A Curcumin‐Modified Coordination Polymers with ROS Scavenging and Macrophage Phenotype Regulating Properties for Efficient Ulcerative Colitis Treatment

Abstract Overexpression of classically activated macrophages (M1) subtypes and assessed reactive oxygen species (ROS) levels are often observed in patients with ulcerative colitis. At present, the treatment system of these two problems has yet to be established. Here, the chemotherapy drug curcumin (CCM) is decorated with Prussian blue analogs in a straightforward and cost‐saving manner. Modified CCM can be released in inflammatory tissue (acidic environment), eventually causing M1 macrophages to transform into M2 macrophages and inhibiting pro‐inflammatory factors. Co(III) and Fe(II) have abundant valence variations, and the lower REDOX potential in CCM‐CoFe PBA enables ROS clearance through multi‐nanomase activity. In addition, CCM‐CoFe PBA effectively alleviated the symptoms of UC mice induced by DSS and inhibited the progression of the disease. Therefore, the present material may be used as a new therapeutic agent for UC.

50 mg CoFe PBA was weighed in the bottle and 5 mL absolute ethanol was added, then the lid was put into the ultrasonicator and dispersed evenly. Then 50 mg curcumin was added and mixxed for 24 h. The material was then used deionized water washing and leaving to dry.

Adsorption energy calculation method
The value of adsorption energy can determine whether the reaction can proceed spontaneously. The calculation method of adsorption energy is as follows: Where, ΔE ads is adsorption energy, kcal/mol; Wateractivated on the E slab / Wateractivated system total energy, kcal/mol; E slab is the total energy of the crystal surface before the reaction, kcal/mol; E waterbate is the energy on the prereaction adsorption, kcal/mol. If the adsorption energy is greater than zero, it cannot proceed spontaneously; if the adsorption energy is less than zero, it can proceed spontaneously. The lower the absolute value of the adsorption energy, the more likely the reaction will occur spontaneously.

Drug loading and drug encapsulation rate
Decompose an appropriate amount of dry CCM-CoFe PBA and dilute to 2 mL of absolute ethanol with 50 μL of hydrochloric acid (2 M). The drug loading and encapsulation efficiency were calculated by the standard curve and calculation formula, and the ultravioletvisible spectrum analysis was carried out at a wavelength of 425 nm: DLE (%) = (amount of loaded drug)/(total amount of feeding drug) ×100% EE (%) = (amount of loaded drug)/(amount of drug loaded NPs) ×100%

Drug release
Dissolve 2 mg of CCM-CoFe PBA in 20 mL of PBS and Tween-20 solution (pH = 5.0), shaking continuously at 37 °C (120 rpm). 1 mL of the solution was centrifuged, and at selected time intervals (0, 1, 2, 3, 5, 18, 36, 48 and 60 hours), replaced with 1 mL of PBS solution. Curcumin release was measured by UV-Vis at 425 nm and combined with a calibration curve. The cumulative percent release was also calculated over the selected time interval.

Colloidal Stability of CCM-CoFe PBA
Add a certain amount of CCM-CoFe PBA to three simulated intestinal fluid environments (pH = 1.2 artificial gastric fluid (ChP), pH = 6.8 artificial intestinal fluid (SIF), pH = 7.4 SIF), as well as H 2 O, PBS, and PBS containing 10% FBS. Prepare a solution of 0.01mg/mL, and evaluate the colloidal stability of CCM-CoFe PBA in different biological buffers by dynamically monitoring particle size changes and Zeta potential changes.
Absorbance at 560 nm was measured immediately after illumination ended.

Determination of CAT-like enzyme activity
Determination of CoFe PBA CAT enzyme activity at room temperature by measuring the solubility of O2 produced at different reaction times (in mg L -1 ) using a specific oxygen electrode on a dissolved oxygen meter (JPBJ-608, Leici, China). Usually, different concentrations of CoFe PBA were mixed with H 2 O 2 in 5.0 mL of PBS buffer (0.1 M, pH = 7.4). Oxygen solubility (mg/L) was monitored with a dissolved oxygen meter.
Kinetic analysis was performed at room temperature in 2 mL reaction buffer solution (0.1 M PBS, pH = 7.4) and 2 mL CoFe PBA solution.
All Michaelis-Menten constants were calculated using the Michaelis-Menten saturation curves from Origin software.

Measurement of POD-like enzyme activity
50 μL CoFe PBA (4 g L -1 ) was mixed with 100 μL hydrogen peroxide (30%, m/m) and 50 μL TMB (0.12 M) in 2.8 mL sodium acetate buffer (0.1 M, pH = 4.0). After incubation for Corporation, Japan). The POD-like activity of CoFe PBA was determined in the presence of hydrogen peroxide using TMB as substrate. To assess capsule-like activity, record the absorbance of the chromogenic reaction (TMB = 652 nm) within a specific reaction time.
Absorbance was recorded over time at 625 nm.

