Dual enzyme-like performances of PLGA grafted maghemite nanocrystals and their synergistic chemo/chemodynamic therapy for human lung adenocarcinoma A549 cells

Advancing nanocatalytic therapies of tumors formed on non-toxic but catalytically active inorganic nanoparticles (NPs) have aroused great interest in tumor therapy recently, but the limited reactive oxygen species within tumors may limit treatment efficiency. Therefore, the combination of chemotherapy and chemodynamic therapy is a promising treatment strategy. Herein, poly(lactic


Introduction
Recently, chemodynamic therapy (CDT) formed on nontoxic but catalytically active inorganic nanozymes for intratumoral generation of high-toxic reactive oxygen species (ROS) has been 3 widely studied in tumor therapy due to its high specificity and diminished invasiveness [1]. Within the period of the CDT procedure, endogenous hydrogen peroxide (H2O2) is decomposed into ROS and hydroxyl radicals (·OH) through the medium acidic tumor microenvironment (TME) by an intratumoral Fenton or Fenton-like reaction by metal catalysts (e.g., Fe, Mn, Cu, etc.). Nanozyme is a kind of nanomaterial with natural mimic enzyme catalytic activity [2,3]. As a new generation of artificial enzyme, nanoenzyme has the advantages of simple synthesis [4], adjustable catalytic activity [5,6], high stability, low cost and easy operation [7,8]. They have become a promising alternative to natural enzymes and have attracted extensive exploration by biomedical researchers [9][10][11].
Maghemite (γ-Fe2O3) and magnetite (Fe3O4) nanoparticles (NPs) are two main iron oxide nanoparticles (IONPs) that are often utilized in a diversity of biomedical functionalities, including magnetic targeting and drug/gene delivery [12][13][14], tumor therapy [15,16], magnetic resonance imaging [17,18], cell labeling and isolation [19][20][21], magnetic biosensors [22,23], and magnetic hyperthermia [24,25]. However, ferrous Fe3O4 may increase the risk of toxicity and chemical instability [26,27]. Therefore, γ-Fe2O3 can be used as a good candidate for long-term biomedical and clinical applications. Recently, it has been reported that Fe3O4 and γ-Fe2O3 NPs have intrinsic enzyme simulation activity and have been developed as catalysts for Fenton reaction, which can catalyze the formation of •OH in situ H2O2 in solid tumors, thus leading to the death of cancer cells [28][29][30]. Moreover, Fe3O4 and γ-Fe2O3 NPs showed pH-dependent peroxidase (POD)-like as well as catalase (CAT)-like performances [31,32]. A typical example of its inherent POD-like activity is that IONPs is able to catalyze the POD oxidation substrates when H2O2 is available in acidic solutions to generate blue products [28]. Mechanism studies have shown that IONPs initially reduce 4 H2O2 to create •OH, which then organizes the oxidation of the studied substrate [33][34][35]. Dissimilar with the natural POD, IONPs mostly lost POD-like performance at neutral pH. However, we found that γ-Fe2O3 NPs directly catalyzed H2O2 to generate H2O and oxygen (O2) under such a condition, which is called CAT-like activity, and can protect cells from the stress of oxidative damage in this study [36].
In the process of cell metabolism, O2 undergoes a series of single-electron reduction to form ROS, including O2 − , O2 2− , ·OH, ·OOH radicals, H2O2, etc [37]. Low-dose ROS play an important role in cell proliferation, signal transduction, differentiation, migration, and body's resistance to the invasion of pathogen [37]. Although, unusually increased ROS levels will devastate the redox homeostasis, result in oxidative stress, and seriously harm the function and infrastructure of cellular macromolecules. The systems of enzyme including glutathione peroxidase (GPx), superoxide dismutase (SOD), and CAT protect cells from ROS damage by regulating intracellular ROS levels.
Nanozyme can also regulate intracellular ROS levels [38,39]. The ROS scavenging ability of nanozyme mainly comes from the simulation activity of SOD, which converts superoxide into H2O2 and then into O2 and H2O, thus reducing intracellular ROS level and enhancing cell activity. ROS is produced by converting H2O2 into •OH free radicals through its POD-like activity. The reaction of iron-mediated Fenton turns endogenous H2O2 into highly toxic •OH, leading to irreversible oxidative damage against tumor cells.
CDT-based Fenton reaction has been proposed as an efficacious strategy for treatment of cancers.
However, the limited H2O2 concentration in tumor cells severely limits the efficacy of CDT [40].
Thus, combining with CDT with other therapeutic methods, including chemotherapy [40], and photothermal treatment [41], is a marvelous way to improve the anticancer impact. Here, we 5 combined CDT with chemotherapy drug DOX to effectively treat lung adenocarcinoma A549 cells.
As shown in Scheme 1, NPPLGA was first prepared with superparamagnetic γ-Fe2O3 NPs as the core, followed by the surface modification with poly (lactic-co-glycolic acid) (PLGA) and the loading of the chemotherapy drug DOX. This formed nanocatalyst drug DOX-NPPLGA, through a reaction similar to Fenton under acidic TME, will show POD-like activity, produce highly toxic •OH, induce the death of cancer A549 cells, augment the sensibility of A549 cells to DOX, and enhance the therapeutic effect of CDT. In the neutral TME, the nanocatalyst exhibited CAT-like activity and could decompose H2O2 into H2O and O2, thus reducing the oxidative damage of H2O2 to A549 cells.
Furthermore, the synergistic anti-tumor effect and related mechanism of NPPLGA and DOX-NPPLGA on A549 cells were further studied in detail. technology Co., Ltd (Beijing, China). Other chemicals and specimens were from local commercial providers and the grade of the analytical reagents, unless otherwise mentioned.

