# Preparation of hybrid SPIONs and nanodiamonds in view of neurodegenerative diseases treatments

Nimisha Singh, Thomas Nury, Leya Liu, Julien Boudon, Lionel Maurizi, Vivien Pires, Gérard Lizard, Nadine Millot

► To cite this version:

Nimisha Singh, Thomas Nury, Leya Liu, Julien Boudon, Lionel Maurizi, et al.. Preparation of hybrid SPIONs and nanodiamonds in view of neurodegenerative diseases treatments. Colloids and Surfaces B: Biointerfaces, 2026, 257, pp.115170. 10.1016/j.col surfb.2025.115170. hal-05296064

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# Preparation of hybrid SPIONs and nanodiamonds in view of neurodegenerative diseases treatments

Nimisha Singh<sup>a</sup>, Thomas Nury<sup>b</sup>, Leya Liu<sup>a</sup>, Julien Boudon<sup>a</sup>, Lionel Maurizi<sup>a</sup>, Vivien Pires<sup>b,c</sup>, Gérard Lizard<sup>a,b</sup>, Nadine Millot<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

BV-2 cells

SPIONs

Nanodiamonds

PEG

SKL peptide

Colloidal stability

Cytotoxicity

## ABSTRACT

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in biomedical applications due to their unique magnetic and physicochemical properties. Surface stabilization and functionalization further expand their applicability in biological environments and help overcome the challenges of achieving targeted therapy. Here, nanodiamonds (NDs) decorated with SPIONs were developed for a dual therapy approach combining magnetic and optical properties: NDs offer excellent properties for optical imaging while SPIONs serve as effective contrast agents. In the present study, amino-PEGylated SPIONs were covalently linked to carboxylated NDs via carbodiimide conjugation, to enhance the colloidal stability of the nanoparticles (NPs) and improve their biocompatibility and stealth properties. The functionalization process was evaluated using various analytical techniques, including TGA, XPS, FTIR, DLS, and zeta potential measurements, which confirmed and validated successful functionalization in each consecutive step. Given that organelle dysfunction plays a critical role in neurodegeneration, and in an effort to specifically target peroxisomes, the serine-lysine-leucine (SKL) peptide was thus anchored on ND-PEG-SPIONs surface with the remaining free amines. To further assess the biological response of these nanohybrids, their colloidal stability was compared in different biological media. Strikingly, based on the *in vitro* data obtained from BV-2 cells, we demonstrate that the nanoparticles accumulate intra-cellularly without inducing cell death or mitochondrial dysfunction, a result that fulfills a crucial prerequisite for their future application in targeted therapeutic delivery of antioxidant molecules to peroxisomes. This work thus paves the way for the development of in pexotherapy, opening new avenues for the treatment of neurodegen-erative diseases associated with peroxisomal dysfunction.

## 1. Introduction

With increasing lifespan, age-related diseases — particularly Alzheimer's disease (AD) — are becoming increasingly prevalent. This neurodegenerative disorder is characterized by progressive memory loss and cognitive decline, which gradually undermines patients' mental health [1,2]. Pathologically, AD progression is marked by the accumulation of abnormal neuritic plaques and neurofibrillary tangles in the brain, processes associated with inflammation, diminished antioxidant defenses, and elevated oxidative damage to lipids, proteins and DNA [3,4]. In the pathogenesis of AD, oxidative stress emerges as a critical factor in both the onset and progression of the disease [5]. Analysis of the brains of AD patients has revealed significant alterations in

mitochondrial morphology compared to those of age-matched control individuals [6]. Multiple lines of evidence have demonstrated reduced ATP production, elevated levels of reactive oxygen species (ROS), and pronounced respiratory dysfunction in mitochondrial preparations derived from various AD mouse models [7,8]. Furthermore, peroxisomes (PER), which are involved in lipid metabolism and redox status regulation, are both topographically and functionally closely linked to mitochondria. Emerging evidence now indicates that peroxisomal dysfunction can precipitate mitochondrial dysfunction — and vice versa [9–11]. Elevated ROS levels have also been associated with peroxisome damage. Additionally, multiple studies support the involvement of PER in certain neurodegenerative diseases [12,13]. Currently, there is growing evidence supporting the involvement of mitochondrial and

<footnote><sup>a</sup> Université Bourgogne Europe, CNRS, Laboratoire Interdisciplinaire Carnot de Bourgogne ICB UMR 6303, Dijon 21000, France</footnote>

<footnote><sup>b</sup> Université Bourgogne Europe, Laboratoire Bio-PeroxIL EA7270, Dijon 21000, France</footnote>

<footnote><sup>c</sup> Université Bourgogne Europe, Institut Agro, CNRS, INRAE, UMR CSGA, Dijon 21000, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: nadine.millot@ube.fr (N. Millot).</footnote>
peroxisomal dysfunction in AD [14,15]. Consequently, identifying effective treatments that target mitochondria and peroxisomes – such as mitotherapy [16] and pexotherapy [17] – has emerged as a critical priority. In response to these challenges, our group proposes a nano-particle (NP)-based approach.

