Two-photon responsive porphyrinic metal-organic framework involving Fenton-like reaction for enhanced photodynamic and sonodynamic therapy

Designing new oxygenation nanomaterials by oxygen-generating or oxygen-carrying strategies in hypoxia-associated anti-tumor therapy is a high priority target yet challenge. In this work, we fabricated a nanoplatform involving Fenton-like reaction, Pd@MOF-525@HA, to relieve tumor hypoxia via oxygen-generating strategy for enhanced oxygen-dependent anti-tumor therapy. Thereinto, the porphyrinic MOF-525 can produce singlet oxygen (1O2) via light or ultrasonic irradiation for photodynamic and sonodynamic therapy. Notably, the well-dispersed Pd nanocubes within MOF-525 can convert H2O2 into O2 to mitigate the hypoxic environment for enhanced therapy outcome. Moreover, the two-photon activity and cancer cell specific targeting capability of Pd@MOF-525@HA gave rise to deeper tissue penetration and near-infrared light-induced fluorescence imaging to achieve precise guidance for cancer therapy. This work provides a feasible way in designing new oxygenation nanomaterials to relieve tumor hypoxia for enhanced cancer treatment. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-022-01436-3.


Introduction
As the second leading cause of human death, cancer poses a grievous threat to human health [1,2]. Facing the characteristics of infinite proliferation and easy metastasis of tumor cells, traditional radiotherapy and chemotherapy in combating cancer cells show unsatisfactory treatment results due to the multidrug resistance and severe side effects [3][4][5]. In this sense, researchers have made great efforts to develop more effective treatment methods, such as sonodynamic therapy (SDT) and photodynamic therapy (PDT), which are noninvasive tools with high selectivity for local cancers given the generated reactive oxygen species (ROS) from the photo/sonosensitizers by laser/ultrasound irradiation in an aerobic environment [6][7][8][9][10][11][12]. Unfortunately, the hypoxic environment of the tumor significantly limits their efficiencies [13][14][15][16]. To this end, it is imperative to develop a valid PDT/SDT system that can relieve tumor hypoxia.
Presently, the PDT agents used in clinic are mainly porphyrin derivatives, the excitation wavelength of which are located in the visible region (400-700 nm) leads to poor penetration limiting their further application [17,18]. However, it is worth mentioning that these porphyrin derivatives can be employed as sonosensitizers to effectively compensate for the barrier of shallow penetration depth in PDT [19,20]. Notably, the above two cancer treatment methods are highly oxygen dependent, the severe hypoxic environment of tumor greatly limits the corresponding outcome [21][22][23][24]. To face this grand challenge, various solutions have been developed to fabricate new oxygenation nanomaterials [25][26][27]. Among of them, the introduction of oxygen-generating units triggered by Fenton/Fenton-like reaction was recognized to be one of the most promising approaches [28][29][30]. Therefore, the search for platforms involving Fenton/Fenton-like reaction based on porphyrin derivatives, which can relieve tumor hypoxia via oxygen-generating strategy, to achieve enhanced therapy outcome is an extraordinary desired target.
Based on the above considerations, we fabricated a porphyrinic MOF-based nanoplatform involving Fenton-like reaction, Pd@MOF-525@HA (Scheme 1), for enhanced oxygen-dependent anti-tumor therapy

