Synthetic Biohybrids of Red Blood Cells and Cascaded‐Enzymes@ Metal–Organic Frameworks for Hyperuricemia Treatment

Abstract Hyperuricemia, caused by an imbalance between the rates of production and excretion of uric acid (UA), may greatly increase the mortality rates in patients with cardiovascular and cerebrovascular diseases. Herein, for fast‐acting and long‐lasting hyperuricemia treatment, armored red blood cell (RBC) biohybrids, integrated RBCs with proximal, cascaded‐enzymes of urate oxidase (UOX) and catalase (CAT) encapsulated within ZIF‐8 framework‐based nanoparticles, have been fabricated based on a super‐assembly approach. Each component is crucial for hyperuricemia treatment: 1) RBCs significantly increase the circulation time of nanoparticles; 2) ZIF‐8 nanoparticles‐based superstructure greatly enhances RBCs resistance against external stressors while preserving native RBC properties (such as oxygen carrying capability); 3) the ZIF‐8 scaffold protects the encapsulated enzymes from enzymatic degradation; 4) no physical barrier exists for urate diffusion, and thus allow fast degradation of UA in blood and neutralizes the toxic by‐product H2O2. In vivo results demonstrate that the biohybrids can effectively normalize the UA level of an acute hyperuricemia mouse model within 2 h and possess a longer elimination half‐life (49.7 ± 4.9 h). They anticipate that their simple and general method that combines functional nanomaterials with living cell carriers will be a starting point for the development of innovative drug delivery systems.

Characterization methods.Scanning electron microscopy (SEM) analyses and energydispersive X-ray spectroscopy (EDS) elemental mappings were performed on a fieldemission scanning electron microscope (Merlin, Zeiss, Germany).Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) imaging were carried out using a Talos L120c transmission electron microscope (Thermo Fisher Scientific, USA) at 120 kV.The X-ray diffraction (XRD) was measured by an X-ray diffractometer (Rigaku Miniflex, Japan).Nitrogen adsorption-desorption isotherms and the pore size distribution were determined at 77 K by N 2 adsorption-desorption measurement (BSD-PM1, 3 BSD instruments, China).The UV-Vis absorption measurements were recorded on a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan).Fluorescence emission measurements were carried out using a fluorescence spectrometer (Shimadzu RF-6000).Fourier transform infrared spectroscopy (FT-IR) spectra were measured by an FT-IR spectrometer (Spectrum Two, PerkinElmer, USA).Fluorescent images were obtained using the LAS X software on a DMi8 microscope (Leica, Germany) operated in channel mode.AFM was used to observe the morphology of RBC in contact mode, the probe was triangular silicon nitride cantilever (OXFORD instruments, PNP-TR-Au) with a nominal spring constant of 0.08 N/m, and the morphological images were analyzed and processed with the software of AFM instrument AR 16.25.226.

Section S2. Nanoparticles synthesis
ZIF-8 NPs synthesis.ZIF-8 NPs were synthesized according to the reported approach with slight modifications [1] .Prepare a solution of 229.6 mg/mL of 2-MIM and 11.9 mg/mL of Zn(NO 3 ) 2 •6H 2 O. 2 mL of 2-MIM was mixed with 2 mL of Zn(NO 3 ) 2 •6H 2 O, stirred at room temperature for 15 min, and centrifuged at 8000 rpm for 10 min to obtain a white precipitate, which was washed several times with deionized water.

UCZ synthesis.
Based on the previous method, UOX, CAT, and 2-MIM solutions were initially mixed, followed by the addition of Zn(NO 3 ) 2 •6H 2 O solution, and the mixture was agitated at room temperature for 15 min.A white precipitate was produced and washed multiple times with deionized water.UCZ with different ratios of UOX and CAT were prepared by the same procedure.

Section S3. Enzyme activity test
Degradation kinetics of UA.In brief, 2 mg UA was dissolved in 2 mL borate buffer (pH 8.5, 100 mM) and sonicated until completely dissolved.Then, the UA working solution was formed by diluting the above solution 20 times with borate buffer.The enzyme solution to be tested was added with UA working solution, respectively.The reaction system was incubated at 37 ºC, and then the absorbance of the mixtures at 290 nm was determined using UV-Vis.