NPs-based theranostic agents for brain disorders have been designed to cross the blood-brain barrier (BBB) and regulate oxidative stress at the peroxisomal level. To achieve this, nanodiamonds (NDs) and superparamagnetic iron oxide nanoparticles (SPIONs) are combined and functionalized with the consensus peroxisomal targeting sequence: the serine-lysine-leucine (SKL) peptide [18,19]. NDs belong to the unique class of carbon-based NPs that have garnered considerable interest in recent years [20]. They exhibit outstanding biocompatibility, chemical inertness of their diamond cores, and rich surface chemistry, along with robust optical and physical properties. One of the most promising recent advancements has been the application of NDs in drug delivery and diagnostic systems [21]. Their excellent optical properties of NDs can therefore facilitate the measurement of the intraneuronal vesicular transport by enabling the tracking of individual particles [22]. SPIONs, on the other hand, complement NDs with their strong magnetic properties and minimal toxicity, making them highly suitable for *in vivo* application. Indeed, these unique nanocrystals can, generate contrast signals several orders of magnitude higher than those of gadolinium chelates for example [23,24]. SPIONs, which are already clinically approved, were also selected for their ability to i) cross the BBB [25], ii) purify the final construct (via magnetic separation) and iii) enable quantification within the cells (using ICP or magnetic measurements). When functionalized with specific targeting moieties, SPIONs can be also employed for *in vivo* imaging of molecular markers associated with disease progression [26–28]. A wide range of surface coatings for SPIONs has been developed using synthetic and natural polymers to enhance biocompatibility and colloidal stability in biological environment. These polymer shells on SPIONs offer multiple grafting sites for the covalent immobilization of peptides, including the SKL peptide sequence and NDs in our study. PEGylation – a process involving the coating of nanoparticle surfaces with polyethylene glycol (PEG) through covalent or non-covalent conjugation – is one of the most widely used NPs surface modification methods in the biomedical field due to the high hydrophilicity of PEG chains [29]. Consequently, a phosphonate PEG derivative is employed to coat the SPIONs, yielding amine-terminated SPIONs that enable covalent bonding to carboxylated NDs.

To develop a nanoplatform capable of targeting peroxisomes, we utilized the peroxisomal targeting sequence (PTS) Ser-Lys-Leu (SKL) [18,30]. This was accomplished by conjugating the carboxylated SKL peptide to PEG-functionalized ND-SPIONs hybrids. In biochemical protein targeting, a peroxisomal targeting signal (PTS) is a specific region of a peroxisomal protein that binds to its receptor, ensuring that proteins containing this motif are directed to and localized within the peroxisome [31,32]. Literature indicates that the carboxyl-functionalized SKL motif acts as a topogenic signal during protein translocation into peroxisomes, requiring the complete tripeptide sequence with a free alpha-carboxyl (COOH) group at the C-terminus [33].

In this study, we present a novel multifunctional nanoplatform designed for dual-modal imaging—enabling *in vitro* tracking *via* optical imaging (thanks to nanodiamonds, NDs) and *in vivo* monitoring through MRI (using superparamagnetic iron oxide nanoparticles, SPIONs)— while specifically targeting peroxisomes *via* the grafting of the SKL peptide. This innovative approach combines, for the first time, the unique properties of NDs and SPIONs with peroxisome-targeting capabilities, offering a versatile tool for both diagnostic and therapeutic applications.

To evaluate the potential of this nanoplatform, we conducted a comprehensive study using murine BV-2 microglial cells, focusing on two key aspects: i) the intracellular accumulation of nanoparticles, visualized and confirmed by Perls staining, and ii) their biological impact, assessed through a panel of complementary techniques

including phase contrast microscopy, fluorescein diacetate (FDA) assay, MTT assay, and May-Grünwald-Giemsa (MGG) staining. Our findings provide critical chemical and toxicological insights into the behavior of hybrid SPIONs and nanodiamonds functionalized with the SKL peptide. These data constitute a mandatory prerequisite for the study of the therapeutic delivery of antioxidant molecules to peroxisome.

## 2. Material and methods

### 2.1. Chemicals and reagents

N-hydroxysuccinimide (NHS) (98 %) and N-(3-dimethylamino-propyl)-N'-ethylcarbodiimide hydrochloride (EDC) sodium hydroxide (NaOH, ≥97 %), hydrochloric acid (HCl, 37 %), 2-(N-morpholino)ethanesulfonic acid (MES) were obtained from Sigma Aldrich (Saint-Quentin-Fallavier, France). Iron (II) chloride tetrahydrate (FeCl₂·4 H₂O, 98 %) and iron (III) chloride hexahydrate (FeCl₃·6 H₂O, 97 %) were purchased from Alfa Aesar (Karlsruhe, Germany). PBS 1× solution (Fisher Bioreagents, France), dimethyl sulfoxide (DMSO, ≥99.7 %) (Acroseal), peptide (C-terminal SKL sequence, > 95 %) were also purchased from Fisher Chemicals (Leicestershire, UK). Amino PEG Phosphonic acid hydrochloride (n = 45) were obtained from specific polymers (France), and nanodiamonds (CAS 7782-40-3) were obtained from Van Moppes (Geneva Switzerland).

The ultrafiltration stirred cell (Model 8400, 400 mL) and membranes (ref. #PLHK07610, regenerated cellulose 100 kDa) were purchased from Merck Millipore (Darmstadt, Germany). All other chemicals used were of analytical grade and without further purification.

### 2.2. Preparation of iron oxide nanoparticles

Bare iron oxide nanoparticles (Fe₃O₄, SPIONs) were prepared using previously reported chemical co-precipitation method using FeCl₃·6 H₂O (34.6 g) and FeCl₂·4 H₂O (12.7 g), in the molar ratio of 2:1. They were dissolved in water (1.5 L) [34]. SPIONs were then rapidly precipitated by adding ammonia solution (120 mL, 28 %) at 22 °C. The resulting black suspension was stirred for 10 min. SPIONs were then magnetically decanted using a strong magnet and washed thoroughly with deionized (DI) water until the solution reached pH 8. To obtain the stable suspension, the precipitate was kept in nitric acid solution (1 mM, pH 3) and dialyzed against nitric acid solution (10 mM) for 48 h. The dialyzed nanoparticles were then centrifuged for 15 min at 20,000 g with deceleration. The supernatant was then used for further experiments.

