Co-Modication With Mesenchymal Stem Cells Membrane and PDA Prevents Fe3O4-Induced Pulmonary Toxicity in Micevia AMPK-ULK1 Axis

Background: Fe 3 O 4 nanoparticles are widely used in the diagnosis and treatment of diseases, but the toxicity should not be ignored. It has been reported that PDA modication can reduce the toxicity of Fe 3 O 4 and increase the biocompatibility. However, a better modication method is still worth studying. We have developed a new method to coat Fe 3 O 4 @PDA nanoparticles with mesenchymal stem cells membrane (MSCM) and evaluated the lung toxicity of the modied particles to mice. Result: We found that MSCM modication signicantly reduced the lung injury induced by Fe 3 O 4 nanoparticles in mice. Compared with Fe 3 O 4 @PDA nanoparticles, co-modication with MSCM and PDA modication signicantly reduced autophagy and apoptosis of mouse lung tissue, and reduced the activation of autophagy pathway AMPK-ULK1 axis. Thus, co-modication with MSCM and PDA prevents Fe 3 O 4 -induced pulmonary toxicity in mice by inhibiting the AMPK-ULK1 derived autophagy. Conclusion: MSCM coated Fe 3 O 4 @PDA nanoparticles were demonstrated to prevent lung damage from autophagy and reduce the toxicity of iron oxide nanomaterials. The co-modication of PDA and MSCM can improve the biocompatibility and facilitate their further bioapplication. of LC3-II/I can estimate the level of autophagy. We detected the LC3-II/I ratio of lung in four groups of mice by Western blotting. The results showed that MSCM-Fe 3 O 4 @PDA signicantly inhibited the increase of LC3-II/I ratio induced by Fe 3 O 4 .IHC staining of Beclin1 p62 and LC3-II(Figure.6D) protein in mouse lung was consistent with Western blot. These results indicate that co-modication with MSCM and PDA prevents Fe 3 O 4 -induced autophagy of lung tissue.


Background
Nano-sized Fe 3 O 4 has many specialproperties, such as superparamagnetism,which enables it to be used as magnetic resonance imaging contrast agents [1], targeted drug delivery systems [2]and hyperthermic agents [3].However, the negative effects of nano-sized Fe 3 O 4 in practical application should not be ignored. Some studies have shown that metal oxides,including Fe 2 O 3 ,Fe 3 O 4 and ZnO can induce autophagy in vivo [4,5]. In addition, it has been reported that in the process of drug treatment with nanoparticles, nanoparticles not only induce autophagy of speci c lesions, but also induce autophagy of normal cells, leading to poisoning of healthy cells [6].
As the most abundant organ of blood ow, pulmonary macrophages absorb nanoparticles through endocytosis and cause lung damage [7][8][9].Nano titanium exposure induces dose-and size-dependent cytotoxicity on human epithelial lung cells [10].Nano NiO can lead to lung injury in rats which may be related with nitrative stress reaction based on pulmonary in ammation [11].Little is known about the role of Fe 3 O 4 in pulmonary toxicity, northe mechanisms of Fe 3 O 4 -induced autophagy.
Before being used in medicine, the nanoparticles must be modi ed with some molecular layers to produce biocompatibility. Dopamine is one of the main pigments of melanin, which is rich in neurotransmitter in human body. This neurotransmitter can self-aggregate to form polydopamine (PDA) under speci c conditions. Because of the high biocompatibility and biodegradability, PDA has been widely used in the camou age of nanoparticles and various biomedical applications [12].PDA-coated magnetic nanostructureshave good thermal stability, photothermal conversion e ciencies, pH responsiveness,biocompatibility and low genotoxicity [13][14][15].Some studies have shown that the use of PDA modi cation can signi cantly improve the biocompatibility of Fe 3 O 4 , but it still shows a certain degree of cytotoxicity. Therefore, the development of new modi cation methods is an urgent need for the clinical application of Fe 3 O 4 .
The natural stem cell membrane camou aged nanoparticles were used as new drug delivery carriers [16]. The new drug delivery carrier rst separated the mesenchymal stem cell membrane, and then obtained the nanoparticles camou aged by the physical extrusion method. By passing the "passport" to these nanomaterials, the immune system avoids external rejection of particles, thereby avoiding the occurrence of immune responses [17]. The nanoparticles camou aged by stem cell membrane have the advantages of uniform particle size, good stability and biocompatibility.
Herein, wehave developed a new surface modi cation method of Fe 3 O 4 , that is, the surface of Fe 3 O 4 is modi ed by mesenchymal stem cell membrane (MSCM) after it is coated with PDA. Compared with Fe 3 O 4 @PDAparticles, this modi ed method has better biocompatibility and less lung damage. In addition, we also explored the mechanism of lung injury induced by Fe 3  nanoparticles were separated by a magnet, then washed with n-hexane and ethanol back precipitation three times, and then dispersed in toluene. Then disperse Fe3O4 nanoparticles and super particles in Tris buffer solution (10mM, pH8.5), add PDA hydrochloride monomer and stir for 3h, and then wash the centrifugal washing to remove excess self-polymerized dopamine in the solution to obtain Fe 3 O 4 @PDA superparamagnetic nanoparticles.