Synthesis and characterization of NPPLGA
The magnetic γ-Fe2O3 NPs were synthesized by chemical coprecipitation method [42,43], then PLGA was grafted to prepare NPPLGA. The morphology, size, crystal structure and stability of γ-Fe2O3 NPs and NPPLGA were characterized in our previous study [44].

The dual POD-like and CAT-like activity of NPPLGA
In our previous work, the POD-like activity and steady-state kinetics of NPPLGA were studied in detail [44]. The CAT-like performance of NPPLGA was evaluated by CAT assessment kit conforming to the protocols of the manufacturer. In brief, 10 μL of NPPLGA solution (1 mg mL −1 ) or CAT solution (1 mg mL −1 ) was added to 50 μL buffer (T-S buffer, 33 mM phosphoric acid, 33 mM citric acid, 23 mM boric acid, pH 7.0) when H2O2 is available at different concentrations. After 5 min of reaction, the dilution of residual H2O2 was performed 50 times with T-S buffer solution and detected with 520 nm UV-VIS spectrophotometer (UV-1000, Shanghai, China). The kinetic parameters of NPPLGA were assessed utilizing the following the plot of Lineweaver-Burk (a): Where V states the primary velocity, Vmax represents the maximum velocity of reaction, S represents the concentration of the substrate, and Km states the constant of Michaelis-Menten, which is equal to the concentration of substrate at which the conversion rate is half of Vmax and represents the enzyme affinity [45]. Vmax was measured as the molar alternation by the absorbance of UV 8 based on the following Eq. (b): Further, A represent the absorbance, ε shows the coefficient of absorbance, l shows the distance length, and c represents the molar concentration with = 3.9 × 10 4 −1 −1 and = 10 mm [46]. Loading content (%) = × 100% Encapsulation efficiency (%) = × 100% Where Wt shows the weight of DOX in NPPLGA, Ws states the weight of NPPLGA, and W0 represents the primary weight of DOX in the procedure.

Cell culture and cytotoxicity assessment
The lung adenocarcinoma A549 cells were routinely cultured in an RPMI-1640 medium including 9 1% streptomycin (100 μg mL −1 ), 1% penicillin (100 U mL −1 ) and 10% heat-inactivated FBS, in a humidified incubator at 37 ℃ with an atmosphere of 95% air and 5% CO2. The cells were typically passaged at a ratio of 1:3 every 3 days to retain the growth stage exponentially.
After A549 cells reached the exponential growth stage, the cells were harvested to provide cell

Apoptosis assessment
To assess the apoptotic cells, A549 cells processed with different formulations were stained with the solution of Hoechst H33258 (2 μg mL −1 ) at room temperature (RT) for 10 min. The stained cells 11 were observed using an inverted fluorescence microscope, and unprocessed A549 cells utilized as the control.

