Size and crystallinity effects on enzymatic activity and
anti-inflammatory properties of cysteine-assisted
Prussian blue nanozymes

Yilin He, Alberto Bianco, Cécilia Ménard-Moyon

► To cite this version:

Yilin He, Alberto Bianco, Cécilia Ménard-Moyon. Size and crystallinity effects on enzymatic activity and anti-inflammatory properties of cysteine-assisted Prussian blue nanozymes. Journal of Colloid and Interface Science, 2024, 679, pp.930-938. 10.1016/j.jcis.2024.10.008. hal-05227647

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Distributed under a Creative Commons Attribution 4.0 International License
Size and crystallinity effects on enzymatic activity and anti-
inflammatory properties of cysteine-assisted Prussian blue
nanozymes

Yilin Heª, Alberto Biancoª,*, and Cécilia Ménard-Moyonª,ᵇ*

E-mail: a.bianco@ibmc-cnrs.unistra.fr, c.menard@ibmc-cnrs.unistra.fr

<footnote>ª CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572,
University of Strasbourg, ISIS, 67000 Strasbourg, France</footnote>

<footnote>ᵇ University of Strasbourg Institute of Advanced Study (USIAS), 67000 Strasbourg,
France</footnote>

<footnote>* Corresponding author</footnote>
**Abstract**

Prussian blue nanoparticles (PB NPs) exhibit multiple enzymatic activities, such as superoxide dismutase-like, catalase-like, and peroxidase-like activities, which enable them to effectively scavenge reactive oxygen species (ROS) and demonstrate anti-inflammatory effects. To further enhance the enzymatic activity of PB NPs, it is crucial to explore the relationship between their physicochemical properties, such as size and crystallinity, and their enzymatic performance. In this study, PB NPs were synthesized using different pH and varying concentrations of cysteine (Cys) as stabilizer. As the size decreases, crystallinity is gradually reduced, and defects increase. Cys-PB NPs with a smaller size and lower crystallinity exhibited high peroxidase-like activity, effectively reducing inflammation and scavenging intracellular ROS *in vitro*. Additionally, the stability of Cys-PB NPs plays a critical role in their anti-inflammatory properties, with higher stability favouring anti-inflammatory effect.

**Keywords:** Prussian blue nanozyme, Anti-inflammatory, Size and defects-dependent enzymatic activity
# 1. Introduction

Prussian blue nanoparticles (PB NPs), known for their favourable biocompatibility and unique properties, have garnered significant attention in the biomedical research. For example, PB NPs have been developed as effective agents for biomedical imaging (e.g., magnetic resonance imaging and photoacoustic imaging). [1,2] They have shown great potential for drug delivery, photothermal therapy (PTT) and chemodynamic therapy (CDT) in anticancer therapy. [3,4] Notably, PB NPs exhibit enzymatic-like activities including catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD), making them highly effective in scavenging reactive oxygen species (ROS). [5-7] Their enzymatic properties, attributed to the various redox states of PB, present huge potential for treating different diseases, such as inflammatory bowel disease, wound healing and osteoarthritis. [8-12]

Nanozymes are nanomaterials and nanoparticles that display enzyme-like characteristics. With their high catalytic activity, low cost and good stability, nanozymes have drawn significant interests in exploring their diverse applications. [13] Interestingly, studies have revealed that the enzymatic activity of many nanozymes is greatly dependent on their particle size, shape and defects. [14-16] It is worth noting that the framework of PB NPs usually contains $[Fe(CN)_6]$ vacancies occupied by coordinated $H_2O$ molecules, and these lattice defects have a significant impact on their properties, such as the electrochemical performance. [17-19] The ROS scavenging enzyme activity of PB NPs can be effectively regulated by controlling the size, with smaller-sized NPs exhibiting the strongest anti-inflammatory activity and wound healing effects. [20,21] To improve the activity of PB nanozymes, it is necessary to understand the relationship between their structural parameters and enzymatic activities. Therefore, there is a significant need to study the factors that affect the enzymatic activity of PB
through a controlled synthesis.