### 2.3. Preparation of amino PEG phosphonic acid terminated SPIONs

Amino PEG phosphonic acid (60 mg) was first dissolved in ultrapure water (5 mL). SPIONs (20 mg, 5.3 mL) were added into the above solution. The reaction was kept at pH 4 and 22 °C overnight. The modified PEG-SPIONs were then dialyzed (12–14 kDa) against DI water for 48 h. PEG-SPIONs were then stored in fridge at 4 °C for further use. These conditions were optimized after analyzing different mass ratio of PEG and SPIONs (1:1, 1:2 and 1:3 w/w of SPIONs: PEG) prepared over different time periods (3 h, 6 h, 9 h and 24 h) and pH conditions (pH 4 and pH 5.5).

### 2.4. Conjugation of carboxylated nanodiamonds with PEG-SPIONs

Commercial NDs-COOH (50 nm) were used for conjugation. Prior to their use, the commercial NDs were centrifuged (20,000 g, 30 min) to optimize their size and stability. The resulting supernatant suspension of NDs was then used for conjugation with SPIONs by EDC/NHS covalent chemistry. In this method, EDC (0.02 M, 0.1 M MES pH 5) was first added to NDs suspension (10 mg, 0.1 M MES pH 5) to activate the carboxylic group on the surface. After 10 min, NHS (0.04 M, 0.1 M MES
pH 5) was added to the previous solution to avoid the formation of unstable intermediates. The suspension was then added to a PEG-SPIONs suspension (5 mg, 0.1 M PBS pH 7.4) via a dropping funnel to avoid aggregation and ensure effective conjugation. The reaction was then left at 22 °C for 24 h. The carboxylic group of NDs were conjugated by some amine groups of PEG on SPIONs. The ND-PEG-SPIONs suspension was then magnetically separated, washed with DI water and stored at 4 °C before further use.

## 2.5. Immobilization of SKL peptide on ND-PEG-SPIONs

The SKL peptide (Ser-Lys-Leu) (1 mg, 0.1 M MES pH 5) was immobilized onto ND-PEG-SPIONs using the same EDC/NHS covalent chemistry as previously used. The carboxylic group of the SKL peptide was first activated by EDC and NHS, and then added dropwise into the suspension of ND-PEG-SPIONs (4.8 mg, 0.1 M PBS pH 7.4). The SKL peptide was then grafted onto the remaining NH₂ groups at the end of the PEG chains. The resulting ND-PEG-SPIONs-SKL were then magnetically separated, washed with DI water and stored at 4°C until biological assay.

## 2.6. Nanoparticles characterization techniques

The surface morphology and chemical functionality of the developed nanop platform were analyzed sequentially by TEM and XPS analyses. The TEM instrument was a Hitachi HT7800. Samples were prepared on carbon-coated copper grids using a dilute suspension of NPs in DI water before air drying. ImageJ software was used to estimate the average nanoparticle size (100 nanoparticles were analyzed per sample).

XPS was performed to understand the chemical functionality on the NP surface using Omicron ESCA (Electron Spectroscope for Chemical Analysis) from Oxford Instruments (Germany). An aluminium anode with an energy of 1486.7 eV was used to analyze samples, with an acquisition time of 0.1 s. Casa XPS software was used to analyze the obtained data. The C1s carbon peak at 284.5 eV was used as the reference in the neutralization to minimize charging effects and Gaussian (70 %) - Lorentzian (30 %) profiles were used to subtract a Shirley background. Full widths at half maximum (FWHM) were set between 1.4 and 1.6 eV for O 1 s, 1.6-1.7 eV for C 1 s and 1.7-1.8 eV for N 1 s.

FTIR was used to confirm peptide functionalization and immobilization, using a Shimadzu 8400s spectrophotometer with a range of 4000-400 cm⁻¹ and a resolution of 4 cm⁻¹ with a total of 45 scans per measurement. Samples were prepared from 2 mg of sample ground together with KBr powder. The resulting mixture was then pressed to form a transparent pellet before being measured by transmission.

The quantity of grafted molecules as well as the quantity of NDs was quantified using ThermoGravimetric Analysis (TGA, TA instrument, Discovery TGA) under air at a flowrate of 25 mL min⁻¹. After an isotherm at 100 °C for 15 min, TGA was performed with a heating rate of 10 °C min⁻¹ from 100 °C to 700 °C.

The Malvern Zetasizer NanoZSP (Malvern, UK) assisted by the DTS Nano V8.01 software was used to measure the hydrodynamic diameter. The NPs suspension was first ultrasonicated for 15 min in 10⁻² M NaCl aqueous solution. The isoelectric point was calculated by zeta potential measurements, varying the suspension pH from 3 to 11 by adding HCl (0.1 M) or NaOH (0.1 M and 0.01 M) solutions. The mean diameter was determined by Dynamic Light Scattering (DLS) curves derived from the number distribution calculation on the same instrument.

## 2.7. Cell culture and treatments

BV-2 cells are good preliminary in vitro model to evaluate the biological activity of different compounds on oxidative stress and inflammation, as well as on organelles (mitochondria, peroxisome), that are affected in microglial cells for several degenerative diseases [35]. Murine BV-2 microglial cells were cultured in RPMI medium with 10 % heat-inactivated fetal calf serum (FCS) and antibiotic/antimycotic

solution as described previously [36,37]. For cytotoxic studies, based on the assessment of cell death, cells were seeded in appropriate density and cultured overnight as described previously. Cells were then exposed to nanoparticles for 24 h at concentrations from 3 to 38 µg/mL depending on the initial amounts of nanoparticles available in distilled water: SPIONs 3.8 mg/mL, PEG-SPIONs 2.3 mg/mL, ND-PEG-SPIONs 0.8 mg/mL, ND-PEG-SPIONs-SKL 0.6 mg/mL, and bare NDs 1 mg/mL. The highest concentrations used for all studied nanoparticles were obtained at an initial dilution of 1/100 in the culture medium.

