# Ensuring colloidal stability of cisplatin-loaded mesoporous silica nanoparticles: from synthesis to cytotoxicity assays

Eduardo Hernando Abad, Florence Bouyer, Igor Bezverkhyy, Rémi Chassagnon, Nicolas Geoffroy, Lucien Saviot, Frédéric Bouyer

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Eduardo Hernando Abad, Florence Bouyer, Igor Bezverkhyy, Rémi Chassagnon, Nicolas Geoffroy, et al.. Ensuring colloidal stability of cisplatin-loaded mesoporous silica nanoparticles: from synthesis to cytotoxicity assays. International Journal of Pharmaceutics, 2026, 687, pp.126395. 10.1016/j.ijpharm.2025.126395. hal-05387462

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# Ensuring colloidal stability of cisplatin-loaded mesoporous silica nanoparticles: from synthesis to cytotoxicity assays

Eduardo Hernando Abad<sup>a, *, †</sup>, Florence Bouyer<sup>b, c</sup>, Igor Bezverkhyy<sup>a</sup>, Rémi Chassagnon<sup>a</sup>, Nicolas Geoffroy<sup>a</sup>, Lucien Saviot<sup>a</sup>, Frédéric Bouyer<sup>a, *</sup>

## ARTICLE INFO

### Keywords:

Mesoporous silica nanoparticles

Nanocarrier

Drug delivery

Colloidal stability

Copolymer

## ABSTRACT

Mesoporous silica nanoparticles (MSNs) have attracted significant attention as a promising drug delivery platform for cancer treatment. However, their colloidal stability often remains overlooked, despite the numerous drawbacks associated with its absence. In this study, the enhancement of the colloidal stability of thoroughly characterised (TEM, DLS, zeta potential, TGA, XRD, N₂ physisorption) monodisperse MCM-41 MSNs in physiological conditions through the grafting a comb-like copolymer was successfully accomplished (preservation of the hydrodynamic diameter and the polydispersity index for at least 3 days). The loading of a therapeutic agent, cisplatin, was then achieved (12 wt%) without compromising their colloidal stability, and drug release kinetics were evaluated in cell culture medium, observing a complete release of the cargo after 48 h. Cytotoxicity studies conducted before and after drug loading confirmed the innocuousness of unloaded MSNs and demonstrated cytotoxic effects comparable to those of free cisplatin for the loaded nanoparticles ($IC_{50} = 23$ and 13 µM CDDP for loaded MSNs versus 24 and 11 µM CDDP for free cisplatin in, respectively, SW480 and A549 cell lines). Particular emphasis was placed on ensuring the reproducibility of the results (e.g., hydrodynamic diameter and polydispersity index were measured across 49 independent synthesis) and on thoroughly evaluating the colloidal stability of the nanoparticles throughout the study, from synthesis to cytotoxicity assays. To the best of our knowledge, this work represents the first systematic study of the conditions required to develop stable colloidal suspensions of monodispersed mesoporous silica nanoparticles loaded with cisplatin, achieving high reproducibility, which constitutes a novel and robust contribution to the field.

## 1. Introduction

Since Vallet-Regí et al. first proposed the use of mesoporous silica as a drug delivery system in 2001 (Vallet-Regi et al., 2001), mesoporous silica nanoparticles (MSNs) have attracted significant attention in the field of nanomedicine (Ahmadi et al., 2022; Manzano and Vallet-Regí, 2020; Wang et al., 2015). Their high specific surface area, biocompatibility, tuneable particle and pore sizes, and easily modifiable surface chemistry for the incorporation of different functional groups make MSNs one of the most promising candidates for drug delivery and other biomedical applications.