During the synthesis of PB NPs, stabilizers can be used as surface capping agents to control the size and prevent agglomeration. Given the strong coordination ability of thiol and carboxyl groups with iron, cysteine (Cys) was selected as a stabilizer in this study. We found that the concentration of Cys had an effect on the shape of PB NPs. The control of the reaction conditions allowed tuning the [Fe(CN)₆] vacancies and crystallinity of Cys-PB NPs. Interestingly, Cys-PB NPs with a highly defective structure exhibited high POD enzymatic activity. Moreover, Cys-PB NPs displayed an excellent biocompatibility and defect-dependent anti-inflammatory effect in vitro, providing a potential approach for the design of PB NPs for future treatment of anti-inflammatory diseases.

## 2. Experimental section

### 2.1 Materials

Potassium hexacyanoferrate(II) trihydrate ($K_4[Fe(CN)_6]$, ≥ 99.5%), iron(III) chloride hexahydrate ($FeCl_3$, ≥ 99.5%), phorbol 12-myristate 13-acetate (PMA, ≥ 98.0%), 3,3',5,5'-tetramethylbenzidine (TMB, ≥ 98.0%), SOD assay kit, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, ≥ 94%) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride ($[Ru(dpp)_3]Cl_2$, ≥ 98.0%) and Cys (≥ 99.0%) were from Alfa Aesar. Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 medium, PBS and fetal bovine serum (FBS) were ordered from Corning Incorporated. MTS was bought from Promega Corporation and cytokine ELISA kits from BD Biosciences. The water used in this work was Milli-Q® water.
## 2.2 Synthesis of Cys-PB NPs

Cys-PB NPs were prepared using a co-precursor method by controlling the pH and amount of Cys. In detail, 0.25 mmol (or 0.5 mmol) of Cys was dissolved in 20 mL of 1.0 mM $FeCl_3$ solution and stirred at 60°C. Subsequently, the same amount of cysteine was dissolved in 20 mL of 1.0 mM $K_4Fe(CN)_6$ solution and added dropwise to the $FeCl_3$ solution. The pH was adjusted to 1 (or 2). As a result, four types of Cys-PB NPs were obtained: those synthesized with a total 0.5 mmol of Cys at pH 1 (denoted as Cys-PB-1), 1.0 mmol of Cys at pH 1 (denoted as Cys-PB-2), 0.5 mmol of Cys at pH 2 (denoted as Cys-PB-3) and 1.0 mmol of Cys at pH 2 (denoted as Cys-PB-4).

## 2.3 Characterization

Transmission electron microscopy (TEM) was performed on a Hitachi 7500 (Tokyo, Japan) with an accelerating voltage of 80 kV, equipped with an AMT Hamamatsu camera (Tokyo, Japan) to observe the morphology and size of Cys-PB NPs. X-ray diffraction (XRD) patterns (Bruker D8 X-ray diffractometer) were collected to study the crystal structure of Cys-PB NPs. Dynamic light scattering (DLS) and zeta potential measurements were recorded using a DTS1070 cell on a Malvern Zetasizer Nano ZS (Malvern Instruments) at 25°C to evaluate the hydrodynamic diameter and stability of Cys-PB NPs. Elemental analyses were carried out using a Thermo Flash 2000, Thermo Scientific. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) analyses were performed on a Varian 720 ES instrument. UV-Vis absorption spectra were done on a Cary 5000 spectrophotometer. Thermogravimetric analysis (TGA) was performed using a TGA1 Mettler Toledo apparatus using platinum pans. The TGA temperature ranged from 40 to 600°C with a ramp of 10°C/min under a flow rate of $N_2$ at 50 mL/min. A Thermo Scientific KAlpha X-ray photoelectron spectrometer was employed to perform X-ray photoelectron spectroscopy (XPS). Cys-PB NPs were
lyophilized to get a powder and deposited onto a scotch tape (3MTM EMI Copper Foil Shielding Tape 118) with a spot size of 400 µm. The C 1s photoelectron binding energy was set at 284.8 eV and served as the reference for aligning other peak positions. The vacuum chamber was maintained between 10⁻⁸ and 10⁻⁹ bar, with an Al anode utilized as the X-ray source emitting at 1486 eV. Survey spectra were averaged from 10 scans, employing a pass energy of 200.00 eV and a step size of 1 eV. High-resolution Fe 2p, N 1s, C 1s and O 1s spectra were obtained from an average of 20 scans, with a pass energy set at 50.00 eV and a step size of 0.1 eV. Three repeated measurements were performed on each sample. Fourier-transform infrared spectroscopy (FTIR) spectra were collected on a FT-IR 4700 Fourier Transform Infrared Spectrometer (JASCO) equipped with an attenuated total reflection diamond.