Preparation of Cells and MSCM-Fe 3 O 4 @PDA
MSCs were purchased from American Type Culture Collection (Manassas, VA, USA). They were cultured in DMEM α supplemented with 10% FBS in a humidi ed atmosphere of 5% CO 2 at 37°C. The suspended cells were washed 3 times with PBS and centrifuged. Then, the cell pellet was suspended in a mixture of 225 mM d-mannitol, 20 mM Tris-HCl (pH 7.5), 75 mM sucrose and 0.2 mM EGTA for 24 hours to allow them to completely dissolve. The cells were then disrupted using a Dounce homogenizer at 4 °C. After centrifuging at 3500 g for 5 min at 4 °C, the supernatant was kept and centrifuged at 20,000 g for 30 min at 4 °C. The supernatant was discarded and the membrane of MSCs was collected as an off-white precipitate for subsequent experiments.The MSCM were fully dispersed in PBS, and cell membrane vesicles were prepared using Avanti micro extruder. MSCM vesicles and Fe 3 O 4 @PDA core were mixed at a weight ratio of polymer to membrane protein of 2: 1 and coextruded through a PDA membrane with a pore size of 200 nm for 10 times to obtain MSCM-Fe 3 O 4 @PDA.

Animal experiment
As shown in Figure.1, 48 ICR mice weighing 18-22 g were purchased from SPF Biotechnology (Beijing, China) and were randomized into four groups: saline, Fe 3 O 4 , Fe 3 O 4 @PDA and MSCM-Fe 3 O 4 @PDA exposure groups. Drug was injected into mice via tail vein. The dosage of Fe 3+ was 45 mg/kg.kw/d (1/10 LD 50 ).After four weeks, the mice were euthanized. According to the previously described method, the lungs were taken out and lavaged using 500 μL PBS for three times to obtain a total of 1500 μL of bronchoalveolar lavage uid (BALF).Then half of the lungs of each group were stored at -80 degrees to extract protein and mRNA, and the rest were xed with 4% formalin and stored at room temperature for staining.

H&E staining
Para n sectionswere dewaxed to water, dyed with Harris hematoxylin for 3-8 min and washed with water.Then the sectionswere immersed 1% Hydrochloric acid alcohol for several seconds and washed with water.Next, the sections were dyed in eosin solution for 1-3 min, then dehydrated gradiently and sealed with gum. The sections were observed under microscope and the images were collected.

Western blotting
The mice lung tissues were lysed with ice-cold RIPA lysis buffer containing phosphatase-protease inhibitor cocktails (Beyotime Biotechnology, Shanghai, China). The concentration of protein was measured by BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein lysates were subjected to SDS gel electrophoresis, immunoblotted with primary antibodies, and then the matched secondary antibodies. Western blot results were quanti ed by using the Image J software. Antibodies

RNA isolation and qRT-PCR
Total RNA was extracted from tissues using TRIzol (Invitrogen) following the manufacturer's instructions. 1mg of total RNA was subjected to reverse transcription using the One-Step cDNA Synene, Beijing, China) was used for RT-qPCR. The PCR primer sequences are shown as Table 1. Tissue slides were depara nized and rehydrated, then incubated with primary antibodies overnight at 4 °C. Next, the sections were incubated with matched secondary antibody for 30 minutes at room temperature, stained with DAB, and counterstained with hematoxylin.

Statistical analysis
All statistical analyses were carried out using the SPSS 19.0 statistical software package (SPSS Inc., Chicago, IL, USA). The data were presented as the mean ± standard deviation (SD) at least three independent experiments. For the analysis of IHCpictures, 5 visual elds were randomly selected to take photos, and the average score was calculated.Data were analyzed using one-way analysis of variance for comparison between groups.p<0.05 was statistically signi cant.

Co-modi cation with MSCM and PDA prevents Fe 3 O 4 -induced lung injury in mice
The total amount of protein and neutrophils in BALF can re ect the lung damage of mice. As shown in Table 2

Co-modi cation with MSCM and PDA prevents Fe 3 O 4 -induced activation of AMPK-ULK1 axis
Ulk1 complex can be used as a bridge between upstream energy sensing protein AMPK and downstream autophagy formation. Ulk1 is highly phosphorylated by AMPK, and then Beclin1 is activated, which is necessary for autophagy initiation.We then examined whether co-modi cation with MSCM and PDA attenuated