Graphical Abstract
Scheme 1 Scheme illustration showing the preparation of Pd@ MOF-525@HA, highlighting the process of enhanced photodynamic and sonodynamic therapy outcome. Thereinto, the porphyrinic MOF, namely MOF-525 (Zr 6 (OH) 4 O 4 (TCPP-H 2 ) 3 ) [31][32][33], was chosen as the target support based on the following considerations: (1) The porphyrin moiety within framework can act not only as PDT and SDT agents but also as two-photon responsive unit for near infrared (NIR) light-induced PDT with deeper tissue penetration due to its large π-conjugated system and rigid planar structure [34][35][36]; (2) The highly dispersed Pd nanocubes can be readily encapsulated, which can catalyze and react with over-expressed H 2 O 2 in cancer cells to produce hydroxyl radicals (•OH) and O 2 through Fenton-like reaction, respectively, and then triggers cell apoptosis and greatly alleviates the tumor hypoxia for enhanced oxygen-dependent cancer therapy [37][38][39][40]; (3) The hyaluronic acid (HA)-wrapping through surface modification gives rise to considerable biocompatibility and cancer cell-specific targeting ability [41][42][43]. The results show that the obtained twophoton responsive nanoplatform, Pd@MOF-525@HA, possesses deeper tissue penetration, considerable light/ ultrasonic-induced singlet oxygen ( 1 O 2 ) generation capacity, efficient oxygen generation and cancer cell specific targeting ability. Besides, we further combined twophoton fluorescence imaging to realize the combined precision anti-cancer treatment via PDT/SDT/CDT method.