## 2.8. Analysis of cell morphology by phase contrast microscopy

BV-2 cells were seeded in 12-well plates at an initial seeding density of 5 × 10⁴ cells/well. Cells were exposed to nanoparticles for 24 h. Cell morphology was assessed under a phase contrast microscope (Axiovert 40 CFL, Zeiss) as previously described [38]. Digital images were taken with an Axiocam ICm1 camera (Zeiss).

## 2.9. Analysis of nanoparticles cytotoxicity with the FDA assay: impact at the plasma membrane level

BV-2 cell viability was assessed using a lipophilic fluorochrome, fluorescein diacetate (FDA) (Sigma-Aldrich / Merck, L'Isle d'Abeau, France)) as previously described [39]. In the presence of plasma membrane esterases of living cells, the non-fluorescent FDA is converted into a green fluorescent metabolite "fluorescein" which accumulates inside cells [40]. Thus, FDA permits the assessment of cytotoxic damages at the plasma membrane: dead cells are FDA-negative cells while living cells are FDA-positive cells [41]. At the end of the treatment (24 h), cells were incubated in the dark with FDA (15 µg/mL, 5 min, 37 °C), washed twice with phosphate-buffered saline (PBS 0.1 M, pH 7.4), and then lysed with 10 mM Tris-HCl solution containing 1 % sodium dodecyl sulfate (SDS). Fluorescein fluorescence intensity was measured with excitation at 485 nm and emission at 528 nm using a microplate reader (Sunrise spectrophotometer, TECAN, Lyon, France) to quantify live cells. All assays were performed in two independent experiments carried out in triplicate. Results were expressed as % of control: Fluorescence (assay) / Fluorescence (control) × 100.

## 2.10. Analysis of nanoparticles cytotoxicity with the MTT assay: impact at the mitochondrial level

Cell viability was assessed based on mitochondrial activity using MTT assay (3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as previously described [37,40,42]. Cells were seeded in 12-well plates at an initial seeding density of 5 × 10⁴ and incubated overnight at 37 °C, 5 % CO₂. After exposure to nanoparticles (24 h), cells were harvested by centrifugation at 200 × g for 5 min and then incubated with MTT (0.05 mg/mL) for 3 h in the dark. In metabolically active cells (viable cells with mitochondrial activity), MTT salt is reduced to formazan by succinate dehydrogenase, a mitochondrial enzyme belonging to complex II of the mitochondrial respiratory chain, to form formazan blue [43]. The formed formazan crystals were solubilized using 200 µL of DMSO (Sigma-Aldrich / Merck) and the absorbance was read at 570 nm with a microplate reader (Sunrise TECAN, Switzerland). Final results were obtained by subtracting the absorbance of control wells (without cells) from the absorbance of treated samples to avoid nanoparticle interference with the MTT results. Results were expressed as % of control: absorbance (assay) / absorbance (control) × 100.

## 2.11. May-Grünwald-Giemsa staining procedure

May-Grünwald-Giemsa staining allows visualization of morphological details. May-Grünwald contains an acid dye (eosin) and a basic dye (methylene blue); Giemsa, on the other hand, contains eosin and a basic dye (methylene azure). Under these staining conditions, two colors are
observed: the nucleus is stained pink and the cytoplasm blue-violet. This staining procedure allows distinguishing viable and dead cells, such as apoptotic cells [44]. At the end of the treatment in the presence or absence of nanoparticles, BV-2 cells (approximately 40,000 cells) were deposited on glass slides by cytocentrifugation (5 min) using a cytospin (Shandon, Sheshire, UK) then fixed with 100 % methanol (5 min). The cell deposits were then stained by May-Grünwald (5 min) and subsequently incubated with Giemsa at room temperature (15 min). The preparations were then washed with distilled water (2 x × 15 s), dried at room temperature (30 min), and immersed in xylene (30 s). Assembly was carried out in EUKITT between slide and coverslip, and the samples were stored at +4 °C until observation under a microscope (Axiovert 40 CFL, Zeiss). Digital images were taken with an Axiocam ICm1 camera (Zeiss).

## 2.12. Perls staining procedure

Perls staining is commonly used to detect iron accumulation inside cells (iron accumulation appears as blue dots of various size and shapes, and nuclei are stained dark pink) [37,45]. BV-2 cells treated with SPIONs (including SPIONs, PEG-SPIONs, ND-PEG-SPIONs, and ND-PEG-SPIONs-SKL) were first washed in PBS. Then, cells were spun down onto glass slides using cytospin (Shandon). After fixation in 5 % paraformaldehyde (10 min), cells were stained with a 5 % potassium ferrocyanide / 5 % hydrochloric acid solution diluted in distilled water (30 min). The nuclei were counterstained with hematoxylin and the slides were washed to observe the resulting Prussian blue staining. SPIONs in BV-2 cells were detected by brightfield microscopy (Axi-o-Skope A1, Zeiss) and digitalized images were obtained with an Axiocam camera (Zeiss).

## 2.13. Statistical analysis

All experiments were performed in triplicate and values were expressed as mean ± SD. Statistical comparison between the control (untreated cells) and experimental values was done using the Mann-Whitney test. Data with p < 0.05 were considered statistically significant.