Characterization of NPPLGA
The core-shell structure of NPPLGA can ensure the dispersion stability of γ-Fe2O3 NPs, enhance its enzyme-like activity (Fig. 1A), and improve the bio-compatibility of NPPLGA for further intracellular application. The structure and morphology of the γ-Fe2O3 NPs and NPPLGA were characterized employing the Fourier-transform infrared spectrum (FT-IR), transmission electron microscope (TEM), and X-ray diffractometer (XRD). The details can be found in our previous study [44]. The TEM images illustrated that the prepared nanoscale γ-Fe2O3 NPs had uniform morphology, the diameter range was 10-15 nm [44], and the NPPLGA exhibited a mono-dispersed sphere with a 40-50 nm uniform size (Fig. 1B). Moreover, the XRD data demonstrated that the crystalline properties and the peaks conform to the standard γ-Fe2O3 reflection, but the α-Fe2O3 phase was not observed.
Furthermore, these γ-Fe2O3 NPs were successfully modified by PLGA according to the FT-IR data [44]. To accurately analyze the elements of the NPPLGA, EDS spectroscopy characterization was performed to confirm the presence of O and Fe (Fig. 1C). Thermogravimetric analysis was used to determine the change of NPPLGA mass with temperature increase. The results showed that the weight loss of NPPLGA was severe at 200-400℃, nearly 70% at 400℃, and stable at 550℃ at about 28.8% (Fig. 1D). 14 The POD-like activity and steady-state kinetic parameters of the NPPLGA for TMB oxidation were studied in detail as we did previously [44]. The maximum primary velocity (Vmax) and the contant of Michaelis−Menten (Km) were measured utilizing the Lineweaver-Burk plot of the double reciprocal line related to the equation of the Michaelis-Menten. Kinetic analysis showed that NPPLGA ( = 0.9) had a greater affinity for TMB compared to POD ( = 1.98) at acidic pH [44]. Furthermore, the outcomes revealed that for H2O2, the Km value of NPPLGA ( = 4.41) was greater than POD ( = 0.30), proposing that NPPLGA needed a higher concentration of H2O2 to describe the same POD activity as natural POD.
We further examined the CAT-like activity of NPPLGA. As shown in the Fig. 2B, it is obvious that O2 was produced in the NPPLGA and natural CAT groups when H2O2 is available, exhibiting that both NPPLGA and natural CAT is able to catalyze H2O2 to create O2. In order to measure the enzyme parameters, we investigated the steady-state kinetics of NPPLGA through calculating the primary rates as a function of the concentration of H2O2. The catalytic procedure followed the normal Michiaelis-Menten reaction, and the Lineweaver−Burk diagram was shown in Fig. 2

Effect of CAT-like activity of NPPLGA on H2O2-induced cellular oxidative damage
We first evaluated the cytotoxicity of NPPLGA to A549 cells by MTT assay, and A549 cells were processed for 24 h with various concentrations of NPPLGA. The results indicated the processed A549 cells still maintained a high survival rate even when the concentration of NPPLGA reached 400 μg·mL -1 , indicating that NPPLGA possessed a minor impact on the ability of A549 cell proliferation (Fig. 3A).
H2O2 is a common ROS produced in cellular metabolism, and CAT and POD have progressed to preserve cells against oxidative damage induced by H2O2 [47]. This study speculated that NPPLGA with CAT-like activity had a protective effect on human lung cancer A549 cells against against oxidative damage induced by H2O2. MTT assessment was utilized to study the effect of NPPLGA at different concentrations and 5 mM H2O2 on H2O2-induced oxidative damage of A549 cells in neutral TME. The results indicated that the rate of survival for A549 cells increased with the increase of NPPLGA concentration (Fig. 3A). In addition, we further confirmed the effect of NPPLGA on H2O2-induced oxidative damage of A549 cells with various treatments (Fig. 3B). The results suggested that the A549 cells co-treated with NPPLGA and H2O2 displayed higher viability than those treated with H2O2 alone. This is because NPPLGA could decompose H2O2 to generate H2O and O2 under neutral TME conditions, thereby reducing the toxicity and side effects on cells. These results further confirmed that NPPLGA could reduce the oxidative damage induced by H2O2.