Among human diseases, cancer stands out as a pathology for which nanoparticles may improve the risk-benefit ratio of anticancer drugs. This study focuses on the vectorisation of cisplatin

(cis-diaminedichloroplatinum(II), CDDP), a widely used chemotherapeutic for several digestive and genital cancers (Han et al., 2022). Nanoparticle-based delivery of cisplatin can enhance its accumulation in tumour tissues while reducing drug resistance and toxicity (Anable, 2016). Although numerous organic and inorganic nanomaterials have been developed to vectorise platinum drugs (Cheng et al., 2014; Isaac et al., 2018; Min et al., 2012; Murakami et al., 2011; Paraskar et al., 2012; Stathopoulos and Boulikas, 2012), only a few studies focus on the loading of cisplatin into MSNs (Ahn et al., 2013; Gu et al., 2010; Varache et al., 2019; Ortiz-Islas et al., 2020; Zarkesh et al., 2022; Siraj et al., 2025). MSNs are promising candidates for cisplatin delivery, as they can improve the solubility of poorly water-soluble drugs, promote higher intracellular drug accumulation, and minimise side effects. However, cisplatin is insoluble in most organic solvents, except for N,

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

<footnote><sup>b</sup> Université Bourgogne Europe, Dijon, France</footnote>

<footnote><sup>c</sup> INSERM, Therapies & Immune Response Canc Team, Bioact Mol & Hlth Res Grp, Res Ctr UMR1231, Dijon, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: eduardo.hernando.abad@gmail.com (E. Hernando Abad), frederic.bouyer@ube.fr (F. Bouyer).</footnote>
N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC) and dimethyl sulfoxide (DMSO) (Riley and Sternson, 1986). The toxicity of DMF and DMAC precludes their use to load cisplatin in MSNs. DMSO, although commonly used to solubilise cisplatin, forms a very stable complex with the drug that exhibits significantly reduced cytotoxicity. Indeed, DMSO has been reported to inhibit cisplatin's ability to induce cell death, and should therefore be strictly avoided during the loading process (Fischer et al., 2008; Hall et al., 2014). For this reason, aqueous-based loading methods are preferable.

One of the main challenges in developing MSNs for biomedical applications is achieving a colloidalily stable suspension, particularly since these nanoparticles are most often administered intravenously. Aggregation can lead to uncontrolled biological reactions, potential toxicity, immune responses or blockage of blood vessels (Lin et al., 2012; Moore et al., 2015; Siegrist et al., 2019). Therefore, maintaining colloidal stability from synthesis to administration -including all intermediate steps such as surface functionalisation and drug loading- is essential. In this context, it is crucial to evaluate the colloidal stability of MSNs not only in water but especially under physiological conditions, where high ionic strength promotes nanoparticle aggregation. To the best of our knowledge, only a limited number of studies address the dispersion and stability of MSNs in biologically relevant environments. The literature often assumes these systems are colloidalily stable and overlooks their potentially instability in physiological media (da Schneid et al., 2020). Dynamic light scattering (DLS) is a key technique for accurately assessing the colloidal stability of a nanoparticle suspension. While electron microscopy is commonly used, it does not necessarily reflect the overall sample since only a limited number of nanoparticles are analysed. In contrast, DLS offers a more comprehensive view of the colloidal state of the sample. However, many studies report only a single value for the hydrodynamic diameter (or the size distribution from which this value was calculated) typically measured immediately after synthesis or surfactant removal. Haynes et al. (Lin et al., 2012) critically noted that most works assess colloidal stability at only one point, failing to monitor changes after each modification step or following redispersion from the dried state. Consequently, such studies often overextend their conclusions regarding long-term stability without supporting data. In many cases, DLS measurements are taken over seconds or minutes, with very few studies performing multi-day stability assessments in water, in even fewer in physiological conditions. Da Cruz Schneid et al. reviewed 27 studies evaluating the colloidal stability of silica nanoparticles in biologically relevant fluids (Da Cruz Schneid et al., 2022). Interestingly, in most of these studies, stability was assessed only once. While the study of long-term particle dispersion is essential to demonstrate the true stability of MSNs, only a limited number of studies in the literature address this issue (Lin et al., 2011b; da Schneid et al., 2020; Wang et al., 2010; Zhang et al., 2014). From a practical perspective, drying nanoparticles is convenient for transport, storage and weighing. Nevertheless, only a few studies evaluate the efficiency of redispersion using DLS (Lin et al., 2011b), despite its importance for the proper execution of subsequent steps. Often, the redispersion method is either vaguely described (e.g., stating only that stirring or sonication was used) or not mentioned at all. This lack of methodological detail is frequently accompanied by the absence of any validation of the redispersion efficiency, with suspensions used directly in subsequent steps without confirming their colloidal state (Lin et al., 2012). Moreover, many studies fail to specify the type of DLS-derived size reported, which could be the intensity-weighted mean hydrodynamic size calculated from the cumulant analysis (Z-Ave) or from other models (Contin, NNLS, etc.), or the number- (or volume-) weighted mean hydrodynamic size, merely stating that it is the hydrodynamic diameter. This, combined with the fact that size distributions are not systematically shown, makes it difficult to assess colloidal stability. Indeed, the presence of aggregates (and thus additional distributions at larger sizes) leads to a skewed Z-Ave value, which no longer reflects the primary distribution of the MSNs. Similarly, reporting only the intensity- (or volume-, or number-)