## 2.4 Enzyme-like activity evaluation

The SOD-like enzymatic activity of Cys-PB NPs at various concentrations was evaluated using a commercial SOD test kit ordered from Sigma-Aldrich, following the manufacturer's instructions. The test kit provides a colorimetric method for quantitatively measuring SOD activity. In the assay, O₂⁻ is generated by xanthine oxidase in the presence of xanthine. The O₂⁻ anions react with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1), producing a yellow formazan dye with a specific absorbance at 450 nm. SOD-like enzymatic activity of Cys-PB NPs can scavenge O₂⁻, thus decreasing the amount of produced formazan dye. Therefore, the absorbance at 450 nm can be used to quantify the SOD activity.

CAT-like enzymatic activity of Cys-PB NPs was investigated by evaluating generated oxygen from H₂O₂ decomposition. [Ru(dpp)₃]Cl₂ was used as an oxygen probe, as its fluorescence is quenched by oxygen. Typically, Cys-PB (100 µL, 100
µg/mL) was mixed with different concentrations of H₂O₂ in a 96-well plate reader, followed by the addition of [Ru(dpp)₃]Cl₂ (10 µL, 10 µg/mL). The fluorescence intensity was measured before and after a 10-min incubation (excitation at 488 nm; emission at 620 nm).

POD-like enzymatic activity was measured using TMB as a substrate. Cys-PB NPs (10 µL, 4 µg/mL) and TMB (10 µL, 10 mg/mL) were added into 230 µL of acetic acid-sodium acetate (HAc-NaAc) buffer solution containing 1000 mM H₂O₂. The absorbance at 650 nm was measured over time by a microplate reader (Multiskan FC, Thermo Fisher Scientific). The steady-state kinetic assays were performed by using either H₂O₂ or TMB as the substrate. In detail, the steady-state kinetic assays of Cys-PB NPs with H₂O₂ as the substrate was conducted by adding Cys-PB NPs (10 µL, 4 µg/mL) and TMB (10 µL, 10 mg/mL) into 230 µL of HAc-NaAc buffer solution containing various concentrations of H₂O₂ (0, 50, 100, 200, 400, 800 and 1000 mM). The steady-state kinetic assays of Cys-PB NPs with TMB as the substrate was conducted by adding Cys-PB NPs (10 µL, 4 µg/mL) and various concentrations of TMB (0, 0.5, 1, 2, 4, 6, 8 and 10 µL, 10 mg/mL) into 230 µL of HAc-NaAc buffer solution containing 1000 mM H₂O₂. The absorbance was recorded at different time points. Then, the Michaelis–Menten equation was used to fit the data and calculate the turnover number $k_{cat}$, which indicates the catalytic activity of the enzyme. The calculation of $k_{cat}$ is detailed in Supporting Information.

## 2.5 Stability evaluation

The stability of Cys-PB NPs in PBS was evaluated at 37°C by UV-Vis-NIR spectroscopy. Briefly, Cys-PB1, Cys-PB2, Cys-PB3 and Cys-PB4 NPs were dispersed in PBS at a concentration of 100 µg/mL, followed by incubation up to 14 days. The absorbance was measured at day 0, 1, 3, 5, 7 and 14. The normalized absorbance at λmax
at different time points was calculated.

## 2.6 Cell culture and cell viability test

Mouse macrophage RAW 264.7 cells and human monocytic THP-1 cells were cultured in DMEM and RPMI 1640 cell culture medium with 10 mM N-(2-hydroxyethyl)-piperazine-N''-ethane sulfonic acid (Lonza BioWhittaker), 10 µg/mL gentamycin (Lonza BioWhittaker), 0.05 mM β-mercaptoethanol (Lonza BioWhittaker) and 10% FBS. Cells were maintained under a humidified atmosphere of 5% CO₂ at 37 °C, and the culture medium was changed every 2 days. Briefly, RAW 264.7 cells were seeded in 96-well plates at a density of ~1×10⁴ cells per well. THP-1 cells were seeded in a plate at a density of ~1×10⁴ cells per well and pre-incubated with 200 nm PMA for 72 h to differentiate THP-1 from monocyte state into macrophages.[22] Then, the cell culture medium was replaced with fresh one containing Cys-PB NPs with different concentrations. The materials were incubated for 24 h and washed with PBS twice. MTS reagent was diluted 10 times in a cell culture medium and then added into the plates at 100 µL per well. Then, the cells were incubated at 37°C for 1 h. The absorbance at 490 nm was measured by a microplate reader (Multiskan FC, Thermo Fisher Scientific).

