Dual Size/Charge‐Switchable Nanocatalytic Medicine for Deep Tumor Therapy

Abstract Elevating intratumoral levels of highly toxic reactive oxygen species (ROS) by nanocatalytic medicine for tumor‐specific therapy without using conventional toxic chemodrugs is recently of considerable interest, which, however, still suffers from less satisfactory therapeutic efficacy due to the relatively poor accumulation at the tumor site and largely blocked intratumoral infiltration of nanomedicines. Herein, an ultrasound (US)‐triggered dual size/charge‐switchable nanocatalytic medicine, designated as Cu‐LDH/HMME@Lips, is constructed for deep solid tumor therapy via catalytic ROS generations. The negatively charged liposome outer‐layer of the nanomedicine enables much‐prolonged blood circulation for significantly enhanced tumoral accumulation, while the positively charged Fenton‐like catalyst Cu‐LDH released from the liposome under the US stimulation demonstrates much enhanced intratumoral penetration via transcytosis. In the meantime, the co‐released sonosensitizer hematoporphyrin monomethyl ether (HMME) catalyze the singlet oxygen (1O2) generation upon the US irradiation, and deep‐tumoral infiltrated Cu‐LDH catalyzes the H2O2 decomposition to produce highly toxic hydroxyl radical (·OH) specifically within the mildly acidic tumor microenvironment (TME). The efficient intratumoral accumulation and penetration via the dual size/charge switching mechanism, and the ROS generations by both sonosensitization and Fenton‐like reactions, ensures the high therapeutic efficacy for the deep tumor therapy by the nanocatalytic medicine.

a rotary evaporator at 60 °C for 1 h, and the resultant thin films were dried overnight under vacuum. Then, 5 ml PBS containing Cu-LDH (4 mg mL -1 , pH = 7.4) was added into the flask, and the mixture was rotated for another 1 h to fully emulsify the thin film. Finally, the vesicles were obtained by a repeated extrusion process on a high-pressure extruder (ATS Engineering Inc, Jiangsu, China) using 1000-and 400-nm membranes, respectively, and then further purified by dialysis.

Characterization
The hydrodynamic size distribution and zeta potential were determined by a Malvern Zetasizer Nano series (Malvern Panalytical, Malvern, UK). X-ray diffraction measurements (XRD Bruker D8 Focus, Bruker, Billerica, MA, USA; 2θ ranging from 10° to 90° Cu K α1 ) were performed on as-synthesized fresh LDH and Cu-LDH powder. At an accelerating voltage of 200 kV, transmission electron microscopy (JEM-2100F, Tokyo, Japan) and scanning electron microscopy (JEOL Ltd., Tokyo, Japan) imaging coupled and energy-dispersive X-ray spectroscopy (EDS) elemental analysis were applied to capture their morphology and chemical components, respectively. The valence of copper doped in LDH was analyzed from the X-ray photoelectron spectroscopy (XPS) spectrum which gets by a Thermo ESCALAB250i spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Electron spin resonance (ESR) measurements were performed on a jeol-fa200 spectrometer at room temperature with the following settings: microwave frequency = 9.425 GHz, microwave power = 0.998 mW, modulation frequency = 100.00 kHz, and modulation amplitude = 2.00 G. DMPO and TEMP were used as the spin trap of ·OH and 1 O 2 , respectively. All UV−Vis absorption spectra were obtained from a UV-3600 Shimadzu spectrometer. And all the quantitative element analysis was determined by inductively coupled plasma optical emission spectrometer (ICP-OES, Agilent Technologies, US). The encapsulation efficiency of HMME in the Lips was measured by UV-vis spectra technology. The amount of Cu-LDH encapsulated in the Lips was determined by ICP-OES.

In vitro cellular-uptake
4T1 cells were cultivated in confocal dishes (2×10 5 cells per well) for 12 h. Then, the cells were incubated with free FITC labeled Cu-LDH, HMME@Lips, FITC @Lips, and FITC-Cu-LDH/HMME@Lips for 20 min, respectively. After washing with PBS three times to move the residual materials, the cellular uptake was monitored by CLSM and quantitatively detected by flow cytometry.