# 3. Results and discussion

Various types of nanoparticles (SPIONs, PEG-SPIONs, ND-PEG-SPIONs, ND-PEG-SPIONs-SKL) were synthesized (schematic representations of selected nanoparticles are shown in Fig. 1) and characterized using complementary chemical, physical, and biological methods. The morphology and size of all prepared nanoparticles were analyzed by TEM, as presented in Fig. 2. The as-synthesized SPIONs (Fig. 2 exhibited a nearly spherical shape with an average size of 8.3 ± 2.1 nm, and a narrow size distribution. These nanoparticles are consistent in size with SPIONs reported in the literature, which demonstrate superparamagnetic properties [29,46,47]. NDs showed an average TEM size of 24.1 ± 7.8 nm (distribution of TEM sizes given in Figure S1, supporting information). Before conjugation, the NDs hydrodynamic size

was reduced from around 50 nm to below 20 nm by centrifugation resulting in a dispersed and stable suspension from the commercially available NDs (Figure S2, supporting information). It is worth noting that by grafting NDs onto PEG-SPIONs, NDs surround SPIONs creating a flower-like structure – as depicted in Fig. 2 as inset of the TEM pictures – suggesting successful grafting of NDs onto PEG-SPIONs surface as there is no single NDs in the image.

By modifying the surface of SPIONs with NH₂-terminated PEG phosphonate derivatives, the particles were found to be stable in suspension. DLS data acquired with each successive coating showed an increasing hydrodynamic size of nanoparticles, as shown in Fig. 3. DLS measurements showed hydrodynamic sizes of 21.2 ± 0.4 nm for bare SPIONs and 37.8 ± 0.2 nm for PEG-SPIONs suggesting the presence of a polymer coating.

However, prior to these optimized conditions of grafting PEG on SPIONs, different sets of experiments were performed and analyzed in order to find the best concentration and parameters to prepare the stable PEG-SPIONs nanohybrid to achieve the targeted drug delivery (Fig. 3). Thus, the hydrodynamic diameter and zeta potential on the studied parameters were evaluated as also shown in Fig. 4.

From the studied parameters, a 1:3 w/w ratio of SPIONs to PEG was chosen to react for 24 h at pH 4 to obtain the stable core-functionalized NPs. The chosen parameters lead to nanohydrens with uniform size distribution. This is justified in the curve of number distribution (Fig. 3). The zeta potential value was of ~14 mV.

Observing the zeta potential values after each surface modification at the physiological pH (7.4), the value increases from -9 mV to -22 mV from PEG-SPIONs to ND-PEG-SPIONs-SKL. The negative charges imply the presence of hydroxyl and carboxylic groups on the surface.

The zeta potential values also indicate that the nanoparticle stability increases with each surface functionalization step. Zeta potential values greater than +20 mV or lower than -20 mV are typically associated with enhanced colloidal stability, which is favorable for biomedical applications.

To understand the surface chemistry at each grafting step, FTIR was performed on the samples to explore the different coating steps on the NPs' surface (Fig. 5).

First, a peak was observed at approximately 1785 cm⁻¹, corresponding to the stretching vibration of the protonated carbonyl group characteristic of ND structures (grey line, Fig. 5) [48]. The carboxylic bonds from PEG appeared at 1740 cm⁻¹ [49]. Bare SPIONs (green line) exhibited characteristic bands in the low IR region, specifically between 500 and 700 cm⁻¹, attributed to the stretching of Fe-O bonds [50,51]. In the spectra for PEG-grafted SPIONs (black line), absorption peaks were identified, including the bending and stretching of C-H bonds at 1460 cm⁻¹ and a broad band between 2850 and 3000 cm⁻¹ [52]. An adsorption peak at 960 cm⁻¹, corresponding to phosphonate groups from PEG, was also detected [53]. The same vibrational peaks for C-H bonds, originating from PEG, were observed at identical wavenumbers in the FTIR spectra of both ND-PEG-SPIONs (red line) and ND-PEG-SPIONs-SKL (blue line), confirming the presence of PEG on the ND-SPION hybrids. Additionally, the two characteristic vibration bands for NDs (C=O at 1785 cm⁻¹) and SPIONs (Fe-O at 500-700 cm⁻¹) were present in these ND-SPION spectra, indicating interactions between the two types of nanoparticles. Ideally NDs grafting on the surface introduce secondary amine and an additional C=O band in the spectrum. However, C=O bond peak and NH band peak overlap making it difficult to conclude about the grafting using FTIR. Thus, the data is further validated on fitting the XPS spectra as can be seen in the carbon-fitted XPS spectra: an additional peak appears at 285.8 eV (Fig. 6B) of C-N/C=O suggesting the formation of covalent bond of CO-NH which was absent in PEG-SPIONs spectrum (Fig. 6A) [54]. Certainly because of its low concentration on ND-PEG-SPIONs, FTIR did not prove the presence of SKL peptide on this nanohybrid as no differences were observed on FTIR spectra between ND-PEG-SPIONs and ND-PEG-SPIONs-SKL (red and blue lines in Fig. 5 respectively). However, in XPS significant differences

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematics of the different NPs used in this study. The SKL (Serine-lysine-leucine) peptide sequence should allow peroxisomes to be labelled.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 2. TEM images of the nanoparticles presented in this study. From left to right: NDs: Nanodiamonds; ND-PEG-SPIONs: Nanodiamonds Pegylated SPIONs (Superparamagnetic iron oxide Nanoparticles) grafted on NDs; SPIONs (Superparamagnetic iron oxide Nanoparticles).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 3. A-C. DLS number distribution curve of PEG-SPIONs nanohybrids for different functionalization conditions and D-F. Zeta potential values at different mass ratios, duration and pH. (The insets of Figure A-C show comparative charts of the different measured sizes).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 4. A. DLS results on as prepared nanoparticles and B. zeta potential curves as a function of pH for all the prepared nanohybrid platforms at 25 °C in 10⁻² M NaCl aqueous solution.</figcaption></figure>
<figure><img src="image_7.png" /></figure>