## 2.7 Anti-inflammation in vitro

The anti-inflammatory ability of Cys-PB was evaluated on inflammatory model RAW 264.7 cells stimulated with LPS (1 µg/mL). Firstly, RAW 264.7 cells were seeded in 96-well plates at a density of ~1×10⁴ cells per well. Then, the cells were incubated together with LPS and different concentrations of Cys-PB for 24 h. The cell supernatant was collected to determine the level of tumour necrosis factor (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) using ELISA test kits in accordance with the
manufacturer's instructions. Meanwhile, the cell viability was tested using MTS assay,
as described above.

DCFH-DA was used to study ROS scavenging ability of Cys-PB. Briefly, RAW
264.7 cells were seeded in 96-well plates at a density of ~1×10⁴ cells per well, following
incubation of Cys-PB (50 µg/mL) and LPS (1 µg/mL). After 24 h of incubation, the
cells were washed with PBS 2 times and 50 µL of a solution of DCFH-DA (5 µM) in
PBS were added in each well. After incubation for 30 min at room temperature, the
fluorescence intensity was detected by a microplate reader (Multiskan FC, Thermo
Fisher Scientific) (excitation at 480 nm; emission at 530 nm). The cells were also
observed by fluorescence microscopy using ZEISS Axio Observer Z1 spinning disk
confocal microscope. The images were treated with ImageJ software.

## 2.8 Statistic

The experiment data were calculated from at least three independent
experiments and presented as means ± SD. Student's t-test was done to assess statistical
significance.

# 3. Results and discussion

Cys-PB NPs were synthesized through a traditional co-precipitation method involving FeCl₃ and K₄[Fe(CN)₆] in an aqueous solution. Cys was added to control the size of PB NPs due to the strong chelating effect of the thiol, amine and carboxyl groups of Cys to Fe³⁺. The reaction rate of Fe³⁺ with K₄[Fe(CN)₆] can be effectively slowed down by Cys, resulting in a slower nucleation growth. Indeed, due to the protonation of the carboxyl and amino groups, their chelating ability decreases at low pH. Therefore, pH (1 or 2) and the molar ratio of Cys/FeCl₃/K₄[Fe(CN)₆] (25:1:1 or 50:1:1) was
controlled to obtain PB NPs of different sizes and crystallinities, namely Cys-PB1 (pH 1, 25:1:1), Cys-PB2 (pH 1, 50:1:1), Cys-PB3 (pH 2, 25:1:1) and Cys-PB4 (pH 2, 50:1:1). TEM images revealed that the size of PB NPs decreased significantly with the reduction in pH and Cys concentration (Fig. 1a). Cys-PB1 and Cys-PB2 exhibited an irregular particle shape, while Cys-PB3 and Cys-PB4 showed a cubic morphology. The size distribution by TEM of Cys-PB NPs was assessed. Due to the irregular and disordered assemblies in Cys-PB1 and Cys-PB2, it was impossible to obtain accurate measurements of their size by TEM. Therefore, the size of only Cys-PB3 and Cys-PB4 was assessed using TEM. Cys-PB3 exhibited a narrow size distribution, with 71% of NPs ranging from 80 to 120 nm (Fig. S1). In contrast, Cys-PB4 displayed a larger size, with 91% of NPs in the range 180-240 nm. XRD analysis indicated that the four Cys-PB NPs exhibited similar diffraction peaks, corresponding to the standard pattern of PB crystals (Fig. 1b). The intensity of the peaks increased with the particle size, while the peak width narrowed. The results indicated that the crystallinity of Cys-PB NPs improved as the size increased, with Cys-PB1 exhibiting the lowest degree of crystallinity.
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** (a) TEM images and (b) XRD patterns of Cys-PB1, Cys-PB2, Cys-PB3 and Cys-PB4 NPs.</figcaption></figure>