Cytotoxicity test
The cell counting kit-8 (CCK-8) assay (Beyotime, China) and calcein acetoxymethyl ester/propidium iodide (Calcein AM/PI) cell viability/cytotoxicity assay kit (Beyotime) to detect the effects of different concentrations (16-1000 μg mL -1 ) of pure curcumin, CoFe PBA and CCM-CoFe PBA on the growth of RAW264.7 (mouse macrophages) and L929 (human epithelial cells). Co-cultivate cells and material for 3 days, and CCK-8 was detected every 24 hours. After the CCK-8 reagent was incubated with the cells for 2 hours, the absorbance was measured with a microplate reader at 450 nm, so as to detect the cell viability. After 3 days, the calcein AM/PI cell viability/cytotoxicity was detected by a kit for detecting cell viability.
Live cells (green) or dead cells (red), observed under a fluorescent microscope after incubation with CalceinAM/PI buffer for 30 min.

Intracellular ROS clearance rate detection
The DCFH-DA fluorescent probe was used to monitor intracellular ROS levels, and the excitation wavelengths were 488 nm and 525 nm, respectively. RAW264.7 cells were cultured in a 24-well plate at a density of 100,000 cells ml -1 per well, 1 mL per well, and incubated at 37 °C for 24 hours. Then, 500 ng mL -1 LPS was mixed to induce the production of intracellular ROS for 24 hours. Then, 1 mL of 3 mg L -1 CoFe PBA, 5 mg L -1 CoFe PBA, and CCM-CoFe PBA were added to the corresponding Wells for 24 h. Wash 1 time with serum-free cell culture medium, stain with DCFH-DA for 30 minutes, and wash 3 times with serum-free cell culture medium. Then, 300 μL of PBS was added to each well and observed under a fluorescent inverted microscope.

RNA isolation and real-time PCR
RAW264.7 was inoculated into a 6-well plate at a seeding density of 100,000 mL -1 , 2 mL per well, and incubated for 24 hours. To determine the effect of CCM, CoFe PBA, and CCM-CoFe PBA on proinflammatory cytokines, 200 ng mL -1 of LPS and 30 ng mL -1 of IFNγ were added to RAW264.7 cells and incubated for 24 h. Then added CCM (6 μmol L -1 ), CoFePBA (10 mg L -1 ), CCM-CoFePBA (4.5 mg L -1 ), and cultured with RAW264.7 cells for 24 hours. Total RNA was extracted using Trizol reagent (Takara) according to the manufacturer's instructions. Then 200 μL chloroform was added, violently shaken for 15 s, and then left for 5 min. Centrifuge the sample at 12,000 rpm for 15 minutes at 4 °C and transfer the supernatant to a new EP tube. After adding 500 μL of isopropanol to a new EP tube, the mixture was shaken, centrifuged (12000 rpm, 10 min, 4 °C), and the sediment was washed once with 75% ethanol. The concentration and purity of RNA was analyzed by measuring the absorbance at 260 nm and 280 nm on a NanoDropone (ThermoScientific).
CDNA was synthesized using PrimeScript_II First Strand CDNA Synthesis Kit (Takara). The primers used were synthesized by bio-synthetics, and their sequence is shown in Table S2

Measurement of cytokines
RAW264.7 was inoculated into a 6-well plate at a seeding density of 100,000 mL -1 , 2 mL per well, and incubated for 24 hours. Cells were stimulated with 200 ng mL -1 of LPS and 30 ng mL -1 of IFN-γ for 24 h and then treated with CCM (6 μmol L -1 ), CoFe PBA (10 mg L -1 ), and CCM-CoFe PBA (4.5 mg L -1 ) for 24 h, respectively. The supernatant was collected, and the concentrations of IL-1β, IL-6 and TNF-α were measured with corresponding ELISA kits (Invitrogen by Thermo Fisher Scientific).

Flow cytometry analysis
RAW264.7 was inoculated into a 6-well plate at a seeding density of 300,000 mL -1 , 2 mL per well, and incubated for 24 hours. Cells were stimulated with 200 ng mL -1 of LPS and 30 ng mL -1 of IFN-γ for 24 h. Cells were then treated with CCM (6 μmol L -1 ), CoFe PBA (10 mg L -1 ), and CCM-CoFe PBA (4.5 mg L -1 ) for 24 hours, respectively. Cells were then washed twice with PBS, and stained with CD68 antibody (rabbit, Proteintech, 1:1000) and CD163 antibody (rabbit, Proteintech, 1:1000) at 4 °C for 30 minutes. When the incubating is over, the primary antibody was washed twice by centrifugation with PBS. And then incubated with PBS (5%) containing serum for 30 min. Then the fluorescent secondary antibody goat antirabbit IgG labeled by Alexa 488 (Abcam, 1:2000) was incubated and diluted with serumcontaining PBS (5%) for 30 min at 4 °C. When the incubating is over, the secondary antibody was washed again with PBS, then resuspended with 500 μL PBS, and stored at 4 °C away from light. Flow cytometry analysis was performed using a Beckman Coulter (Life Science, USA) platform. Data analysis and plotting were performed using Flowjo software V10.