<table><caption>Fig. 5. FTIR spectra and major surface functional groups of each grafting on SPIONs surface and Table summarizing the peaks at different coating steps.</caption><thead><tr><th rowspan="2">Nanohybrids</th><th colspan="6">Characteristic Peaks (cm<sup>-1</sup>)</th></tr><tr><th>VFe-O</th><th>VP-OH</th><th>δCH<sub>1</sub></th><th>V(C=O) COOH</th><th>V<sub>C=O</sub> carbonyl</th><th>VCH<sub>2</sub></th></tr></thead><tbody><tr><td>Naked SPIONs</td><td>500-700</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ND-PEG-SPIONs-SKL</td><td>500-700</td><td>960</td><td>1460</td><td>1740</td><td>1785</td><td>3000-2850</td></tr><tr><td>ND-PEG-SPIONs</td><td>500-700</td><td>960</td><td>1460</td><td>1740</td><td>1785</td><td>3000-2850</td></tr><tr><td>PEG-SPIONs</td><td>500-700</td><td>960</td><td>1460</td><td>1740</td><td></td><td>3000-2850</td></tr><tr><td>Naked NDs</td><td></td><td></td><td></td><td>1740</td><td>1785</td><td></td></tr></tbody></table>

were observed between ND-PEG-SPIONs and ND-PEG-SPIONs-SKL. In the nitrogen-fitted XPS spectrum of ND-PEG-SPIONs-SKL, two peaks were observed at 400.7 eV for NH₂ and 398.3 eV for NH (Fig. 6F) [54] which were not detected on PEG-SPIONS (Fig. 6E). This might be due to the low abundance of the free amine groups in the PEG-SPIONS stage (Fig. 6D), although N/Fe ratio can track the amine content, the amount is still too low for its detection. This can also be attributed to a low/weak N1s signal, evidenced by comparing the noise level of C1s and N1s spectra of ND-PEG-SPIONS (Fig. 6B and 6E) and ND-PEG-SPIONS-SKL [55] (Fig. 6C and 6F). Once the peptide has been grafted on ND-PEG-SPIONS, the amine content increased significantly as shown by the nitrogen content in Table 1 and a sharp peak was observed at 400.7 eV. Moreover, on fitting the carbon spectra of both samples, an additional C–O bond peak appears at 284.7 eV of the peptide and the peak position changes slightly compared to the previous grafting of ND-PEG-SPIONS (Fig. 6B) [56]. With increasing nitrogen concentration and decreasing phosphorus content at each successive step relative to the other elements, successful functionalization of each molecule is thus established.

The organic content on the surface of SPIONS was quantified using TGA analysis, based on the observed weight loss, as illustrated in Fig. 7. Following PEG grafting onto SPIONS (where bare SPIONS exhibit a 6 % weight loss attributed to hydroxyl groups), the total weight loss increased to 62 %, indicating that 56 % of this loss corresponds to the polymer content. Subsequent grafting of nanodiamonds (NDs) onto PEG-SPIONS resulted in a total weight loss of 66 %, with an additional 4 % organic content, confirming successful functionalization at each step. The interaction between NDs and PEG-SPIONS increases the decomposition temperature, as shown in Fig. 7. Furthermore, the weight loss profile of bare NDs reflects the stability of their 3D structure and a higher degradation temperature. Given that NDs are composed solely of carbon atoms, their degradation yields only CO₂. This behaviour is similarly observed when NDs are conjugated to PEG-SPIONS, maintaining a comparable degradation temperature. The results align with weight loss profiled reported in the literature [57,58].

To further investigate the colloidal stability of all nanoparticles,

additional experiments were conducted in various biological media, including PBS (0.1 M and 1 M), NaCl (10⁻² M) and albumin (60 mg/mL in DI water). Stability was assessed over time by monitoring changes in hydrodynamic diameter. DLS was used to evaluate nanoparticle size and detect aggregate formation, which is indicated by a shift in the particle size distribution toward higher values, as illustrated in Fig. 8. Un-doubtedly, the hydrodynamic size increases with each successive organic functionalization step, and similar trends observed during stability tests in various biological media. Among the NPs tested, PEG-SPIONS exhibited the smallest and most stable hydrodynamic diameter (Z<sub>average</sub>) in PBS (0.1 M, and 1 M) and albumin solutions, with no detectable deviation over time. However, in NaCl, all nanohybrids – PEG-SPIONS, ND-PEG-SPIONS, and ND-PEG-SPIONS-SKL – demonstrated a comparatively larger hydrodynamic diameter. When NDs were incorporated onto the NPs surface, the resulting nanohybrids remained stable in PBS (0.1 M) and albumin, maintaining a constant Z<sub>average</sub> over time. In contrast, exposure to PBS (1 M) and NaCl (10⁻² M), led to an increase in the Z<sub>average</sub> value, accompanied by a non-uniform size distribution as a function of time. This behavior may be attributed to the higher ionic strength of PBS, where electrostatic interactions between the nanohybrids play a critical role in determining colloidal stability. According to the literature, increasing ionic strength reduces the Debye length, thereby diminishing the repulsive forces between NPs and promoting their aggregation [59]. A similar trend was observed for the peptide-coated NP: aggregation and size variation occurred over time in NaCl and PBS (1 M). However, in PBS (0.1 M) and albumin, the NPs size remained stable, although the hydrodynamic diameter was slightly larger than that measured in water. Additionally, the peptide-coated nanohybrids are dominated by negatively charged surface groups, which enhance their stability in low-ionic strength PBS, as also demonstrated in Fig. 8. The observed aggregation may be related to the complex biological environment, which is influenced by enzymes, salt concentration, and pH [60]. These studies contribute to our understanding of the drug delivery properties of NPs, as particle size directly affects their residence time in biological systems [61]. This underscores the importance of conducting *ex vivo* stability studies and emphasizes
<figure><img src="image_8.png" /><figcaption>Fig. 6. XPS fitted spectra of carbon in A. PEG-SPIONs, B. ND-PEG-SPIONs, C. ND-PEG-SPIONs-SKL samples and nitrogen in D. PEG-SPIONs, E. ND-PEG-SPIONs and F. ND-PEG-SPION-SKL.</figcaption></figure>

<caption>**Table 1**</caption>

Concentration of the elemental composition calculated from XPS of each nanohybrid system prepared.