Consistent with TEM results, DLS measurements showed that the size of Cys-PB increased with the increase in pH and Cys concentration (Fig. 2a and S2). The intensity-weighted size distribution for Cys-PB3 and Cys-PB4 exhibited similar trends as the number-weighted size distribution, while Cys-PB1 and Cys-PB2 showed larger sizes in the intensity-weighted distribution. This is likely due to the fact that Cys-PB1
and Cys-PB2 tend to form disordered assemblies of small NPs (as observed by TEM). Larger particles contribute more to light scattering than smaller particles, which makes DLS more sensitive to the presence of larger particles, leading to an overestimation in the intensity-weighted distribution results.[23] Therefore, the number-weighted size distribution of Cys-PB1 and Cys-PB2 assessed by DLS corresponds more closely to the size determined by TEM. The polydispersity index indicated that Cys-PB NPs possessed a homogeneous size distribution (Table S1). The zeta potential was in the range of – 25.6 to – 32.9 mV, which is generally associated with a good colloidal stability (Fig. 2b and S3). Cys-PB NPs were then analysed by ICP-AES and elemental analysis allowing to calculate the [Fe(CN)₆] vacancies, which corresponded to 0.33, 0.28, 0.18 and 0.13 for Cys-PB1, Cys-PB2, Cys-PB3 and Cys-PB4 NPs, respectively (Table S2).[17,18,24] The defect content of Cys-PB2 was consistent with the defect content of PB NPs reported in the literature.[17] The results showed that by increasing the amount of Cys and raising the pH, the defect content can be effectively reduced. Cys-PB NPs with a lower crystallinity and smaller size have more [Fe(CN)₆] vacancies in their crystal framework. This can be explained by the fact that during the synthesis of PB NPs, the rapid process due to the lower pH and lower amount of Cys generally induces the formation NPs with a higher content of [Fe(CN)₆] vacancies compared to a slower process, leading to a certain amount of lattice defects in the crystal structures.[18,19] Besides, Cys-PB NPs were also characterized by UV-Vis-NIR spectroscopy showing a maximum absorption at 700, 707, 740 and 772 for Cys-PB1, Cys-PB2, Cys-PB3 and Cys-PB4, respectively, which is attributed to the intervalence charge transfer occurring between Fe²⁺ and Fe³⁺ through the bridging cyanide moiety (Fig. 2c). TGA under inert atmosphere was used to evaluate the water content on Cys-PBs (Fig. 2d). The weight loss between 30°C and 250°C was attributed to the adsorbed
and interstitial water,[25] indicating that Cys-PB1, Cys-PB2 and Cys-PB3 had a similar water content, while Cys-PB4 had a slightly lower water content. The weight loss between 250°C and 400°C was assigned to the decomposition of the PB frameworks. The lower weight loss of Cys-PB4 may be due to the higher crystallinity, which results in more durable crystal structure. Moreover, Cys-PB NPs were characterized by XPS, confirming the presence of C, N, Fe and O atoms (Fig. 2e). Interestingly, the atomic percentage of sulfur assessed by XPS was less than 0.10%, which suggests that cysteine helped controlling the size during the growth process, but the amount of cysteine in Cys-PB NPs was negligible. The high-resolution spectra of the Fe 2p, O 1s, N 1s and C 1s peaks showed a similar composition for all Cys-PB NPs (Fig. S4). FTIR spectra of Cys-PB NPs exhibited the characteristic peak at 2065 cm⁻¹ corresponding to the stretching vibration of the -C≡N bond (Fig. 2f). Additionally, the peaks at 1600 and 1407 cm⁻¹ were attributed to the in-plane bending modes δ(H-O-H) and δ(O-H) of water molecules in PB NPs.[26]

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 2.** (a) Number-weighted size distribution (assessed by DLS), (b) zeta potential, (c) UV-Vis spectra (concentration of 100 µg/mL in water), (d) TGA (under nitrogen atmosphere), (e) XPS survey and (f) FTIR spectra of Cys-PB1, Cys-PB2, Cys-PB3, and</figcaption></figure>
Cys-PB4 NPs.

