Macrophage‐Derived Extracellular Vesicles‐Coated Palladium Nanoformulations Modulate Inflammatory and Immune Homeostasis for Targeting Therapy of Ulcerative Colitis

Abstract Ulcerative colitis (UC) is a chronic inflammatory bowel disease mainly involving the colon and rectum, which features recurrent mucosal inflammation. The excessive production of reactive oxygen species (ROS) is a trigger for pathological changes such as cell apoptosis and disordered immune microenvironments, which are crucial for the progression of UC and can be a promising therapeutic target. Nowadays, the development of targeted therapeutic strategies for UC is still in its infancy. Thus, developing effective therapies based on ROS scavenging and elucidating their molecular pathways are urgently needed. Herein, a biomimetic nanoformulation (Pd@M) with cubic palladium (Pd) as the core and macrophage‐derived extracellular vesicles (MEVs) as the shell is synthesized for the treatment of UC. These Pd@M nanoformulations exhibit multienzyme‐like activities for effective ROS scavenging, excellent targeting ability as well as good biocompatibility. It is verified that Pd@M can regulate the polarization state of macrophages by inhibiting glycolysis, and decrease neutrophil infiltration and recruitment. In this way, the colonic inflammatory and immune microenvironment is remodeled, and apoptosis is prevented, ultimately improving colonic mucosal barrier function and alleviating colitis in the mouse model. This finding provides a promising alternative option for the treatment of UC patients.

MEVs was analyzed by nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical co., Malvern, UK), and zeta potential of MEVs were also confirmed.The encapsulation efficiency of Pd@M was analyzed by randomly selecting ten fields of view, and the percentage of number of Pd nanocubes encapsulated in a single MEV was analyzed and calculated by randomly selecting one hundred Pd@M NPs.Furthermore, CD9 and CD63, the specific expression of two markers on MEVs and Pd@M were validated by western blot.According to a previous article, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to investigate the protein compositions of the macrophages (RAW264.7),MEVs and Pd@M [2].
Meanwhile, we examined the expression of the relevant target protein molecules including CD44 and Mac-1 in RAW264.7 membranes, MEVs and Pd@M, as well as the expression of their corresponding ligands P-selectin and ICAM-1 in colonic tissues before or after inflammatory stimulation by western blot.

Determination of ROS Scavenging Capability:
ESR spectroscopy was used to investigate the •OH scavenging ability of Pd@M biomimetic nanoformulations.In a typical determination, DMPO (5 μL 10 M) as a trapping agent was dissolved in PBS buffer (45 μ 50. 10 mM), followed by the addition of Fe 2+ (20 μ L 50mM) to catalyzes the production of hydroxyl radical (•OH) from H2O2.
The amount of •OH was quantified by intensity of the ESR amplitude in the absence and with the addition of different components of nanozyme.The •O2 − scavenging ability was explored by ESR spectroscopy.Under ultrasonic treatment, 70 μg of KO2 was added to 200 μL of the 18-crown-6 in DMSO solution (0.7 mM) to generate •O2 − and followed by the addition of DMPO (50 μL) as a trapping agent.Then the ESR signal was collected for quantitatively estimate the •O2 − scavenging ability without and with the addition of different components of nanozymes.Furthermore, the Superoxide Dismutase (SOD) Activity Assay Kit (Beijing Boxbio Science & Technology Co., Ltd) was also used to determine SOD-like activity of Pd@M.The reaction system of xanthine and xanthine oxidase can produce superoxide anion, O2reducible Nitrogen blue tetrazolium generates blue formazan, which has a characteristic absorption peak at 560 nm; SOD can scavenge •O2 − and thus reduce the production of formazan, so the lighter color of the reaction solution indicated the higher SOD activity.The change in absorbance at 560 nm spectrum can characterize the activity of SOD.Spectra Hydrogen peroxide content detection kit (Beijing Solarbio Science & Technology Co., Ltd) was used to evaluate the CAT activity of Pd@M biomimetic nanoformulations.The CAT activity of the nano-enzyme removes peroxides, and the remaining peroxides oxidize TiSO4 to form yellow titanium persulfate, which is absorbed at 415nm in UV−vis.As the absorbance decreased, the CAT activity of the nanozyme increased.Furthermore, the dissolved oxygen meter (Leici Co., Ltd.) was also used to assess the CAT ability of Pd@M.The higher the oxygen production rate or the final oxygen production, indicated the higher CAT like enzyme activity of the nano-enzymes.
The kinetic analysis of CAT-like enzyme activity for Pd@M NPs was calculated based on the results of oxygen generation [3].The dissolved oxygen profile of the reaction system was recorded as a function of substrate concentration and time.The enzyme kinetic parameters were acquired using the following Michaelis-Menten equation: V = Vmax[S]/(Km+[S]).In the Michaelis-Menten equation, Vmax is the reaction rate at which Pd@M NPs is saturated with substrate H2O2.[S] is the concentration of substrate H2O2.The Km value is called the Michaelis-Menten constant.
Cellular Uptake: RAW264.7 cells (Institute for Biological Sciences, Shanghai, China) were seeded at 2 × 10 5 cells per well into 6-well plates overnight, and the cells were stimulated by LPS (200 ng/ml) for 24 h and then incubated with FITC labeled Pd@M (50 μg/mL) for 1, 4 and 8 h.The cell uptake of Pd@M was tested by laser confocal microscopy (LSM 880 Basic Operation, Zesiss, Oberkochen, Germany) and flow cytometry (BD Bioscience, Brea, CA, USA).The data of flow cytometry was analyzed using FlowJo (FlowJo 10.8.1).
Similarly, to perform cell viability assay, adherent cells were then divided into six groups, which given glucose-free medium containing LPS (1000 ng/ml) for 4 h, and the control group was replaced with normal medium.Then the culture mediums were replaced with different concentrations of Pd@M (6.25, 12.5, 25 and 50 μg/mL) and co-incubated for 4 h.CCK-8 kit (Beyotime Biotechnology, Shanghai, China) was used to detect cell viability after co-incubation for 2 h, live cells that react with the reagent can be detected by a microplate reader.