Animal experiments
Approved by the Laboratory Animal Welfare Ethics Committee of the Yangzhou University, all animal handling procedures were carried out in accordance with the 《Guidelines for the Care and Use of Experimental Animals of Yangzhou University》. Male Balb/c mice (6-7 weeks old, 18-20 g body weight), 5 mice per cage, were used in this study, housed in a temperature-controlled 22 to 25 degrees Celsius with a standard 12/12-hour light/dark Circulating Specific Pathogen Free (SPF) animal facilities. Mice were randomly divided into four groups (normal control group, CCM-CoFe-PBA drug-only group, DSSinduced colitis group, and DSS-induced CCM-CoFe-PBA-treated colitis group) (Table S2).
group. Dextran sulfate-containing sterile drinking water. Aqueous colitis in mice for 7 days.
The normal control group and CCM-CoFe PBA pure drug group were fed with sterile drinking water normally. CCM-CoFe PBA pure drug group and DSS-induced CCM-CoFe PBA treatment group were given CCM-CoFe PBA (10 mg/Kg) by gavage every day from the first day, and the other two groups received an equal amount of normal saline daily. On day 8, the mice were all sacrificed under EFE anesthesia. Visual stool consistency, weight change, and fecal bleeding were assessed daily for 8 days. Disease activity index (DAI) was measured (Table S3), including stool consistency index (0-3), body weight loss index (0-4), and stool bleeding index (0-3). On the 9 th day, the mice were sacrificed, and their entire colons were excised under isoflurane anesthesia. Measure the length of the colon and wash it gently with salt water. For histological analysis a portion of the colon is prepared. The heart, liver, spleen, lung, and kidney were collected for further pathological analysis.

Evaluation for Inflammatory Bowel Disease
The mice were weighed every day during the experiment. DAI was assessed at the end of the intervention based on the three dimensions of weight change, rectal bleeding, and stool consistency. Use a ruler to determine the length of the colon. Histopathological analysis of the colon was performed by hematoxylin and eosin staining.

Quantitative real-time polymerase chain reaction
According to the manufacturer's instructions, total RNA was extracted from the colon tissue (100 mg) of the experimental animal group using Trizol reagent (Takara). Add 1 mL Trizol reagent, and cut the tissue into pieces with scissors. Cut into pieces and blow repeatedly with a 10 mL syringe needle. Then 200 μL chloroform was added, violently shaken for 15 s, and then left for 5 min. Centrifuge the sample at 12,000 rpm for 15 minutes at 4°C and transfer the supernatant to a new EP tube. After adding 500 μL of isopropanol to a new EP tube, the mixture was shaken, centrifuged (12000 rpm, 10 min, 4 °C), and the sediment was washed once with 75% ethanol. The concentration and purity of RNA was analyzed by measuring the absorbance at 260 nm and 280 nm on a NanoDropone (ThermoScientific). CDNA was synthesized using PrimeScript_II First Strand CDNA Synthesis Kit (Takara). The primers used were synthesized by bio-synthetics, and their sequence is shown in

Western blotting
Cut the colon tissue into small pieces and put them into 1.5 mL EP tubes. RIPA lysate mixture was prepared (1 mL of RIPA lysate was added with 5 μL protease inhibitor, 5 μL PMSF, RIPA: PMSF = 100:1, now for use) and 2 μL nuclease; 1 mL of pre-cooled RIPA lysate mixture was added to each 1.5 mL EP tube. Homogenize with a homogenizer at low speed, 30 seconds each time, and ice bath for 1 minute between two homogenizations, and then placed for 5 cycles of ultrasonic lysis (ultrasonic 5 s, stop 5 s) until the tissue was completely lysed. Lysis solution immediately transfer the supernatant to a new centrifuge tube in a pre-cooled centrifuge at 1200 rpm and 4 °C for 10 minutes to denature the protein quantitatively. Protein concentrations were determined using BCA protein Concentration Assay Kit (Beyotime). The denatured proteins were separated on 10% SDS-PAGE gels and transferred to PVDF membranes (BIO-RAD). The PVDF membrane was incubated with CD68 antibody (rabbit, Proteintech, 1:1000) and CD163 antibody (rabbit, Proteintech, 1:1000) at 4°C after blocking the PVDF membrane with 5% skim milk for 2 hours, followed by overnight incubation. After washing with TBST, they were incubated with goat anti-rabbit IgG (Thermo, USA, 1:5000) for 1 hour at room temperature. After washing 3 times with PBST (PBS containing 0.1% Tween-20), positive signals were detected with ECL highsensitivity chemiluminescence detection kit (Vazyme) according to the manufacturer's instructions, and scanned with an imaging system ChemiScope 6000 chemiluminescence detector.