Results and discussion
The composite Pd@MOF-525 with appropriate size for cellular uptake was constructed by introducing 5,10,15,20-tetracarboxy(4-carboxyphenyl)porphyrin (TCPP), pre-synthesized Zr 6 clusters and Pd nanocubes via in-situ growth method [44]. As shown in Fig. 1A, the morphology of obtained Pd nanocubes was observed by transmission electron microscopy (TEM), which has a regular cubic structure, uniform size (≈ 10 nm) and good monodispersity. Moreover, the lattice spacing of 0.22 nm observed in the high-resolution TEM (HRTEM) image was assigned to the Pd (111) plane, confirming the structure of Pd nanocubes [45]. The TEM and scanning electron microscopy (SEM) images displayed clearly that Pd nanocubes were dispersed within MOF-525 and the size of Pd@MOF-525 increased to 130 nm ( Fig. 1B, C). Simultaneously, the TEM elemental mappings were further performed for the Pd@MOF-525 composite, which proved that the homogeneous distribution of Pd element in the MOF-525 framework ( Fig. 1D and Additional file 1: Fig. S1). Besides, the fabricated MOF-525 and Pd@MOF-525 were further demonstrated by the power X-ray diffraction (PXRD). These peaks could be matched with the parent framework of MOF-525 (Fig. 1E) and the characteristic peaks of Pd nanocubes appeared at high angles of 40.0 and 46.0 (Additional file 1: Fig. S2). In addition, HA was used to further modify the surface of Pd@ MOF-525 to enhance its biocompatibility and cancerspecific targeting ability for further biological applications. The zeta potential analysis ( The successful preparation of Pd@MOF-525@HA inspired us to further study its photophysical properties. From the absorption spectrum of Pd@MOF-525@ HA ( Fig. 2A), the characteristic absorption for MOF-525 could be observed. First of all, considering the large π-conjugated feature of porphyrin moiety within frameworks, we investigated the reverse saturation absorption characteristics by z-scan experiments under near-infrared laser (800 nm) with different pulse energies (Fig. 2B) [46]. The results showed that the absorption intensity gradually enhanced with the pulse energy. Moreover, the relationship between changes of normalized (ΔT 0 , the minimum value of T NL (Z) curves) transmittance with laser pulse energy (E) was fitted in the log-log scale. As illustrated in Fig. 2C, the slope was calculated as 1.02 (the slope of the curve plus 1 is the number of effective photons absorbed) for Pd@MOF-525@HA, illustrating its two-photon absorption feature. It implies that Pd@ MOF-525@HA can serve as an excellent candidate for NIR light-induced two-photon bioimaging.
Secondly, the light-induced ROS production ability of Pd@MOF-525@HA was studied systematically. As shown in Fig. 2D, the typical absorption of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA, 1 O 2 indicator) increased under white light (1 W cm − 2 ) or ultrasonic (1.2 W cm − 2 ) irradiation within 5 min, demonstrating the excellent 1 O 2 generation ability of Pd@ MOF-525@HA. Notably, the well-dispersed Pd nanocubes within MOF-525 can convert H 2 O 2 into O 2 and then further improve the 1 O 2 generation ability of Pd@ MOF-525@HA (Fig. 2E). Meanwhile, the oxygen production capacity of Pd@MOF-525@HA was measured by the dissolved oxygen meter to determine the concentration of O 2 in the solution (Fig. 2F). It was found that the O 2 concentration continued to increase after 15 min of repeated addition of H 2 O 2 , indicating that Pd@ MOF-525@HA can continuously catalyze H 2 O 2 to produce O 2 . On the other hand, the ROS fluorescent probe 2' ,7'-dichlorofluorescein diacetate (DCFH-DA) was used to detect •OH under dark condition (Fig. 2G). The emission spectrum of DCFH-DA increased significantly after adding H 2 O 2 , which proved its excellent catalytic capacity of •OH production due to the catalytic decomposition of H 2 O 2 [47]. Moreover, electron spin resonance trapping measurements using 5,5-dimethyl-1-pyrro-Line-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) as the capture agent were further carried out to verify the type of produced ROS. The typical signal of DMPO-OOH (1:2:2:1 triplet) was observed after adding H 2 O 2 into Pd@MOF-525@HA, indicating the production of •OH (Fig. 2H). In addition, the characteristic signals of 4-oxo-TEMPO (1:1:1 triplet) for Pd@MOF-525@HA was acquired under laser and ultrasonic irradiation, manifesting the production of 1 O 2 (Fig. 2I). In this sense, Pd@ MOF-525@HA provides the possibility to relieve tumor hypoxia via oxygen-generating strategy for enhanced oxygen-dependent anti-tumor therapy.
Encouraged by the two-photon feature and HA-wrapping Pd@MOF-525@HA, cellular uptake experiments with CD44 negative cells of human liver cells (QSG-7701) and CD44 positive cells of human liver cancer cell (tumor cells HepG2) were performed to evaluate its tumor cellspecific targeting and NIR light-induced fluorescence imaging ability. As shown in Fig. 3A, in comparison to the weak fluorescence of QSG-7701 cells incubated with Pd@ MOF-525@HA under 800 nm laser irradiation, HepG2 cells group exhibited strong fluorescence. Moreover, the weak fluorescence of HepG2 cells pre-incubated with HA attributed to the preoccupation of receptor sites of CD44 cells by free HA (Additional file 1: Fig. S6) clearly demonstrated that HA-modified Pd@MOF-525 could effectively target tumor cells with CD44 receptors resulting in two-photon fluorescence imaging. Whereafter, the