Anti-ROS and Anti-apoptotic Effects of Pd@M in
Vitro: RAW264.7 cells were incubated in 6-well plates overnight.After LPS (200 ng/ml) pretreated for 24 h, different concentrations of Pd@M (6.25, 12.5, 25 and 50 μg/mL) were added and incubation for 4 h, then stained with Calcein-AM/PI (Beyotime Biotechnology, Shanghai, China) and DCFH-DA (Beyotime Biotechnology, Shanghai, China), respectively.Fluorescence microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany) were used to obtain fluorescent images of diverse groups.In addition, the proportions of live and dead cells (stained with Annexin-X and PI; Beyotime Biotechnology, Shanghai, China), and intracellular ROS levels (stained with DCFH-DA; Beyotime Biotechnology, Shanghai, China) were also quantified by flow cytometry (BD Bioscience) according to the manufacturer's instructions.

Seahorse Analysis:
The extracellular acidification rate (ECAR) was measured by Seahorse XF Glycolysis Stress Test Kit [4]. 1 × 10 4 cells were collected into the Seahorse XF Flux Pak culture microplate for 24 h and centrifuged after 4 h of treatment with various materials.Seahorse XF24 Analyzer (Agilent, USA) were used to detect the real-time ECAR according to the instruction manual.

Induction of Colitis and Treatment Protocol:
Male C57BL/6 mice (6-8 weeks old, 20 to 25 g; SLRC Laboratory Animal, Shanghai, China) were placed in groups of five mice per cage and acclimatized for 1 week before inclusion in the study.Mice were fed with 3% (w/v) DSS (molecular weight: 36,000 to 50,000, Yeasen Biotechnology Shanghai Co., Ltd) for 5 consecutive days to induce colitis [5].To confirm whether synthetic NPs have an effect on colitis, the mice were randomly divided into four groups: control group, DSS group, DSS+Pd group, DSS+Pd@M group.
The mice with DSS-induced colitis were intravenously injected with different NPs (0.5 mg/kg) for 3 consecutive days (days 6, 7 and 8), whereas the control group drank the same volume of distilled water and injected normal saline.

In Vivo Distribution and Inflammation-targeting Ability:
Normal and colitis mice were administered with Cy5.5-labeled NPs (0.5 mg/kg).Wild-type mice injected with an equal amount of Cy5.5 were set as controls.After 3 and 6 h, mice were sacrificed, the main organs and colons were collected.In Vivo Elite imaging system (Vieworks Co., Ltd, Korea) was used to observe and quantify the fluorescence distribution of NPs in the colon, heart, liver, spleen, lung, and kidney.
Histological Analysis: For HE stanning, the colon sections embedded in Paraffin were stained with hematoxylin and eosin, then analyzed by microscopy.For PAS and ROS staining, both were performed using PAS staining kit and ROS staining kit, following the manufacturer's protocol.
For immunohistochemistry and immunofluorescence assays, paraffin sections of colon sections were dried, deparaffinized, subjected to antigen retrieval and goat serum blockade.The primary antibodies, which included F4/80, CD86, and anti-MPO, were then incubated overnight at 4 °C.The next day, the specimens were rinsed with PBS three times and then incubated with corresponding fluorescent secondary antibody, followed by counterstained with 4′,6-diamidino-2phenylindole (DAPI).Primary antibodies were shown in Table S1.Immunofluorescence images were captured using a fluorescence microscope.
Western Blotting: RAW264.7 cells and colon tissues samples were homogenized in lysis buffer with a complete protease inhibitor cocktail (Roche) and BCA assay was used to measure the concentration of protein.Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), followed by incubation with primary antibodies against Raptor, HIF-1α, HK-2, PFKM, PKM2, LDHA, TNF-α, IL-1β, INF-γ, CD98, Arg-1, IL-10, Ym1 and GAPDH, and then incubation with secondary HRP-coupled antibodies.Quantitative analysis was performed with NIH Image J software.