weighted mean hydrodynamic size for the main distribution may, in some cases, obscure the presence of larger aggregates. Finally, colloidal stability should be verified not only for empty nanocarriers but also after drug loading. Nevertheless, most publications dealing with the colloidal stability of MSNs evaluate nanoparticle dispersion only prior to drug encapsulation, neglecting potential changes induced by the loading process. In conclusion, comprehensive studies evaluating the colloidal stability of MSNs across all processing steps remain scarce. Greater attention must be given to the dispersion behaviour of these nanoparticles after each modification step in order to fully understand their long-term stability and performance in biological settings.

In the present work, a comb-like copolymer containing carboxylic groups and polyethylene glycol (PEG) chains was used to enhance the colloidal stability of MSNs through a combination of steric (via polymer chains) and electrostatic (via charged groups) stabilisation. PEGylation is known to improve the colloidal stability of nanoparticles in blood and other physiological fluids, enhancing their biocompatibility and prolonging their circulation time in the bloodstream (Nair et al., 2024). An organic solvent-free method for loading cisplatin was optimised to preserve the colloidal stability of the nanoparticles during and after cisplatin loading, followed by cytotoxicity evaluation. Colloidal stability was thoroughly assessed by dynamic light scattering (DLS) at each step of the process, from synthesis to cytotoxicity assays. In addition, particular attention was paid to the reproducibility of the results whenever feasible, in order to reinforce the robustness of our observations.

## 2. Materials and methods

### 2.1. Materials

Tetraethyl orthosilicate $Si(OC_2H_5)_4$ (TEOS, M = 208.33 g/mol, Sigma-Aldrich, CAS 78-10-4, ≥ 99.0 %), (3-aminopropyl)triethoxysilane $H_2N(CH_2)_3Si(OC_2H_5)_3$ (APTES, M = 221.37 g/mol, Sigma-Aldrich, CAS 919-30-2, 99 %), cetyltrimethylammonium bromide ($C_{16}H_{33}N(CH_3)_3Br$ (CTAB, M = 364.45 g/mol, Sigma Life Science, CAS 57-09-0, ≥ 99.0 %), 2 M decarbonated sodium hydroxyde NaOH (M = 40.00 g/mol, Fisher Scientific, CAS 1310-73-2), 1 M hydrochloric acid HCl (M = 36.46 g/mol, Fisher Scientific, CAS 7647-01-0), ammonium nitrate $NH_4NO_3$ (M = 80.04 g/mol, Acros Organics, CAS 6484-52-2, > 99 %), comb-like copolymer C33s previously synthesised in our laboratory (Dalas, 2014), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride $C_8H_{17}N_3·HCl$ (EDC-HCl, M = 191.70 g/mol, Sigma-Aldrich, CAS 25952-53-8), N-hydroxysuccinimide $C_4H_5NO_3$ (NHS, M = 115.09 g/mol, Thermo Scientific, CAS 6066-82-6), cisplatin Pt ($NH_3)_2Cl_2$ (CDDP, M = 300.05 g/mol, Aldrich, CAS 15663-27-1), PBS 1X prepared by dissolution of a sachet (Euroimmun) in a bottle of 1 L of sterile water (Versylene® Fresenius), DMEM High Glucose, fetal bovine serum (FBS), Hank's Balanced Salt Solution, trypsin, absolute ethanol, acetic acid and crystal violet were used without further purification. Ultrapure water (Milli-Q, 18.2 MΩ-cm) and technical ethanol were employed as solvents.