cytotoxicity of Pd@MOF-525@HA under different conditions was further researched by standard (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Fig. 3B, C, the cell viability of QSG-7701 cells treated by Pd@MOF-525@HA was higher than that of HepG2 cells groups with/without H 2 O 2 addition demonstrating that Pd@MOF-525@HA was harmless to normal cells and the generated •OH could give rise to decreased cell survival rate. As revealed by Fig. 3D and Additional file 1: Figs. S7-9, obviously cell apoptosis could be observed upon the light and ultrasound irradiation, and the cell viability was further decreased upon H 2 O 2 added due to the production of O 2 . Notably, in the presence of H 2 O 2 , the survival rate of the cells treated by light and ultrasound irradiation was only 10%, which demonstrated the excellent in vitro PDT/SDT synergistic therapeutic outcome.
The in vitro PDT/SDT therapeutic effect was further evaluated through confocal laser scanning microscopy (CLSM) imaging. Firstly, the intracellular 1 O 2 and •OH generated from Pd@MOF-525@HA were detected by singlet oxygen sensor green (SOSG) and aminophenyl fluorescein (APF), respectively. As shown in Fig. 4A, obviously SOSG green fluorescence signal was collected with light/ultrasonic irradiation. Moreover, the enhanced SOSG signal from light and ultrasound worked together could be further improved when H 2 O 2 added, which further unveiled that the light/ultrasonic induced 1 O 2 generation ability of Pd@MOF-525@HA could be effectively boosted by O 2 production from Fenton-like reaction. Simultaneously, the obvious green fluorescence of APF was observed due to the generation of •OH (Fig. 4B) and the enhanced APF signal from H 2 O 2 addition could be effectively quenched after adding •OH scavenger (ascorbic acid, AA). The above results corroborated the ROS generation ability of Pd@MOF-525@HA in cells for further treatment application. In addition, as demonstrated by the one-and two-photon fluorescence imaging in fixed mouse brain tissue (Fig. 4C, D), the deeper penetration depth (60 μm) of Pd@MOF-525@HA were collected upon 800 nm laser irradiation (two-photon fluorescence bioimaging). Notably, one-/two-photon fluorescence imaging of Pd@MOF-525@HA in mice displayed the same result. Compared to the images collected upon 458 nm irradiation (one-photon fluorescence bioimaging), brighter fluorescence signals could be observed under 800 nm laser irradiation. It demonstrated that the Pd@MOF-525@HA could be applied for two-photon fluorescence bioimaging and offer deeper penetration depth (Additional file 1: Fig. S10).
Based on the deep tissue penetration and excellent intracellular ROS generation of Pd@MOF-525@HA, we deployed a thoughtful protocol to evaluate the efficacy of in vitro PDT/SDT therapy. The apoptosis was detected by using Calcein acetoxymethyl ester (Calcein AM, green) and propidium iodide (PI, red). As shown in Fig. 5A, brighter PI signal was collected when light and ultrasound worked together than that of only light or ultrasound treatment groups. Moreover, much brighter PI signal could be observed upon H 2 O 2 addition demonstrating the enhanced in vitro PDT and SDT effect by alleviating hypoxia environment. The above results were further confirmed by Annexin V-FITC/PI treatment assay (Fig. 5B). In addition, the cell apoptosis was analyzed by flow cytometry using annexin V-FITC and PI as indicators under different treatment conditions. Upon light (800 nm, 100 mW cm − 2 ) and ultrasound (100 mW cm − 2 ) irradiation for 15 min, the fraction of late apoptotic cells was 56.68%, which was higher than that of only light (41.99%) or ultrasound (30.82%) treatment group (Fig. 5C) suggesting the outstanding synergistic therapeutic effect of Pd@MOF-525@HA due to its markedly enhanced ROS generation ability upon light and ultrasound worked together. With the addition of H 2 O 2 , the late apoptotic cells reached 87.73% suggesting enhanced oxygen-dependent therapeutic effect due to the generation of O 2 . Furthermore, HepG2 3D multicellular tumor spheroids of 3D cancer model (3D MCTs) were incubated with Pd@MOF-525@HA and stained with Calcein AM/PI to validate the efficiency in deep tissue. As shown in Fig. 5D, in contrast to the faint red fluorescence of the control group, MCTs formed an obvious necrotic core with bright PI fluorescence upon the NIR light and ultrasound irradiation corroborating the outstanding synergistic therapeutic effect. It manifested that Pd@MOF-525@HA can be employed as a PDT and SDT combination therapy platform guided by two-photon fluorescence imaging.
Furthermore, the systematically biosafety of Pd@MOF-525@HA was further evaluated by in vivo experiments. Firstly, we evaluated the effect of Pd@MOF-525@HA on red blood cells. As shown in Additional file 1: Fig. S11, even if the concentration of Pd@MOF-525@HA reached 100 µg/mL, it still showed ignorable influence on the integrity of red blood cells membranes or hemolysis. Secondly, it was proved by blood routine indexes test that no obvious toxicological effects were observed in healthy mice after intravenous injection of Pd@MOF-525@HA within 14 days (Additional file 1: Fig. S12). Moreover, the negligible changes of mice body weight further proved the good biosafety of Pd@MOF-525@HA (Additional file 1: Fig. S13). The above results suggested that Pd@ MOF-525@HA could be an effective therapeutic agent for cancer therapy.