We then evaluated the enzymatic activity of Cys-PB NPs and their capacity to scavenge ROS by evaluating the SOD, CAT and POD activity. SOD-like activity can induce the decomposition of $O_2^{•-}$, while CAT-like and POD-like activity can decompose $H_2O_2$ (Fig. S5). To assess the SOD-like activity of Cys-PB NPs, a commercial SOD test kit was employed, based on the reduction of WST-1 by xanthine oxidase that produces $O_2^{•-}$ in the presence of xanthine. WST-1 is reduced by $O_2^{•-}$ leading to the formation of a yellow dye having a specific absorbance at 450 nm. Enzymes with SOD activity can eliminate $O_2^{•-}$, resulting in the inhibition of WST-1 reduction. As shown in Fig. 3a, all Cys-PB NPs showed an effective SOD-like activity through the inhibition of WST-1 reduction. When the concentration of Cys-PB reached 20 µg/mL, the inhibition rate for Cys-PB1, Cys-PB2, Cys-PB3 and Cys-PB4 was 74.6, 81.6, 60.3 and 66.2%, respectively. Higher inhibition rate implied higher SOD activity, which can be contributed by the higher amount of defects and smaller size of Cys-PB. Compared to a previous publication, Cys-PB1 and Cys-PB2 exhibited higher inhibition rates, indicating a more effective SOD-like activity.[11]

SOD can catalyse the conversion of $O_2^{•-}$ into $H_2O_2$, which is the most stable and abundant ROS in cells.[27] Effectively scavenging $H_2O_2$ is essential to protect organisms from oxidative damage and mitigate inflammation. Enzymes with CAT-like activity and POD-like activity can decompose $H_2O_2$. CAT-like activity of Cys-PB NPs was assessed by measuring the oxygen production from $H_2O_2$ decomposition. For this purpose, $[Ru(dpp)₃]Cl₂$ was used as an oxygen probe, and its fluorescence intensity was monitored, as it decreases in the presence of oxygen.[28,29] As shown in Fig. 3b and S6, these Cys-PB NPs can generate a substantial amount of oxygen by the decomposition
of H₂O₂, resulting in a reduced fluorescence intensity of [Ru(dpp)₃]Cl₂. With the increaser of the concentration of H₂O₂, the amount of generated oxygen increased too. Notably, Cys-PB1 and Cys-PB2 exhibited a slightly higher CAT-like activity at low H₂O₂ concentrations (1 and 10 mM) compared to Cys-PB3 and Cys-PB4. This result suggests that Cys-PB1 and Cys-PB2 may exhibit a higher CAT-like activity at H₂O₂ levels present in tumor cells (up to 50 µM).[³⁰]

Cys-PB NPs also demonstrated POD-like activity under acidic conditions, capable of decomposing H₂O₂ to H₂O and oxidizing TMB, a peroxidase substrate. In the presence of H₂O₂ and Cys-PB, the colourless TMB gradually turned blue over time due to its oxidation (Fig. 3c). Interestingly, the POD-like activity of Cys-PB1 and Cys-PB2 was much higher compared to Cys-PB3 and Cys-PB4.

<figure><img src="image_9.png" /><figcaption>**Fig. 3.** (a) SOD-like activity of Cys-PB evaluated by the inhibition rate of O₂⁻. (b) CAT-like activity of Cys-PB determined by monitoring the fluorescence emission of [Ru(dpp)₃]Cl₂ as an oxygen probe. (c) POD-like activity assessed by monitoring the oxidation of TMB using UV-Vis spectroscopy. Water was used as control group. The experiments were performed in triplicate.</figcaption></figure>

It is noteworthy that the POD-like enzyme activity of Cys-PB1, Cys-PB2, Cys-PB3, and Cys-PB4 exhibited a large difference. Specifically, Cys-PB1 and Cys-PB2, characterized by a smaller size and higher number of lattice defects, showed markedly
higher POD enzymatic activity.[16,31] To further assess the catalytic efficiency and activity of Cys-PB as a POD enzyme, steady-state kinetic assays were carried out using various concentrations of TMB or H₂O₂. The turnover number *k*cat reflecting the catalytic activity was calculated (Fig. 4). Since a high value of *k*cat is indicative of a high catalytic activity, Cys-PB1 and Cys-PB2 with a higher *k*cat value compared to Cys-PB3 and Cys-PB4 showed a stronger POD-like catalytic activity. Compared with other reported PB NPs,[31] Cys-PB1 and Cys-PB2 showed a higher *k*cat and POD-like activity (Table S3).