Discussion
Iron oxide nanoparticles have gained extensive attention in biomedicine because of their excellent magnetic properties [18,19].Tagged molecule can now be directed to a desired location with help of an external magnet [20].
Only precaution one has to take is to select a magnetic material having low toxicity and its ability for binding a biomolecules [21].Unfortunately, unmodi ed metal nanoparticles are often reported to have toxic effects, including hepatotoxicity [22,23], nephrotoxicity [24,25] and myocardial toxicity [26,27]. In this study, we focused on the lung toxicity induced by Fe 3 O 4 and how to use new modi cation methods to reduce the negative effects of Fe 3 O 4 .
The damage of nanoparticles to organs is usually caused by cell apoptosis.Apoptosis is mainly caused by the activation of caspase, a cysteine protease, by signal stimulation in vitro or intracellular [28]. Autophagy, like apoptosis, is an important physiological response of cells [29]. Under normal physiological conditions, autophagy remains at a low basic level, but it can be induced by some speci c cell stress states [30].Autophagy may have different effects on cells. Under speci c circumstances, autophagy can maintain cell survival, which is called protective autophagy [31], and sometimes autophagy can cause secondary apoptosis [32].We injected Fe 3 O 4 into the tail vein of mice and took out the lungs of mice four weeks later for observation. We found that Fe 3 O 4 led to the destruction of mice lungs and the increase of apoptosis of lung cells. This is consistent with Stern's report [33]that demonstrated elevated levels of autophagic vacuoles upon exposure of cells to certain nanomaterials and the interaction between nanomaterials and the autophagy pathway is disruptive, resulting in obvious morphological changes and nally cell death.
In order to further push the limits of nanoparticle performance and function, a paradigm shift to a bionic design strategy has recently emerged [34]. A new class of biomimetic nanoparticle has been reported that combines the advantages of natural, cell membrane-derived vesicles with more traditional synthetic nanoparticulate platforms [35,36]. This new coating technology of grafting the cell membrane onto the surface of nanoparticles has pushed the nanomedicine to a new stage of development. The camou aged nanoparticles can better adapt to the complex physiological environment [37]. Through homologous recognition, they can not only avoid the elimination of the immune system, but also greatly enhance the targeting characteristics, which has great application potential.
These particles generally employ a core-shell design, with a layer of cell membrane coated around a preformed nanoparticle core. Initially, membrane-coated nanoparticles were fabricated using a combination of red blood cell membrane and poly(lactic-co-glycolic acid), a biodegradable polymer, via a co-extrusion approach [38]In this experiment, we used the umbilical cord derived MSCM as a vesicle to wrap PDA modi ed Fe 3 O 4 particles.Given the demonstrated low immunogenicity and strong targeting of stem cell membrane [39], we hypothesized that this new modi cation technology can greatly reduce the organ toxicity of Fe 3 O 4 particles in vivo.
It is worth noting that mice injected with Fe 3 O 4 particles coated with MSCM showed signi cantly lower lung injury, lower apoptosis and autophagy levels, which indicated that the co-modi cation of MSCM and PDA could signi cantly improve the biocompatibility of Fe 3 O 4 particles modi ed by PDA alone.
Recently, it has been established that there is a direct link between AMPK, which is an energy sensor and is activated by glucose starvation [40], and ULK1 in triggering autophagy.Based on their doses and physico-chemical characteristics, nanoparticles have the capability of producing reactive oxygen species (ROS) or otherwise initiating signaling pathways that in addition to regulating autophagy, can eventually modulate different cell fates, including necrosis, necroptosis, apoptosis [41]. The intrinsic pathway of apoptosis is initiated by mitochondrial membrane permeabilization, various types of oxidative stress including hypoxia, DNA damage, and growth-factor deprivation [42]. Mitochondrial ROS production is a tightly regulated redox signal that transmits information from the organelle to the cell [43].It has been proposed that ROS can directly regulate AMPK activity independently of changes in adenine nucleotides [44,45]

Declarations
Informed consent for publication was obtained from all participants. All the authors have agreed to publish this research.

Availability of data and materials
All data generated or analysed during this study are included in this published article. The datasets used or analysed during the current study are available from the corresponding author on reasonable request.  As shown in Figure.1, 48 ICR mice weighing 18-22 g were purchased from SPF Biotechnology (Beijing, China) and were randomized into four groups: saline, Fe3O4, Fe3O4@PDA and MSCM-Fe3O4@PDA exposure groups. Drug was injected into mice via tail vein. The dosage of Fe3+ was 45 mg/kg.kw/d (1/10 LD50).After four weeks, the mice were euthanized. According to the previously described method, the lungs were taken out and lavaged using 500 μL PBS for three times to obtain a total of 1500 μL of bronchoalveolar lavage uid (BALF).Then half of the lungs of each group were stored at -80 degrees to extract protein and mRNA, and the rest were xed with 4% formalin and stored at room temperature for staining.    The results showed that MSCM-Fe3O4@PDA signi cantly inhibited the increase of LC3-II/I ratio induced by