Conclusions
In summary, an intelligent nanoplatform (Pd@MOF-525@HA) involving Fenton-like reaction was fabricated for enhanced oxygen-dependent anti-tumor therapy by oxygen-generating strategy. Thanks to the successful synthesis of the porphyrinic metal-organic framework, it can act not only as photo/sonosensitizers but also as two-photon responsive unit, the excellent 1 O 2 generation ability leads to considerable PDT and SDT outcome with and D Two-photon (800 nm, 0.2 W cm − 2 ) 3D fluorescence images of tissue section deeper tissue penetration. In addition, the therapy effect can be further enhanced due to the O 2 production from Pd nanocubes through Fenton-like reaction. It provides a promising platform to relieve tumor hypoxia via oxygengenerating strategy for enhanced oxygen-dependent antitumor therapy.

Materials
All starting materials were obtained from commercial supplies and used without further purification.  Shanghai Bestbio (China). Ultrapure water was used throughout.

Synthesis of pd nanocubes
PVP (53 mg), AA (30 mg), NaBr (130 mg) were dissolved into ultrapure water (4 mL), the mixture was then stirred at 80 ℃ over 5 min. 30 mg of Na 2 PdCl 4 (dissolved in 1.5 mL of ultrapure water) was poured in the above mixture and stirred for 3 h. After being cooled to room temperature, Pd nanocubes were collected by centrifugation and then stored in 1 mL of DMF solution.

Synthesis of Pd@MOF-525
Three mL of the synthesized Pd nanocubes were added to 2 mL DMF solution containing 100 mg Zr 6 clusters), then the solution was stirred at room temperature for 4 h (solution A). TCPP(50 mg) was then dissolved into DMF solution (5 mL) and dispersed by ultrasound (solution B). Then acetic acid (6 mL) was added to mixed mixture of solution A and solution B, and stirred for 12 h. Finally, the product was collected after centrifugation and washing.

Synthesis of Pd@MOF-525@HA
HA (10 mg) was dispersed in the ultrapure water (100 mL), 5 mg of Pd@MOF-525 was added after ultrasonic. After stirring for 24 h, washed with ultrapure water, and the final product was stored in ultrapure water.

Apparatus
UV-Vis absorption spectra were recorded on a UV-265 spectrophotometer. SEM was detected by REGU-LUS8230*. TEM was carried on a JEM-2100. PXRD patterns were recorded on SmartLab 9KW. Fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer. O 2 concentration was detected by dissolved oxygen meter (DO-958-S). Onephoton and two-photon imaging data acquisition and processing were performed using Lecia TCS SP8 DIVE FALCON equipped with single-wavelength laser and femtosecond laser (adjustable output wavelength: 680-1080 nm, 80 MHz, 140 fs).

Cellular uptake analysis
QSG-7701 cells (CD44-negative) and HepG2 cells (CD44-positive) were seeded onto the cell culture dishes and grown to about 70% confluency for next use. QSG-7701 cells and HepG2 cells were treated with Pd@MOF-525@HA (100 µg mL − 1 , and another dish HepG2 cells were precultured with 10 times of HA before incubation with Pd@MOF-525@HA. And after 8 h of incubation, the cellular uptake ability of Pd@MOF-525@HA was analyzed using CLSM.