The above results revealed that Cys-PB1 and Cys-PB2 with small size and low crystallinity possess a high POD-like activity, in addition to high SOD- and CAT-like activities, which could be due to their smaller size and high amounts of defects, resulting in a larger specific surface area and more exposed sites, thereby enhancing their capacity to react with H₂O₂ and superoxide.
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Kinetic analysis of Cys-PB NPs using different concentrations of (a) H₂O₂ and (b) TMB. The turnover number *k*cat was calculated, showing the catalytic efficiency per Fe atom. The experiments were performed in triplicate.</figcaption></figure>

Next, we studied the stability of Cys-PB in PBS up to 14 days. We observed that the stability of Cys-PB decreased over time as the size decreased (Fig. S7). After 24 h, the normalized maximum absorbance of Cys-PB1, Cys-PB2, Cys-PB3 and Cys-PB4 already decreased to 0.71, 0.90, 0.80 and 0.83, respectively. After 7 days, Cys-PB2,
Cys-PB3 and Cys-PB4 were less degraded than Cys-PB1. By 14 days, the degradation process had reached a stable state. Cys-PB1 with the smallest size degraded faster compared to the other Cys-PB, which may be attributed to its higher specific surface area and numerous defects.

The cytocompatibility of Cys-PB was evaluated on murine RAW 264.7 macrophage cells and human THP-1-derived macrophages using the MTS assay. As shown in **Fig. 5**, there was no drastic difference in the viability of RAW 264.7 and THP-1 cells incubated with the four Cys-PB NPs. For RAW 264.7 cells, Cys-PB NPs exhibited a slight toxicity, with survival rates falling slightly below 80 % at the highest concentration (100 µg/mL). The cytocompatibility of Cys-PB NPs with RAW 264.7 macrophages was satisfactory at concentrations of 10–50 µg/mL. In addition, Cys-PB NPs demonstrated an excellent biocompatibility on THP-1-derived macrophages, with a cell viability higher than 95% even at the highest concentration.

<figure><img src="image_11.png" /></figure>

**Fig. 5.** Cell viability of Cys-PB NPs on RAW 264.7 cells and THP-1 macrophages at increasing concentrations. Difference in statistical significance (Student's *t*-test) is indicated by * = *p* < 0.1, ** = *p* < 0.01, and *** = *p* < 0.001 compared with the control group. The experiments were performed in triplicate.
Next, we investigated the anti-inflammatory and antioxidant effects of Cys-PB
NPs at cellular level. RAW 264.7 cells were stimulated with LPS to obtain an
inflammatory cell model with a high level of ROS and pro-inflammatory cytokines.[32-34] TNF-α, IL-1β and IL-6 are the primary pro-inflammatory cytokines that trigger
macrophage inflammatory responses. Therefore, RAW 264.7 cells were treated with
Cys-PB and LPS for 24 h, and the level of TNF-α, IL-1β and IL-6 was measured by
ELISA. Upon LPS stimulation, large amounts of TNF-α, IL-1β, and IL-6 were
measured (Fig. 6a-6c). Cys-PB1 and Cys-PB2 effectively reduced the production of
TNF-α, which was not the case for Cys-PB3 and Cys-PB4. All four Cys-PB NPs
significantly reduced the secretion of IL-1β by nearly half, while Cys-PB1, Cys-PB2
and Cys-PB4 showed a significant decrease on IL-6 release. To thoroughly compare
these four Cys-PB NPs, a detailed calculation of the fold changes in secreted cytokines
relative to the LPS-stimulated RAW 264.7 cells group was conducted (Table S4).
Although all Cys-PB NPs exhibited similar effects in reducing IL-1β, Cys-PB2 showed
a relatively higher effective anti-inflammatory activity when considering the results for
TNF-α and IL-6.

Interestingly, we observed that RAW 264.7 cell survival significantly decreased
to 60.8% after LPS treatment (Fig. 6d), indicating that LPS-induced inflammation can
affect the cell viability, which is consistent with previous observations.[32] Remarkably,
after the addition of Cys-PB NPs, this decline in cell survival was inhibited, leading to
a concentration-dependent increase in cell viability (Fig. 6d). At the highest
concentration (50 µg/mL), the cell viability of Cys-PB1, Cys-PB2, Cys-PB3 and Cys-
PB4 was 91.3%, 91.8%, 85.5% and 87.8%, respectively. This demonstrates that Cys-
PB significantly inhibited the decrease in cell viability induced by LPS-induced
inflammation.
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 6.** Production of (a) TNF-α, (b) IL-1β and (c) IL-6 and cell viability in LPS-stimulated RAW 264.7 cells with and without treatment of Cys-PB NPs. Difference in statistical significance (Student's t-test) is indicated by * = *p* < 0.1, ** = *p* < 0.01, and *** = *p* < 0.001. The experiments were performed in triplicate.</figcaption></figure>