Colon tissue ATP assay:
The ATP content in the colon was detected using the Enhanced ATP Assay Kit (Beyotime Biotechnology, S0027) in accordance with the manufacturer protocol.Tissues were lysed in the ATP lysis buffer, and the protein in each sample was homogenized with a lysis buffer after protein quantification.The ATP contents were determined by measuring chemiluminescence with a luminometer plate reader (Promega Biotech Co. Ltd., Beijing, China).

Magnetic Resonance Imaging (MRI) of Colitis:
7-Tesla dedicated animal scanner (BioSpec 70/20 USR, Bruker, Ettlingen, Germany) utilizing a one-channel circular polarized volume coil (Bruker BioSpec MRI GmbH) was employed to observe the severity of colitis.Mice were anesthetized by isoflurane inhalation and their respiration was monitored and kept constant between 30-50 breaths/min during the entire examination using 2.0-2.5% isoflurane delivered by mixed oxygen and air (20%: 80%).Prone position was selected in the scanner, and the rectal temperature was monitored and maintained at 37 ± 0.5℃, and respiratory signals were also recorded (SA Instruments Inc., Stony Brook, NY).
Biocompatibility Evaluation: Normal male C57BL/6 mice were divided into control group and Pd@M group, with three mice in each group and intravenously injected with saline and Pd@M (10 mg/kg), respectively.The mice were sacrificed at different times of administration (1, 7 and 30 days) to collect the heart, liver, spleen, lung, and kidney for HE staining.Meanwhile, the blood of the mice was collected for blood routine examination and biochemical analysis.

Figure S2 .
Figure S2.A) Typical TEM image of the obtained MEVs.B) The particle size and zeta potential of

Figure S4 .
FigureS4.A) The mean particle sizes of Pd@M after 1, 3 and 7 days of storage (n = 3).B) Tyndall effect of Pd@M nanocubes in different solutions.

Figure S5 .
Figure S5.A) The O2 generation changes of Pd@M reaction with different concentrations of H2O2 (25, 50, 100, 200 mM) at different durations.B) Michaelis-Menten kinetic analysis of catalase-like activities of Pd@M.Steady-state kinetic curves of Pd@M against H2O2 substrate and and (C) the corresponding double-reciprocal plots (n = 3).Data were expressed by mean ± SD.D) Changes of O2 production from the reaction of Pd@M with H2O2 at different pH.

Figure S6 .
Figure S6.ESR curve of scavenging superoxide anion (•O2 -) of Pd and Pd@M with DMPO as the spin trap.

Figure S7 .
Figure S7.A) Evaluation of the CAT-like activities of Pd and Pd@M by UV-Vis utilizing a CAT kit.B) Oxygen concentration changes of Pd and Pd@M at different time points.

Figure S10 .
Figure S10.Detection of Pd and Pd@M reducing the ROS levels in LPS-stimulated RAW264.7 cells by flow cytometry in vitro.

Figure S11 .
Figure S11.Detection of Pd and Pd@M effects on the ratio of live to dead cells in LPS-stimulated RAW264.7 cells by confocal microscopes in vitro.Bar = 200 μm.

Figure S12 .
Figure S12.Detection of Pd and Pd@M reducing the apoptosis levels in LPS-stimulated RAW264.7 cells by flow cytometry in vitro.

Figure S13 .
Figure S13.Oxygen consumption rate (OCR) plot from Seahorse Mito Stress assay performed on Raw264.7 cells with varied treatments.Bar chart showing the quantification of basal respiration, maximal respiration, and ATP production across the four groups (n = 5).For all panels, data are presented as mean ± SD.Statistically significant differences between groups were identified by oneway ANOVA with Tukey's post-hoc test.***p < 0.001, n.s.implies no significant difference.

Figure S15 .
Figure S15.A-B) Statistical chart of the colon length and spleen weight of four groups on day 9 (n

Figure S16 .
Figure S16.A) Typical macroscopic appearance of colons and statistical chart of the colon length (n = 5).B) The body weight changes of the control, DSS and MEVs treated groups (n = 5).C) The DAI score of mice of each group before day 9 (n = 5).Data are expressed as mean ± SD.Statistically significant differences between groups were identified by one-way ANOVA with Tukey's post-hoc test.n.s.means not significant.