<table><thead><tr><th>Elements</th><th>C</th><th>N</th><th>N/C</th><th>O</th><th>N/O</th><th>Fe</th><th>N/Fe</th><th>P</th></tr></thead><tbody><tr><td>Samples</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>PEG-SPIONs</td><td>65.7</td><td>0.4</td><td>0.01</td><td>32.1</td><td>0.01</td><td>1.2</td><td>0.33</td><td>0.6</td></tr><tr><td>ND-PEG-SPIONs</td><td>78.3</td><td>0.9</td><td>0.01</td><td>18.2</td><td>0.05</td><td>2.5</td><td>0.36</td><td>0.1</td></tr><tr><td>ND-PEG-SPIONs-SKL</td><td>69.5</td><td>3.7</td><td>0.05</td><td>23.3</td><td>0.16</td><td>3.5</td><td>1.06</td><td>-</td></tr></tbody></table>

that colloidal stability must be thoroughly assessed before proceeding to *in vitro* experiments.

First, the toxicity of the NPs – including bare SPIONs, PEG-SPIONs, ND-PEG-SPIONs, ND-PEG-SPIONs-SKL and bare NDs – was evaluated in murine BV-2 microglial cells. Cell viability was assessed using two standard assays: the FDA assay, which detects plasma membrane integrity by measuring membrane esterase activity, and the MTT assay, which evaluates mitochondrial function by assessing succinate dehydrogenase activity in the Krebs cycle. [40]. In addition, cell morphology was considered by visualizing cells by phase contrast microscopy or after staining with May-Grünwald Giemsa (MGG) [37]. Morphologically, no
<figure><img src="image_9.png" /><figcaption>Fig. 7. TGA curves showing the thermal degradation of two functionalized nanohybrids (PEGylated and ND-PEGylated ones) compared to bare NDs or SPIONs.</figcaption></figure>

signs of toxicity or cell death induction were observed. Using phase-contrast microscopy, treated cells exhibited no alterations in morphology – such as membrane rupture, swelling, blebbing, or cytoplasmic disintegration – all of which are typical indicators of cytotoxic effects. Instead, cell size and morphology remained comparable to those of the negative control cells (Fig. 9A). Similarly, MGG staining revealed no evidence of apoptotic cells – such as those with condensed or fragmented nuclei – or cells with abnormal shapes. The size and morphology of the treated cells remained consistent with those of the negative control (Fig. 9B)).

However, the absence of morphological changes does not preclude functional changes. The FDA assay, which assesses membrane esterase activity, revealed no significant toxicity – except a slight effect at a

concentration of 38 µg/mL in the presence of SPIONs. This minor effect may indicate a subtle impact on plasma membrane integrity (Fig. 10A). However, the MTT assay revealed that this concentration of SPIONs did not reduce succinate dehydrogenase activity, indicating an absence of toxic effects on mitochondrial function. In contrast, a significantly lower MTT value was observed with ND-PEG-SPIONs-SKL (3 µg/mL) (Fig. 10B). Collectively, the data obtained across a wide range of concentrations – including elevated levels – using both the FDA and MTT demonstrate that cell viability was not affected by the NPs used.

In agreement with the findings from phase contrast microscopy and MGG staining, the results obtained using the FDA and MTT assays further confirm that SPIONs do not affect cell viability nor induce cell death. No evidence of dead cells – whether apoptotic or necrotic – was observed across the complementary methods employed (phase contrast microscopy, MGG, FDA, and MTT assays). However, at high concentration (38 µg/mL and above), SPIONs may disrupt plasma membrane integrity, potentially with minor effects on mitochondrial function. To evaluate this hypothesis, it would be necessary to have larger quantities of nanoparticles to specify possible toxic effects at higher concentrations. At concentrations below 38 µg/mL, it is also possible to ask whether SPIONs penetrate and accumulate in BV-2 cells. Indeed, using iron staining (Perls staining procedure), which stains intracellular iron blue, only a small percentage of cells (5.5 ± 1.1%) are stained. Blue cells were only detected with the highest concentration of SPIONs (38 µg/mL) (Fig. 11).

While the Perls staining technique is not highly sensitive, it remains a standard reference in routine medical biological analyses. Nevertheless, it provides clear evidence that SPIONs can accumulate within cells without toxicity (i.e. cell death induction). This accumulation may involve specific or non-specific interactions with organelles, such as mitochondria and/or peroxisome. To further validate these findings, the current data will need to be complemented by atomic absorption

<figure><img src="image_10.png" /><figcaption>Fig. 8. Stability profile of A. PEG-SPIONs, B. ND-PEG-SPIONs and C. ND-PEG-SPIONs-SKL as a function of time and measured calculating their hydrodynamic diameters in various biological media (PBS (0.1 M, 1 M), albumin and NaCl (10⁻²M)).</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Evaluation of the cytotoxic effects (cell death induction) of nanoparticles by phase contrast microscopy or by staining with May-Grünwald Giemsa (MGG). BV-2 murine microglial cells were exposed to nanoparticles for 24 h at concentrations from 3 to 38 µg/mL depending on the initial amounts of nanoparticles available in distilled water: SPIONs 3.8 mg/mL. PEG-SPIONs 2.3 mg/mL, ND-PEG-SPIONs 0.8 mg/mL, ND-PEG-SPIONs-SKL 0.6 mg/mL, and bare NDs 1 mg/mL. A: phase contrast microscopy; at the highest concentrations used indicated in the pictures, no cytotoxic effects were observed compared to untreated cells (Control); B: MGG staining: no morphological differences were observed at the nuclear and cytoplasmic levels, supporting the absence of marked cytotoxic effects.</figcaption></figure>

spectrometry, which enables sensitive and precise quantification of intracellular iron content [62]. Additionally, more detailed morphological analyses – using confocal microscopy and transmission electron microscopy – will help elucidate the cellular interactions of the various SPIONs, particularly at the peroxisomal level, thanks to the presence of the SKL sequence.