Kinetic studies of H 2 O 2 generation. The kinetics of H 2 O 2 generation and elimination
were monitored according to the following procedure.Firstly, an ABTS solution (1.0 mg/ mL) was mixed with HRP to obtain a substrate solution containing 0.4 mM ABTS and 0.005 U/mL HRP. 100 μL of the substrate solution was injected into a 96-well plate.
Meanwhile, 20 μL of reaction solution of UA with UOX or UCZ was taken at a fixed time and transferred to the aforesaid 96-well plate.The absorbance change at 415 nm was recorded by a microplate reader.

Section S4. Determination of enzyme concentrations
Enzyme concentrations were determined using a BCA assay kit.Briefly, BCA working solution (BWS) was prepared by mixing 50 volumes of Reagent A and 1 volume of Reagent B and then establishing a standard curve (0.03125, 0.0625, 0.125, 0.25, 0.5, 0.75, and 1 mg/mL) with the corresponding enzymes.At the same time, 25µL of enzyme was added into the 200 µL of BWS and incubated at 37 ºC for 30 min.The absorbance at 562 nm of each sample was read out using BioTek (Agilent Technologies, USA).

Section S5. Fluorescent labeling of enzymes
Synthesis of Cy7 labeled UOX.Firstly, 50 µL of Cy7 in DMSO (10 mg/mL) was gradually added to 1 mL enzyme solution (10 mg/mL UOX containing 50 mM Boric acid buffer, pH 8.5).This reaction was carried out overnight at 4 ºC.The labeled proteins were then dialyzed with Boric acid buffer (50 mM, pH = 8.5) to remove free Cy7 and stored at 4 ºC for further use.

Synthesis of FITC labeled CAT.
First, 50 µL of FITC in DMSO (10 mg/mL) was gradually added to 1 mL enzyme solution (10 mg/mL CAT).This reaction was conducted overnight at 4 ºC.Then the labeled proteins were dialyzed to remove free FITC and stored at 4 ºC for further use.

Section S6. Enzyme stability test of UCZ
The effects of temperature, pH, and trypsin digestion on the activity of UOX were explored.The maximum activity of free UOX and UCZ was taken as 100%, respectively.
The residual activity was defined as the percentage of the maximum activity.For the temperature stability test, UOX and UCZ were mixed with UA (200 μM), respectively.Then these mixtures were incubated at different temperatures (30, 40, 50, 60, and 70 °C) for 1 h, and the absorbance changes at 290 nm were measured to evaluate the residual activities of different samples.Similarly, for the pH stability test, UOX and UCZ were placed in PBS at different pH values (5, 6, 7, 8, 9, 10, and 11) and incubated for 1 h at 37 °C.For the trypsin digestion test, UOX and UCZ were incubated with 1 mg/mL trypsin at 37 °C, and samples were taken at different times (0, 10, 20, 40, and 60 min) to determine the residual catalytic activities.

Section S7. Synthesis of red blood cell superstructures
500 μL 1X PBS (pH 5) solution containing 400 μg/mL UCZ NPs was used to suspend 5 million RBCs.After 10 s swirling and 20 s incubation, 500 μL of 32 μg/mL tannic acid in 1X PBS (pH 7.4) solution were added with 30 s vigorous mixing.After forming the red blood cell superstructures, 1X PBS (pH 7.4) was used to rinse them off and store them.

Section S8. Hemolysis assay
Native RBCs and UCZR were washed with 1X PBS (pH 7.4) solution before being suspended in 1X PBS (pH 7.4) solution at room temperature for 7 days.Water and 1X PBS (pH 7.4) solution were employed as positive controls (100% hemolysis) and negative controls (0% hemolysis).The absorbance of hemoglobin in the supernatant was measured by a BioTek microplate reader (Synergy HTX) at 540 nm to calculate the hemolysis percentage following centrifugation (200 g, 5 min).The hemolysis percentage of each sample was determined using the reported equation.Percent hemolysis (%) = 100×(sample abs 540nm -negative control abs 540nm )/(positive control abs 540nm -negative control abs 540nm )

Section S9. Chemiluminescence
The oxygen carrying capacity of RBCs was assessed using Luminol-based chemiluminescence [2] .Briefly, 5 mL of water was sonicated to dissolve 35 mg sodium perborate, 250 mg sodium carbonate, and 100 mg luminol.The luminol solution was left in the dark for 5 min without being touched.For imaging, 1 mL of luminol solution was added to 4 mL samples (20 million native RBCs or RBC superstructures) in 1X PBS (pH 7.4) solution.
percent cell viability was calculated relative to the untreated control cells.The viability of 4T1 cells in the presence of uric acid oxidation reaction catalyzed by UOX, UCZ, and UCZR was tested using a similar procedure.The native UOX, UCZ, or UCZR (20 mU/well), were first incubated with the cells (10 4 cells) for 1 h.Then equal amount of saturated uric acid working solutions (10 µL) were added into each well and incubated with cells for another 12 h at 37 °C.