Synergistic effect of POD-like activity of NPPLGA combined with DOX on A549 cells
We first evaluated the sensitivity for A549 cells utilizing colorimetry based the POD-like activity of NPPLGA. Various numbers of A549 cells (1~8  10 3 cells) were processed by using 200 μg·mL -1 of NPPLGA. The sediments were gathered and washed three times by PBS to eliminate the unabsorbed 18 NPPLGA. When TMB and H2O2 were available in the studied system, the absorbed NPPLGA could catalyze a color reaction that could be discerned by bare eyes and be quantitatively detected the absorbance at 652 nm. When the number of A549 cells raised, the formation of TMB oxidation products changed rapidly, suggesting that more NPPLGA were absorbed by A549 cells. Utilizing this waytechnique, few cells of about 1  10 3 A549 could be detected (Figs. 4A and 4B). Furthermore, the cytotoxicity of NPPLGA to A549 cells was determined by MTT assay under acidic TME conditions. In comparison with the NPPLGA treatment, the survival fraction of cells after H2O2 treatment under acid TME was significantly reduced (Fig. 4C). The results show that NPPLGA 19 decomposed H2O2 to form •OH under the mild acidic TME of pH 6.0, triggering the production of ROS and further enhancing the toxic effect of H2O2 on cells.
To assess the intracellular •OH production, a ROS fluorescence probe DCFH-DA was employed to estimate the intracellular ROS level. Under the acidic TME, the fluorescence of A549 cells was  GSH protects normal immune system and tissue cells from oxidative damage [48]. Therefore, GSH is an important indicator for studying the effects of ROS, free radicals, and oxides on cells [49]. The results showed that H2O2, NPPLGA, NPPLGA+H2O2 and DOX-NPPLGA treatment groups all consumed the reduced GSH to varying degrees (Fig. 5D). Moreover, this phenomenon is even more obvious in the DOX-NPPLGA and H2O2 co-treated group. These results indicated that the NPPLGA in collaboration with the antineoplastic DOX can significantly consume the reduced GSH in A549 cells, making the tumor cells unable to repair the external oxidative damage, thus increasing the cell death.
We also examined the effect of various concentration DOX-NPPLGA on the cell viability related to the treated A549 cells. According to the Fig. 6A, the cell viability of A549 cells decreased with the rising DOX-NPPLGA concentration. Furthermore, the cell viability of A549 cells processed via NPPLGA was much higher than that of the group treated with NPPLGA and H2O2, and the cell survival rate of A549 cells treated with DOX-NPPLGA and H2O2 was significantly more mitigated compared to the group treated with DOX-NPPLGA (Fig. 6B). These results indicated that the anti-tumor drug DOX could enhance the oxidative damage of NPPLGA to A549 cells under acidic TME conditions, and the combination of the POD-like activity of NPPLGA with DOX could produce a synergistic anti-tumor effect on A549 cells. acidic TME (pH 6.0). Untreated A549 cells employed as control.
To observe the apoptotic cells' nuclear division, the processed A549 cells were stained with the 22 fluorescent dye Hoechst H33258, which could combine with the AT-rich zone of DNA to analyze the DNA of apoptotic cells relatively quantitatively [50]. The results showed that when A549 cells were co-treated with DOX-NPPLGA and H2O2, the alterations including nuclear peripheral accumulation, chromatin condensation, and nuclear segmentation were considerably greater than those in other groups (Fig. 6C).
In order to further investigate the apoptosis mechanism, flow cytometry was used to quantitatively examination the apoptosis level of the processed A549 cells. The A549 cells were incubated with different formulations including H2O2, NPPLGA, NPPLGA+H2O2, DOX-NPPLGA and DOX-NPPLGA+H2O2 under acidic TME conditions (pH 6.0). The number of cells in each quadrant was quantitatively analyzed. The results showed the apoptosis of A549 cells processed with NPPLGA + H2O2 was more obvious compared to the H2O2 group, and while the apoptosis of A549 cells with combined DOX-NPPLGA + H2O2 treatment was much more obvious, indicating that the A549 cells produced extra high toxicity •OH (Fig. 6D). These results also indicated that nanozyme activity of NPPLGA together with anti-tumor drug DOX could induce apoptosis and enhance anti-tumor effect under mild acidic TME.

Conclusion
In summary, we successfully constructed NPPLGA i.e. PLGA grafted γ-Fe2O3 NPs with high dual POD-like and CAT-like activities under different conditions. Under acidic TME conditions, NPPLGA showed POD-like mimetic activity, and could effectively catalyze the decomposition of H2O2 to produce high toxicity of •OH through typical Fenton catalytic reaction, leading to lung adenocarcinoma A549 cell death. At the same time, under the neutral TME condition, NPPLGA exhibited CAT-like simulation activity, and could decompose H2O2 to form H2O and O2, thereby reducing the oxidative damage of H2O2 to lung adenocarcinoma A549 cells. More importantly, NPPLGA of POD-like activity combined with the anti-tumor drug DOX, which can induce the obviously increasing apoptosis rate and enhanced anti-tumor effect for lung adenocarcinoma A549 cells. This present work therefore indicates an essential opportunity towards the development of an effective biomimetic nanoplatform with dual inorganic nanozymes to simulate the catalytic activity of lung tumor treatment.