## 4. Conclusion

The present study proposes an innovative, simple and cost-effective method for preparing ND-PEG-SPIONs-SKL nanohybrids with robust stability. Multiple lines of evidence were collected and analyzed to

achieve the sequentially functionalization of the NPs. The nanohybrids were characterized at each step, and covalent chemistry was employed to anchor both the peptide and PEG to the surface, ensuring strong and stable binding, an outcome further supported by the zeta potential measurements. TEM images reveal that the NDs are surrounded by SPIONs. Furthermore, given that progressive alterations in mitochondrial and peroxisomal functions are now recognized as pivotal events in the semiology of not only Alzheimer's disease but also several other neurodegenerative disorders, the absence of toxicity (i.e. no induction of cell death) in the synthesized NPs – whether they interact specifically or non-specifically with mitochondria and/or peroxisomes – enables us to envision future studies exploring the interaction of other NPs with these
<figure><img src="image_12.png" /><figcaption>**Fig. 10.** Evaluation of the cytotoxic effects of nanoparticles with the FDA and MTT assays measuring esterase activity and mitochondrial succinate dehydrogenase activity, respectively. BV-2 cells were exposed to nanoparticles for 24 h at concentrations from 3 to 38 µg/mL depending on the initial amounts of nanoparticles available in distilled water: SPIONs 3.8 mg/mL. PEG-SPIONs 2.3 mg/mL, ND-PEG-SPIONs 0.8 mg/mL, ND-PEG-SPIONs-SKL 0.6 mg/mL, and bare NDs 1 mg/mL. A: FDA assay was used to evaluate possible cytotoxic effects at the plasma membrane level; B: MTT assay was used to evaluate possible cytotoxic effects at the mitochondrial levels. The comparison between untreated cells (control) and nanoparticles-treated cells was performed with the non-parametric Mann-Whitney test; \*: p values < 0.05 were considered as statistically significant.</figcaption></figure>

organelles. Thus, based on the *in vitro* data obtained from BV-2 cells, the developed nanohybrids offer promising therapeutic potential for neurodegenerative diseases associated with peroxisomal dysfunction and could represent a novel tool in pexotherapy [17,63]. However, this approach may face certain limitations due to the cumulative challenges of crossing the blood-brain barrier (BBB), achieving subcellular targeting, and exerting a therapeutic effect at the target site. Nevertheless, we remain optimistic, as various peptides, known for their ability to either translocate into cells (cell-penetrating peptides, CPP) or cross the BBB (THR peptide), have been synthesized and grafted onto the surface of SPIONs-PEG particles (study in progress, unpublished data). These peptides may also exhibit antioxidant properties.

CRediT authorship contribution statement

**Lionel Maurizi:** Writing – review & editing, Writing – original draft, Validation. **Julien Boudon:** Writing – review & editing, Writing – original draft, Validation, Supervision. **Leya Liu:** Validation, Methodology, Investigation. **Thomas Nury:** Writing – review & editing, Validation, Methodology, Investigation. **Nimisha Singh:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation. **Nadine Millot:** Writing – review & editing, Writing – original draft, Validation, Supervision, Funding acquisition, Conceptualization. **Gérard Lizard:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Conceptualization. **Vivien Pires:** Validation, Methodology, Investigation.
<figure><img src="image_13.png" /><figcaption>**Fig. 11.** Assessment of the intracellular accumulation of nanoparticles (SPIONs) with the Perls staining procedure. BV-2 cells were exposed to nanoparticles for 24 h at concentrations from 3 to 38 µg/mL depending on the initial amounts of nanoparticles available in distilled water: SPIONs 3.8 mg/mL. PEG-SPIONs 2.3 mg/mL, ND-PEG-SPIONs 0.8 mg/mL, ND-PEG-SPIONs-SKL 0.6 mg/mL. Intracellular accumulation of SPIONs was observed only in a few cells (5.5 ± 1.1 %) with the highest concentration of SPIONs.</figcaption></figure>

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nadine Millot reports financial support was provided by EIPHI Graduate School (contract ANR-17-EURE-0002). Nadine Millot reports financial support was provided by FEDER-FSE Bourgogne. Nadine Millot reports financial support was provided by Project ISITE BFC contract ANR-15-IDEX-0003. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work was supported by the EIPHI Graduate School (contract ANR-17-EURE-0002) and the "Conseil Régional de Bourgogne Franche-Comté". This work was also supported by the European Union through the PO FEDER-FSE Bourgogne 2014/2020 programs and by French "Investissements d'Avenir" program, project ISITE BFC contract ANR-15-IDEX-0003. The authors acknowledge Dr. R. Chassagnon and Dr. O. Heintz of ARCEN-Carnot platform (ICB) for TEM observations and XPS investigations. They also acknowledge Prof. François Treussart for fruitful discussions and Dr Imen Ghzaiel for her pioneering work on pexotherapy during her PhD thesis at the Université Bourgogne Europe; (website consulted on august 23, 2025; https://theses.fr/265715091).

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.colSURFB.2025.115170.

## Data availability

Data will be made available on request.

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