Section S13. Detection of reactive oxygen species (ROS)
The reactive oxygen species detection kit utilizes a fluorescent probe 2',7'dichlorofluorescein diacetate (DCFH-DA) to detect reactive oxygen species.DCFH-DA itself has no fluorescence, and can freely permeate the cell membrane.Once inside the cell, it is hydrolyzed by intracellular esterase to produce DCFH.Intracellular reactive oxygen species can oxidize DCFH to generate DCF with fluorescence.4T1 cells inoculated in confocal culture dishes were treated with various materials.Subsequently, the original medium was replaced with a fresh culture medium containing UOX, UCZ, or UCZR mixed with UA.After 12 h, the intracellular ROS levels were monitored by DCFH-DA staining and observed under a fluorescence microscope.

Section S14. In vivo studies of uric acid degradation
Male KM mice (6 weeks old) were purchased from Hunan Slyke Jingda Experimental Animal Co., Ltd.Animal experiments were conducted with reference to animal welfare.
All animal experiments were carried out in the SPF Laboratory of the Animal Experiment Center, South China University of Technology.All animal experiments were approved by the Institutional Animal Care and Use Committee of South China University of Technology (Approval NO.2021011).Before the formal experiment, the animals were fed adaptively for one week, during which they were free to eat and drink.All mice were raised at a controlled temperature (24 °C) and relative humidity (50%) on a 12-h light/dark cycle.The suspension was prepared with 0.5% sodium carboxymethyl cellulose (CMC-Na) as the solvent.Blank group, 100 mg/kg potassium oxonate + 200 mg/kg hypoxanthine model group, free UOX group, UCZ group, and UCZR group were established.Briefly, the mice were perfused with hypoxanthine (200 mg/kg) by intraperitoneal administration, while potassium oxonate (100 mg/kg) was performed to mice by subcutaneous administration.After 1 h of modeling, all drugs were injected into mice via vein tail at a dosage of 25 U/kg (UOX) body weight.Basal uric acid levels in the blood of mice were measured before injection.Blood samples were collected from the tail vein of mice at each time points (2 h, 4 h, and 6 h) and centrifuged at 3500 r/min for 10 min to separate the plasma.The samples were frozen at -20 °C for subsequent analysis, and uric acid levels were assessed using a blood biochemical instrument.For LDH, ALT, AST, BUN, and CRE measurement, blood samples were collected from the orbital venous plexus of mice with blood collection needle.Then the blood samples were stored overnight at 4 °C in a coagulant tube, which allows the blood to coagulate naturally.Afterward, samples were centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected for blood biochemical analyzer on EMO-EXPRES.For WBC measurement, the routine blood tests were completed within 4 h with a BC-5000 vet.The contents of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in serum were assessed by mouse ELISA kits, respectively, following the provided protocols.For H&E staining analysis, the mice were euthanized 24 h after the treatment, and the main organs (heart, liver, spleen, lungs, and kidney) were taken for H&E staining analysis.

Figure S2 .
Figure S2.XRD patterns of UCZ stored at different times.

Figure
Figure S4.a) UA degradation under different ratios of UOX and CAT.b) H 2 O 2 generation under different ratios of UOX and CAT.

Figure S5 .
Figure S5.Standard curve represents the relationship between absorbance and enzyme concentration.a) UOX-Cy7 and b) CAT-FITC.

Figure S10 .
Figure S10.The reversible transfer between the oxygenated and deoxygenated states of UCZR.

Figure S12 .
Figure S12.Representative images of ZIF-8-RBC biohybrids before and after passing through the chip channels (bright field, FITC, merged image, from left to right).Scale bars: 2 μm.

Figure S16 .
Figure S16.Western blotting analysis of Native RBC membrane, UCZR membrane for characteristic CD47 of the membrane marker.

Figure
Figure S18.a) Hemolysis rate test of different concentrations of UCZ.b) Hemolysis rate test of different UCZ concentrations of UCZR.(mean± standard deviation, n = 3).

Figure S19 .
Figure S19.Cell viability assays after treated with UOX, UCZ, and UCZR in the presence of UA.