Pathological evaluation
The colons of the normal control group, CCM-CoFe PBA pure drug group, DSS-induced colitis group, and DSS-induced CCM-CoFe PBA treated colitis group was fixed with 10% formalin, conventionally processed, dried, embedded with OCT cryo-embedding agent, and sections with a thickness of 6 microns were processed with H&E and staining. The stained tissue was examined with a light microscope and photographed.

Immunofluorescence
The colons of the normal control group, DSS-induced colitis group, and DSS-induced CCM-CoFe PBA-treated colitis group were fixed with 10% formalin, conventionally processed, dried, and embedded in the OCT cryo embedding agent. The slices were 6 μm thick. Sections were incubated with 0.25% Triton PBS solution for 25 minutes at room temperature, washed 3 times with PBS for 5 minutes/time. Goat serum prepared in 10% PBS was blocked at room temperature for 30 minutes, and washed 3 times with PBS, 5 minutes each time. Antibody CD68 (rabbit, Proteintech, 1:200) and antibody CD163 (rabbit, Proteintech, 1:200) were diluted in 10% goat serum. The negative control group was incubated with 10% goat serum. Incubated overnight at 4 °C in a humid chamber, then washed with PBS 3 times for 5 min each. Secondary antibodies goat anti-rabbit IgG labeled by Alexa 488 (Abcam, 1:500) and goat anti-rabbit IgG labeled by Alexa568 (Abcam, 1:500) were diluted in PBS. Negative controls were incubated with mixed secondary antibodies, incubated 2 h at 37 °C, and then washed with PBS 3 times for 10 min each. Nuclei were stained with DAPI for 30 minutes and washed 3 times with PBS for 5 minutes each.
Fluorescence was observed by fluorescence inverted microscope (Olympus Corporation) after the tablets were sealed with a fluorescent sealing agent.

In vivo biosafety
The normal control group, CCM-CoFe PBA pure drug group, and CCM-CoFe PBA treated colitis group induced by DSS were observed by pathology. Seven days after gavage, blood was drawn for routine serum chemistry and hematology. Tissues such as heart, liver, spleen, lung and kidney were collected to observe the in vivo toxicity of CCM-CoFe PBA in major organs. These tissues were collected, washed with deionized water and fixed in 10% neutral buffered formalin. Routine processing, drying, OCT low temperature tissue fixation, 6 µm sections, H&E staining, inspection, light micrographs.

In vivo pharmacokinetics and biodistribution
Free CCM solution (100 mg/kg) and CCM-CoFe PBA solution (100 mg/kg) were orally administered to mice. At predetermined time points (1, 4, 8, 24 h), blood samples were collected from the orbit and centrifuged for 10 minutes (4 ℃, 5000 rpm) to obtain plasma.
And then add sodium citrate buffer (50 μL, pH = 3.0) to 150 μL plasma, incubate for 3 min, then add 1.5 mL of methanol and gently rotate to dilute. Centrifuge (4 ℃, 10000 rpm, 10 min) to obtain the supernatant, and measure the CCM content using a microplate reader under excitation/emission at 420/540 nm.
To measure its biological distribution, we sacrificed mice 24 h after oral administration of free CCM and CCM-CoFe PBA by gavage, and collected their main organs, blood, and feces. After washing and weighing, homogenize the sample and then add sodium citrate buffer (500 μL. pH = 3.0). Rotate with 1 mL of methanol for 10 min. After centrifugation to obtain the supernatant, the CCM concentration in each tissue was determined using the above method.
We conducted in vivo pharmacokinetic and biological distribution experiments on the biodegradability of nanoparticles and their clearance ability in vivo. CCM-CoFe PBA solution was orally administered to mice by gavage at a dose of 100 mg/kg of mice body weight.
Collect blood, fecal samples, and various major organs at predetermined time points (1, 4, 8, 24 h). After washing and weighing, homogenize the sample, digest the ground sample with concentrated nitric acid for 24 h, and quantitatively determine the content of Co ions using ICP-MS.

Statistical analysis
Statistical data were analyzed using GraphPad Prism7 and SPSS software. The experimental results are represented by mean ± SEM (standard error of the mean).

Density Functional Theory (DFT) Calculations
In this study, all the calculated results were obtained by using the first-principle method through the Cambridge serial total energy package (CASTEP) module in Materials Studio.