Biodegradable 2D Fe–Al Hydroxide for Nanocatalytic Tumor‐Dynamic Therapy with Tumor Specificity

Abstract Therapeutic nanocatalysis has emerged as an intriguing strategy for efficient cancer‐specific therapy, but the traditional inorganic nanocatalysts suffer from low catalytic efficiency and difficulty in biodegradation, hindering their further clinical translation. Herein, a tumor microenvironment‐triggered, biodegradable and biocompatible nanocatalyst employing 2D hydroxide nanosheet is presented, and is shown to have high catalytic capacity to efficiently produce abundant hydroxyl radicals under the tumor microenvironment and consequently kill tumor cells selectively. A polyethylene glycol (PEG)‐conjugated Fe2+‐containing hydroxide nanosheet is successfully constructed via a facile but efficient bottom‐up approach that concurrently realizes nanosheet synthesis and PEGylation. Importantly, the nanosheets are featured with high catalytic activity to disproportionate H2O2 in tumors, and consequently generate abundant hydroxyl radicals at a high reaction rate under tumorous acidic condition; the highly toxic hydroxyl radicals, as a result, cause the death of tumor cells in vitro and suppress the tumor growth in vivo without the use of any supplementary toxic agent, only with the biocompatible nanocatalysts. Meanwhile, the desirable biodegradation and biocompatibility of the hydroxide nanosheet render a high degree of safety to the organism. Therefore, this work provides the first paradigm of biodegradable 2D nanocatalytic platform with concurrently high catalytic‐therapeutic performance and biosafety for efficient tumor‐specific treatment.


Synthesis of ultrathin LDH nanosheets and bulk LDH nanoparticles
The LDH nanosheets were synthesized by a solvent-free bottom-up method. Solution A (10 mL) containing FeCl 2 (2 mmoL) and AlCl 3 •6H 2 O (1 mmoL) was mixed with Solution B (10 mL) containing NaOH (6 mmoL) with a constant pH value under nitrogen atmosphere, followed by mixing with Solution C containing phosphonic acid terminated poly (ethylene glycol) (0.2 g PEG, 8000 g mL -1 ). The mixture was then hydrothermally treated at 100 o C for 8 h. The PEG/Fe-LDH nanosheets were subsequently obtained, after removing extra salt and unattached PEG via centrifugation. Similarly, the PEG/Mg-LDH was prepared by adopting the aforementioned procedure but replacing FeCl 2 with MgCl 2 •6H 2 O.
The Fe-LDH nanoparticles were synthesized by mixing Solution A and Solution B at a constant pH value under nitrogen protection for 30 min followed by hydrothermal treatment and centrifugation.

Characterizations
X-ray diffraction (XRD) measurements were performed on LDH powder and film samples using a PANalytical expert multipurpose X-ray diffraction system (MPD) instrument operated at 45 kV and 40 mA and fitted with a CuKα source. A scan rate of 0.01°/min was applied with a step size of 2θ = 0.0260°. Size distribution and zeta potential were measured using a Malvern Zetasizer Nano Series. The polydispersity index (PDI) was used to describe particle size distribution. Fourier transform infrared spectroscopy (FTIR) was carried out on a Bruker IFS 66/S single-beam spectrometer. Spectra were obtained at regular time intervals in the MIR region of 4000 -400 cm -1 at a resolution of 4 cm -1 (32 scans). Thermal gravimetric analysis (TGA) was conducted using a TGA Q5000 instrument. Powder samples were placed in a platinum pan and heated in nitrogen atmosphere from room temperature to 800 °C at 20 °C min -1 . Transmission electronic microscopy (TEM) and scanning transmission electronic microscopy (STEM) imaging coupled and energy-dispersive X-ray spectroscopy (EDS) elemental analysis were performed on a FEI Tecnai G2 (200 kV) and JEOL JEM-F200 (200 kV) equipped with an annular dark-field detector and a JEOL windowless 100 mm 2 silicon drift X-ray detector. EDS data processing and analysis were carried out using the Thermo Scientific Pathfinder X-ray Microanalysis Software. The morphology of nanoparticles was observed using a Transmission Electronic Microscope (TEM, FEI Tecnai G2) at an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) was performed using a Thermo ESCALAB250i spectrometer with a monochromatic X-Ray source (AlKα, 1486.68 eV) operated at a 164 W emission power. XPS spectra were analyzed using Avantage 4.88 software. Electron spin resonance (ESR) spectra were obtained at room temperature in perpendicular mode on a Bruker EMX-8/2.7 spectrometer with the following settings: microwave frequency = 9.773 GHz, microwave power = 0.6325 mW, modulation frequency = 100.00 kHz and modulation amplitude = 2.00 G. DMPO was used as a spin trap.

Michaelis-Menten kinetics
TMB assay was conducted to monitor the chromogenic reaction (λ = 650 nm) of LDH/H 2 O 2 system. In a HAc-NaAc buffer solution (800 μL), LDH suspension (2 μg mL -1 ) was mixed with TMB and H 2 O 2 at the final concentrations of 800 μM and 1 mM respectively. The absorbance of the mixture was measured on a UV-vis spectrometer (CARY 300, Varian) equipped with a temperature controller. For the kinetic study, the concentrations of H 2 O 2 (0.01, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2, 4, and 20 mM) were varied while the concentrations of TMB (800 μM) remained fixed. The Michaelis-Menten constant was calculated by using Lineweaver-Burk plots of the Michaelis-Menten equation: , where v represents the initial velocity, V max the maximum reaction velocity, C the concentration of substrate, and K m the Michaelis-Menten constant.