### 2.2. Preparation of MSN-NH₂

Amino-functionalised mesoporous silica nanoparticles (MSN-NH₂) were synthesised via a co-condensation method based on the procedure proposed by Varache et al. (Varache, 2014) with slight modifications. Typically, 200 mL of ultrapure water and 416.6 mg of CTAB (1.14 mmol) were added to a 500-mL three-neck round-bottom flask equipped with a 35x16 mm oval magnetic stirring bar. The flask was fitted with a condenser on the central neck and connected to a Schlenk line through one of the side necks. The oil bath was maintained at 80 °C (resulting in an internal temperature of ~66 °C inside the flask) and the mixture was stirred at 700 rpm. A continuous flow of nitrogen (1 L/min) was passed through the flask and the condenser. Before adding the base, the solution
was purged three times with nitrogen. Then, 1.45 mL of 2 M decarbonated NaOH (2.90 mmol, 14.4 mM after addition) was added under nitrogen flow (1 L/min). The mixture was stirred for 1 h. In parallel, 3.62 mL of TEOS and 0.420 mL of APTES were mixed in a glovebox, and 2.02 mL of this mixture (8.99 mmol of silanes) was added to the flask using a syringe pump at a rate of 1 mL/min. The reaction mixture was stirred for 2 h. To reinforce the structure of MSN-NH₂, the pH was gradually lowered by adding 1 mL of 1 M HCl every 5 min, up to 3 mL. The suspension was stirred overnight (~18 h) at 700 rpm, under static nitrogen, and at a maintained temperature of 80 °C. The resulting suspension was centrifuged in 6 tubes at 10,000 g for 1 h. After removing the supernatants, each pellet was redispersed in 15 mL of ethanol using an ultrasonic probe (Sonics & Materials, Vibra-Cell™, fixed tip made of Ti-6Al-4 V titanium alloy with a length = 254 mm and a diameter = 13 mm, coupled with a VCX 750 generator with a power of 750 W and a frequency of 20 kHz). To remove the CTAB surfactant, the redispersed suspensions were transferred into a 250-mL round-bottom flask containing 2 g of ammonium nitrate dissolved in 50 mL of ethanol. The mixture was stirred at 700 rpm with a 25x12 mm oval magnetic stirring bar and heated under reflux for 1 h. It was then centrifuged in 6 tubes at 10,000 g for 10 min, and the pellets were redispersed in 10 mL of ethanol using the ultrasonic probe. This CTAB extraction process was repeated 3 additional times. Following extraction, 5 ethanol washings were performed to eliminate residual ammonium nitrate. After the final CTAB extraction, each pellet was redispersed in 20 mL of ethanol by sonication and suspensions were centrifuged in 6 tubes at 10,000 g for 20 min. This washing procedure was repeated 4 additional times. After the fifth wash, each pellet was redispersed in 5 mL of ethanol by sonication. The suspensions were combined, and the solvent was removed using a rotary evaporator (BUCHI, R-300, France), yielding a dry white powder, which was stored in a desiccator under vacuum. For comparison purposes, analogue nanoparticles referred to as MSN-OH were prepared using TEOS only (2.01 mL, 8.99 mmol) as the silica precursor, following the same procedure.

## 2.3. Redispersion of powdered MSN-NH₂

Typically, 20 mg of powdered MSN-NH₂ was redispersed in 20 mL of ultrapure water in a plastic flask using an ultrasonic probe (2 s ON, 3 s OFF, 70 %, 1 min 30 s). The resulting suspension was allowed to cool to room temperature before further use.