Cytotoxicity assays in cells
The PDT/CDT/SDT effect of Pd@MOF-525@HA was studied by the methylthiazolyldiphenyltetrazolium bromide (MTT) assay. The Pd@MOF-525@HA stock solution is diluted with fresh medium to the required concentration (0, 30, 60, 90, 120, 150 µg mL − 1 ). Before the experiment, HepG2 cells were cultured for 24 h in 96-well plates. Then exchange the cell culture medium with different concentrations of Pd@MOF-525@HA medium solution. They were incubated at 37 °C for 8 h in 5% CO 2 atmosphere, and then irradiated by laser (800 nm, 1 W cm − 2 ) and ultrasound (1.2 W cm − 2 ) for 15 min. 100 µL of fresh medium were used to exchange the cell medium solutions 20 µL (5 mg mL − 1 ) MTT solution were added to each well following. The cell plates were then incubated for another 4 h. After removing the MTT medium, the formazan crystals were dissolved in DMSO (100 µL well − 1 ) and the absorbance was detected at 490 nm using a microplate reader. And duplicated experiments have been tested.

Live/dead assay with calcein AM/PI
After the HepG2 cells were washed with PBS solution twice, Pd@MOF-525@HA (100 µg mL − 1 ) was added to the above medium and incubated for 8 h, and then the cells were treated under different conditions. Calcein AM and PI were added to detect the cells vitality of HepG2 cells. Fluorescence images are collected by CLSM.

Determination of annexin V-FITC and PI
HepG2 cells were incubated with Pd@MOF-525@ HA (100 µg mL − 1 ) at 35 °C and 5% CO 2 for 8 h. After adding H 2 O 2 , they were irradiated with laser (800 nm, 1 W cm − 2 ) and ultrasound (1.2 W cm − 2 ) for 15 min. Then, the Annexin V-FITC (1 µM) and PI (1 µM) were added and incubated for 20 min. Fluorescence images of the cells were collected by a confocal laser scanning microscope.

Flow cytometry study
Cells seeded into the 6-well plates were incubated for 24 h, the medium containing Pd@MOF-525@HA (100 µg mL − 1 ) was used. After irradiated with laser (800 nm, 1 W cm − 2 ) and ultrasound (1.2 W cm − 2 ) 15 min, the cells were collected after centrifugation and then resuspended in binding buffer containing Propidium Iodide (PI, 10 µL) and Annexin-V FITC (5 µL) for 15 min in darkness. The signal was collected by a BD FACS Calibur flow cytometer (Beckaman/Gallios).

The one/two-photon fluorescence imaging study of Pd@ MOF-525@HA
A Lecia TCS SP8 DIVE FALCON which equipped with single-wavelength laser and femtosecond laser (adjustable output wavelength: 680-1080 nm, 80 MHz, 140 fs) was employed to achieve one/two-photon fluorescence imaging. HepG2 cells were treated with Pd@MOF-525@HA for 8 h. And then, slices were prepared from cardiac muscle tissue in Balb/c mice. The tissue sections were cut to 200 mm thickness. The tissue sections were incubated with Pd@MOF-525@HA for 30 min. The one-photon fluorescence emission was observed excitation at 458 nm (0.2 W/cm 2 ). The two-photon fluorescence emission was observed excitation at 800 nm (0.2 W/cm 2 ).

Hemolysis assay
The mice red blood cells were collected from removing serum from the whole blood by centrifugation and washing. Whereafter, the Pd@MOF-525@HA was dispersed in phosphate-buffered saline with ascending concentration series (2,5,10,20,50, and 100 µg/ mL), followed by adding into the mice red blood cells, respectively. Simultaneously, ultrapure water and phosphate-buffered saline were used as positive and negative groups, respectively. After the mixtures were incubated at 37 ℃ for 2 h and centrifugation at 2000 rpm for 10 min, the supernatant was collected and measured the absorbance at 540 nm. The hemolysis ratio was calculated using the following formula: A nanoplatform involving Fenton-like reaction, Pd@ MOF-525@HA, to relieve tumor hypoxia via oxygengenerating strategy for enhanced oxygen-dependent anti-tumor therapy has been fabricated.