LPS-stimulated RAW 264.7 cells also produce high ROS levels. To assess the
ROS scavenging ability of Cys-PBs in vitro, the cells were stained with DCFH-DA,
which exhibit green fluorescence after oxidation by ROS. Fluorescence microscopy
revealed that LPS-stimulated RAW 264.7 cells exhibited a strong green fluorescence,
confirming high ROS content (Fig. 7a). In contrast, RAW 264.7 cells treated with Cys-
PB NPs showed a reduced green fluorescence intensity, suggesting a decrease in ROS
levels. A quantitative analysis of the fluorescence intensity was performed using a
microplate reader (Fig. 7b). The results were consistent with the microscopy images,
demonstrating that Cys-PBs effectively scavenged ROS. Cys-PB1 and Cys-PB2
displayed a higher ROS scavenging capability than Cys-PB3 and Cys-PB4.
Overall, Cys-PB NPs with different size and defect content all possess anti-inflammatory ability. Among the four Cys-NPs investigated in this study, Cys-PB2 can be considered as the most effective NPs. This could be due to the fact that Cys-PB2 has a higher enzymatic activity than Cys-PB3 and Cys-PB4, because of a larger amount of defects, and compared to Cys-PB1 owing to a better stability.

<figure><img src="image_16.png" /></figure>

**Fig. 7.** (a) Fluorescence microscopy images of RAW 264.7 cells incubated with LPS and Cys-PBs. The cells were stained with DCFH-DA to visualize ROS. LPS was used to stimulate the cells to generate high levels of ROS. (b) Normalized fluorescence intensity of DCFH-DA measured using a microplate reader. Cys-PB NPs at a concentration of 50 µg/mL were incubated with LPS-treated RAW 264.7 cells. The scale bar is 200 µm. Difference in statistical significance (Student's *t*-test) is indicated by * = *p* < 0.1, ** = *p* < 0.01, and *** = *p* < 0.001. The experiments were performed in triplicate.

**4. Conclusion**

In summary, four different types of cysteine-stabilized PB NPs with different sizes and crystallinities were synthesized. The decrease in the size and crystallinity led to an increase in vacancy defects in the crystal structure. Although previous studies
explored enzyme-like activities of PB NPs, such as SOD, CAT and POD, our study provides insights in the relationship between the structure of PB NPs and their nanozyme properties. Indeed, the POD-like activity of Cys-PB NPs was size- and defect-dependent. As the size decreased and defects increased, the POD-like activity of Cys-PB NPs significantly increased. Additionally, Cys-PB NPs with a higher biodegradability displayed a lower POD-like activity. Furthermore, Cys-PB NPs effectively inhibited significantly the production of cytokines and scavenged intracellular ROS in inflammatory cells. This work demonstrates that PB NPs with a high stability and large amounts of defects possess better anti-inflammatory effects. Such a comparative study can facilitate the future design of PB NPs as nanozymes for biological applications as anti-inflammatory materials.

**CRediT authorship contribution statement**

**Yilin He**: Conceptualization, Methodology, Investigation, Data curation, Writing - Original Draft preparation. **Alberto Bianco**: Supervision, Writing - Review & Editing. **Cécilia Ménard-Moyon**: Conceptualization, Supervision, Writing - Review & Editing.

**Declaration of Competing Interest**

The authors declare no conflict of interest.

**Acknowledgements**

This work was supported by the Centre National de la Recherche Scientifique
(CNRS), by the Agence Nationale de la Recherche (ANR) through the Interdisciplinary
Thematic Institute SysChem via IdEx Unistra (ANR-10-IDEX-0002) within the
program Investissement d'Avenir and by Jean-Marie Lehn Foundation. The authors
wish to thank Cathy Royer from the "Plateforme Imagerie In Vitro de l'ITI Neurostra"
CNRS UAR 3156, University of Strasbourg (Strasbourg, France) for TEM analysis, as
well as Haijun Peng and Jean-Daniel Fauny for help with XRD and confocal
microscopy, respectively. YH is indebted to the Chinese Scholarship Council for
supporting her PhD.

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