## 2.4. Grafting of comb-like copolymer C33s (MSN-NH₂@C33s)

9.3 mg of comb-like copolymer C33s (1.66·10⁻⁵ mol of COO⁻ groups) was dissolved in 4 mL of ultrapure water. Under stirring, 15.9 mg of EDC (8.30·10⁻⁵ mol) dissolved in 0.5 mL of ultrapure water was added to the copolymer solution. Subsequently, 19.1 mg of NHS (1.66·10⁻⁴ mol) dissolved in 0.5 mL of ultrapure water was added and the solution was stirred at 500 rpm at room temperature for 50 min. The activated C33s solution was then added dropwise to a freshly prepared MSN-NH₂ suspension (see Section 2.3), and the pH was adjusted to 7 using 0.1 M NaOH. The final suspension was stirred at 500 rpm using a 12x6 mm oval magnetic stirring bar at room temperature for 20 h. The resulting nanoparticles were then purified by centrifugation using a centrifuge tube equipped with a 100 kDa or a 50 kDa filter (Merck Millipore, Amicon® Ultra-15, Ultracel®, regenerated cellulose) followed by washing with ultrapure water as described in Protocol S1. The washed nanoparticles were recovered from the filter in a final volume of 10 mL of ultrapure water and redispersed using an ultrasonic probe (2 s ON, 3 s OFF, 50 %, 2 min). The suspension was dried, if required, using a rotary evaporator (pressure = 175 mbar, bath temperature = 60 °C). A reference sample was prepared by treating nanoparticles under identical conditions in the absence of C33s, EDC and NHS. Additionally, and an alternative protocol was performed without coupling agents (MSN-NH₂/C33s). The mass concentration of the nanoparticle suspension was

determined by diluting 8 mL of the obtained suspension with 8 mL of ultrapure water, and drying three aliquots of 5 mL for each sample in small pre-weighed glass vials in an oven at 100 °C. The mass before and after drying was measured, and the concentration was calculated using the Expression S1.

## 2.5. Colloidal stability evaluation of MSN-NH₂ and MSN-NH₂@C33s

The colloidal stability of redispersed powdered MSN-NH₂ at 0.1 mg MSN-NH₂/mL and freshly prepared non-dried MSN-NH₂@C33s at 0.09 mg MSN-NH₂@C33s/mL (equivalent to 0.07 mg MSN-NH₂/mL when excluding the mass contribution of the copolymer C33s) was evaluated under various conditions listed in Table 1. These conditions were selected to investigate the effects of physiological ionic strength and pH by adding NaCl and PBS respectively. Each sample condition was prepared in duplicate, and each replicate was analysed by DLS 9 times per day without filtration. The pH of each suspension was measured after DLS analysis. Suspensions were stored without stirring and only shaken for a few seconds prior to DLS measurements and stirred during pH measurement each day. Each reported result corresponds to the average of 2 sample values, each being the average of 9 measurements, and is presented along with its standard deviation. Additionally, redispersed powdered MSN-NH₂ at 1 mg MSN-NH₂/mL was also evaluated under condition A.

## 2.6. Loading of CDDP in MSN-NH₂@C33s

A given amount of cisplatin (8, 12, 16, 20 or 24 mg) was mixed with 4 mL of PBS (0.1X, 0.2X or 0.4X, prepared by dilution from PBS 1X) in a flat-bottomed glass vial. The mixture was stirred at 500 rpm using a 12x6 mm oval magnetic stirring bar and protected from light for 1 h at room temperature. Subsequently, 4 mL of MSN-NH₂@C33s suspension at 1.8 mg MSN-NH₂@C33s/mL (equivalent to 1.4 mg MSN-NH₂/mL) was then added and the mixture was stirred under the same conditions for 72 h. The suspension initially prepared with 12 mg of cisplatin and 0.4X PBS was then centrifuged using a 50–mL tube equipped with a 100 kDa filter at 2000 g for 20 min in 0.2X PBS, obtaining 8 mL of suspension, which were homogenised with a micropipette. This suspension was used immediately for cytotoxicity assays. The platinum concentration in the MSN-NH₂@C33s@CDDP suspension was quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using an iCAP PRO XP DUO (Thermo Electron) instrument. Platinum was measured at λ = 214.423 nm after sample treatment with aqua regia and mineralisation with HF.