Biodegradation of PEG/Fe-LDHs
The PEG/Fe-LDH nanosheets were suspended in a dialysis tube (MWCO 3500Da) in the buffer of pH 5, 6.5 or 7.4. An aliquot of leachate (2 mL) was sampled at a certain time point, and 2 mL fresh buffer was then added to replace the extracted solution in the suspension. The iron content in the leachate aliquots were measured on a PerkinElmer OPTIMA 7300 inductively coupled plasma optical emission spectroscopy (ICP-OES). The proportion of iron ions released was calculated as: where i represents the sampling time; c i (mg/mL) the sample content; c 0 (mg/mL) the initial metal content. The leachate (containing released iron ions Fe 2+ and Fe 3+ ) was also sampled to monitor •OH generation from the released solution via ESR.
To collect the releasing suspension, the PEG/Fe-LDHs were directly suspended in the buffers of different pH values, and suspension aliquots containing both released ions and PEG/Fe-LDH particles were collected for TEM observation and ESR test. The relative ESR intensity = (ESR signal intensity at each time points at different pH values / maximum ESR signal intensity at pH 5.0).

Cell Culture
The breast cancer cells (4T1 cells and MCF-7 cells) and fibroblast (Hs27 cells) were cultured in a growth medium (RPMI1640, DMEM and RPMI1640 with glutamine respectively) supplemented with 10% fetal calf serum (FCS), streptomycin (100 mg mL -1 ) and penicillin (100 units mL -1 ). The cells were cultured at 37 °C in a humidified atmosphere with 5% CO 2 in air.

In vitro anti-cancer effect evaluation
The 4T1 cells were seeded (1×10 4 cells in 100 μL RMPI1640 per well, pH 7.4) in 96-well microplates and allowed to adhere overnight. Cell culture media at pH 6.5 was used to simulate the extracellular microenvironment in a solid tumor. Hydrochloric acid was added to the RPMI (pH = 7.4) in order to acidize it to a pH of 6.5. The culture medium was then replaced with fresh RPMI (pH 6.5 or pH 7.4) containing PEG/Fe-LDHs at concentrations of 0,

Intracellular ROS detection
The 4T1 cells were seeded at a density of 1 × 10 5 in RPMI of pH = 7.4 in the φ 15 CLSMexclusive culture disk and allowed to adhere overnight. After incubation with DCFH-DA (20 μM in FCS-free RPMI) at 37 o C in 5% CO 2 for 20 min, the culture medium was replaced with medium of pH 6.5 containing 6 µg mL -

MCF-7 cells were seeded in a 24-well plate with coverslips at a density of 5×10 4 cells per
well. At semi-confluency, culture medium was replaced with medium containing PEG/LDH-FITC or LDH-FITC (10 µg mL -1 ). After a given period of time (

In vitro toxicity study
The Hs27 fibroblast cells were seeded (1×10 4 cells in 100 μL RMPI1640 with glutamine per well) in 96-well microplates. When the culture reached 50% confluency, the culture medium was then replaced with fresh RPMI1640 with glutamine containing PEG/Fe-LDHs at concentrations of 0, 0.03, 0.15, 0.3, 1.5, 3, 6 and 12 µg mL -1 . After 24 h incubation, the culture media were replaced by with RPMI media containing 10% alamarBlue and incubated for 1 h. Cell viability was determined by comparing the absorbance at λ 570 nm to the control group. The absorbance of samples was measured on a microplate reader 5 (FLUOstar, Omega, Germany). The experiments were carried out in triplicate, and the values from each experiment calculated from 3 wells.

In vivo toxicity study
All animal experiment operations were performed with approval of the Aminal Ethics Committees of University of New South Wales and Chongqing Medical University. The 7- week female Balb/c mice (~ 20 g) were intravenously injected with PEG/Fe-LDH saline solution (150 µL, 10, 40 and 100 mg kg -1 Fe) with the same volume of saline as the control, the body weight of mice was measured every two days (n = 5 for each group). At 30 days, blood was collected for hematological and biomedical indexes analysis. Mice were then sacrificed to collect their major organs (heart, liver, spleen, lung, and kidney) in a 10% formalin solution for histopathology analysis using a typical hematoxylin and eosin (H&E) staining assay.

In vivo anti-cancer effect evaluation
Xenografted tumors were generated in female Balb/c mice at 7 weeks of age (~ 20 g) by subcutaneously injecting 1×10 6 4T1 cells suspended in PBS (100 μL) into the mouse rear leg.
Once the tumor reached a volume of 100 mm 3 , 5 mice per group were randomly allocated for different treatments. As a proof-of-principle assessment of Fe-LDH as a catalytic therapeutic agent, 50 μL of PEG/Fe-LDH saline solution (15 mg kg -1 Fe) was injected intratumoral into the tumor, with the control groups undergoing an intratumoral injection of saline solution (50 μL) or PEG/Mg-LDH saline solution (50 μL, 15 mg kg -1 Mg). Tumors were measured every two days with a digital caliper. The tumor volumes (V) were calculated as V = L × W 2 / 2, and normalized to the initial volume (V 0 ) to obtain the relative tumor volume (V/V 0 ). The pathological tissue sections of tumors were collected in 12 h post-treatment for H&E staining assay. Mice were euthanized once tumor volume reached 1000 mm 3 .

Statistical Analysis
Quantitative data are presented as mean ± SD and analyzed by one-way ANOVA with TUKEY post-tests using GraphPad Prism software; a P-value < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, and ***P < 0.001).    absorbance at pH 5 than that at pH 6.5 and 7.4 ( Figure S4a). However, a typical 1:2:2:1 signal of the DMPO•OH adduct was clearly shown in the ESR spectra with the similar signal intensity at pH 5, 6.5 and 7.4 ( Figure S4b).