the Ki67 expression decreased in the TiC-PVP@DOXjade-treated tumors compared with the other treatment groups. This reduction is consistent with the high antitumor activity TiC-PVP@DOXjade provides under conditions of photo-irradiation.

Conclusion

In conclusion, here we proposed a “dual-therapeutic prodrug nanomedicine” approach for iron chelation chemo-photothermal therapy. Especially, to realize this goal, a new and novel tumoral acidic microenvironment responsive dual-therapeutic conjugate DOXjade that incorporates in the clinically approved iron chelator deferasirox and DOX was rationally designed. After loading onto the engineered ultrathin 2D Ti3C2 MXene nanosheets, resulting TiC-PVP@DOXjade not only enables the inherent iron chelation and chemotherapeutic functions of DOXjade to be selectively activated at the tumor sites, but imparts a robust photothermal potential with the photothermal conversion efficiency up to 40%. Remarkably, TiC-PVP@DOXjade also displays an outstanding drug loading efficiency. Antitumor mechanism investigations reveal the iron depletion-induced TfR protein down-regulation and apoptotic cell death under multimodal treatments. With these unique merits, sensitive tumor pH-responsive iron chelation/PTT/chemotherapy antitumor effect was achieved both in vitro and in vivo. This study therefore represents a promising iron chelation phototherapeutic paradigm toward cancer precise therapy.

CRediT authorship contribution statement

Yunjie Xu: Project administration, Conceptualization, Data curation, Investigation, Software, Formal analysis, Writing – original draft. Yingwei Wang: Investigation, Data curation, Resources, Writing – original draft, Funding acquisition. Jusung An: Investigation, Data curation, Formal analysis. Adam C. Sedgwick: Investigation, Resources, Data curation, Formal analysis, Writing – review & editing. Mingle Li: Writing – review & editing, Funding acquisition. Jianlei Xie: Methodology. Weibin Hu: Methodology. Jianlong Kang: Investigation. Sajal Sen: Investigation, Writing – review & editing. Axel Steinbrueck: Investigation, Writing – review & editing. Bin Zhang: Methodology. Lijun Qiao: Methodology. Swelm Wageh: Methodology, Writing – review & editing. Jonathan F. Arambula: Supervision, Writing – review & editing. Liping Liu: Supervision. Han Zhang: Conceptualization, Supervision. Jonathan L. Sessler: Project administration, Conceptualization, Supervision, Funding acquisition, Writing – review & editing. Jong Seung Kim: Project administration, Conceptualization, Supervision, Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare no conflict of interest.