Cellular internalization and excretion of Cu-LDH
4T1 cells were seeded into a 12-well plate overnight to allow cells to adhere. Then, the cells (n = 3) were incubated with Cu-LDH@Lips and PEG-Cu-LDH@Lips for different times (0, 0.5, 1, 2, 3, 4, and 5 h), respectively. After washed with PBS, the cells were then digested, resuspended, and counted. The metal contents (Cu, Mg, and Al) in the cell suspensions were determined by ICP-OES. The intracellular contents of Cu-LDH were calculated accordingly.
In the excretion process, 4T1 cells (n = 3) that had been incubated with Cu-LDH@Lips (200 μg mL -1 ) and PEG-Cu-LDH@Lips for 5 h were recultivated in fresh DMEM for varied times (0, 0.5, 1, 2, 3, 4, and 5 h). At each time point, cells were harvested using trypsin after washed with PBS 3 times, resuspended, and counted. The remaining contents of Cu-LDH in cells were determined by ICP-OES. The internalization and excretion of Cu-LDH nanosheets in cells were further monitored by bio-TEM directly. Typically, the ultrathin sections of 4T1 tumor cells were made after coincubated with Cu-LDH/HMME@Lips for varied times (2, 4, and 8 h) and in-situ observed by bio-TEM directly.

Migration of Cu-LDH/HMME@Lips in a transwell system
To observe the migration of Cu-LDH with positive zeta potential and HMME with negative zeta potential between 4T1 cells, a migration model in a transwell system (Corning, USA) was established. A 3 μm-diameter microporous polyester membrane was used to block the migration of cells. In this case, blank 4T1 cells were seeded onto the basolateral compartment of the transwell system and cultured for overnight to allow cell adhesion. Additional blank cells were firstly incubated with FITC labeled Cu-LDH/HMME@Lip for 4 h. After washing with PBS 3 times, cells were digested and added back to the apical compartment and US irradiation for 5 min and followed by incubation for another 4 h. Finally, DAPI was used to stain the cell nucleus and was observed by multiphoton CLSM.

In vitro antitumor activity
For in vitro antitumor activity evaluation, 4T1 cells were plated in 96-well plates (1 × 10 5 cells per well) and cultured for 24 h. In the blank HMME@Lips group, the cells were treated with HMME@Lips (0, 12.5, 25, 50, 100, and 200 μg mL −1 ) in 100 μL of complete DMEM. To prove the indispensability of H 2 O 2 in the action of Cu-LDH@Lips, 4T1 cells were incubated with Cu-LDH@Lips (Cu 2+ : 0, 1.25, 2.5, 5, 10, and 20 μg mL −1 ) in 100.0 μL of complete DMEM or pre-treated with catalase (CAT) before incubation with Cu-LDH@Lips. The sonotoxicity against cancer cells was also further evaluated. 4T1 cells were initially incubated with Cu-LDH@Lips, Cu-LDH/HMME@Lips, or HMME@Lips for 12 h. Subsequently, they were exposed to US irradiation (1.0 MHz, 1.5 W cm -2 , 50% duty cycle, 1 min) and then incubated for another 12 h. After incubation, all of the relative cell viability was measured using the standard CCK-8 assay based on the absorbance at the wavelength of 450 nm.
The fluorescence signals of IR783 were detected by an ex/in vivo imaging system (VISQUE Invivo Smart-LF, Korea). The mice were sacrificed in 24 h after injection. The major organs (heart, liver, spleen, lung, kidneys) and tumors were collected for semiquantitative biodistribution analysis and imaging using the ex/in vivo imaging system.

In vivo anti-cancer effect evaluation
BALB/c nude mice were used to establish xenografted tumors models. In this experiment, tumors were planted by subcutaneously injecting 4T1 cells (1×10 7 cells suspended in PBS) into the mouse rear leg. Once tumor volumes reached 100 mm 3 , mice were divided into 7 groups randomly (n = 7) including: (1) pure saline, (2) pure saline + US, (3) HMME@Lips, (4) HMME@Lips + US, (5) Cu-LDH@Lips, (6) Cu-LDH@Lips + US, (7) Cu-LDH/HMME@Lips + US. They were i.v. injected into animals at the same doses of HMME (14 mg kg -1 ) on 0 and 7 th day. US irradiations in the above groups were performed in 6 and 12 h post-injection. Their body weight and tumor volumes were monitored every other day after administration. At the end of the treatment, mice were sacrificed and their tumors were excised, weighed, and photographed.
The pathological tissue sections of tumors were collected in 24 h post-treatment for H&E TUNEL and Ki-67 staining assay.

In vivo ROS evaluation
4T1 tumor-bearing mice were treated by pure saline, pure saline + US, HMME@Lips, HMME@Lips + US, Cu-LDH@Lips, Cu-LDH@Lips + US, and Cu-LDH/HMME@Lips + US, respectively. These mice were killed after injection for 12 h, and tumor tissues were immediately collected, and sectioned and DCFH-DA stained for CLSM observation.

Statistical analysis
Data were analyzed by OriginLab statistical software. Quantitative data are expressed as mean ± s.d. The sample size n for each group is 5. Statistical comparisons were conducted by using Student's two-sided t-test as *P < 0.05 (significant), **P < 0.01 (moderately significant) and ***P < 0.001 (highly significant).