## 2.7. CDDP release kinetics

2.5 mL of MSN-NH₂@C33s@CDDP suspension at an equivalent concentration of 4.5 mg MSN-NH₂/mL and 454.46 mg Pt / L was introduced into dialysis membrane tubing (Cellu Sep® T3, dry diameter = 6.37 mm, nominal MWCO = 12000–14000). The tubing was immersed in a closed Schott bottle containing 47.5 mL of DMEM High Glucose supplemented with 10 % FBS placed on a bath at 37 °C. The system was magnetically stirred to provide a homogeneous dispersion of

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

Experimental conditions used for colloidal stability evaluation. NaCl and PBS were added to investigate the effect of physiological ionic strength and pH, respectively. The NaCl concentration in condition B corresponds to 0.9 wt%.

<table><thead><tr><th>Condition</th><th>[PBS] (X)</th><th>[NaCl<sub>additonal</sub>] (M)</th><th>[Salt]<sub>total</sub> (M)</th></tr></thead><tbody><tr><td>A</td><td>0</td><td>0</td><td>0</td></tr><tr><td>B</td><td>0</td><td>0.15</td><td>0.15</td></tr><tr><td>C</td><td>0.10</td><td>0</td><td>0.02</td></tr><tr><td>D</td><td>0.10</td><td>0.15</td><td>0.17</td></tr><tr><td>E</td><td>0.05</td><td>0</td><td>0.01</td></tr><tr><td>F</td><td>0.05</td><td>0.15</td><td>0.16</td></tr></tbody></table>
free cisplatin inside and outside the dialysis membrane, and the bottle was shielded from light. The platinum concentration outside the dialysis membrane was monitored over time by ICP-AES using an iCAP PRO XP DUO (Thermo Electron) instrument. Platinum was measured as described previously. The test was performed in duplicate. Results are expressed as the amount of cisplatin released (calculated with Expression S2) as a function of time.

To investigate the mechanism of drug release, the experimental data were fitted to several mathematical models: zero-order, first-order, Higuchi, Hixson-Crowell and Korsmeyer-Peppas (Khalbas et al., 2024; Zarkesh et al., 2022).

* The zero-order model describes a system in which the drug is released at a constant rate over time. It can be expressed as $F = K_0 \cdot t$, where F is the fraction of drug released at time t, and $K_0$ represents the zero-order release constant.

* The first-order model assumes that the release rate is proportional to the remaining amount of drug and is given by $\ln(1-F) = K_1 \cdot t$, where $K_1$ is the first-order release constant.

* The Higuchi model is expressed as $F = K_H \cdot t^{1/2}$, where $K_H$ is the Higuchi release rate constant.

* The Hixson-Crowell model can be written as $1 - \sqrt[3]{1 - \bar{F}} = K_{HC} \cdot t$, where $K_{HC}$ is the Hixson-Crowell release constant.

* Finally, the Korsmeyer-Peppas model is expressed as, $\ln F = \ln K_{KP} + n \cdot \ln t$, where $K_{KP}$ is the Korsmeyer-Peppas release constant and n represents the drug release mechanism from the nanocarrier.

F is calculated as $(n_t - n_0)/(n_\infty - n_0)$, where $n_t$ is the percentage of cisplatin released at time t, $n_\infty$ is the percentage of cisplatin released at t = 48 h (when the entire cargo has been released) and $n_0$ is the percentage of cisplatin released at t = 1h. To fit the data to the different models, values corresponding to burst release phase (t < 1h) and those corresponding to the plateau after complete release (t > 48 h) were excluded.

## 2.8. Cell lines and culture conditions

Human colorectal cancer cell line SW480 and human lung adenocarcinoma cell line A549 were obtained from the American Type Culture Collection (Manassas, VA, United States). Both cell lines were cultured in DMEM High Glucose medium supplemented with 10% FBS under 5% CO₂ atmosphere at 37 °C. SW480 and A549 cells were grown as exponentially proliferating monolayers in mycoplasma-free culture conditions, which were regularly confirmed by polymerase chain reaction (PCR) analysis using the PCR Mycoplasma Test Kit I/C (PromoKine, PromoCell, France).

## 2.9. Cytotoxicity assays in cancer cell lines

Cancer cell lines were seeded in 96-well plates at densities of 10,000 cells per well for SW480 and 7000 cells per well for A549 to reach subconfluency on the day of treatment. Cells were then exposed for 72 h to concentrations ranging from 0 to 200 µg/mL of the different MSNs tested: MSN-NH₂, MSN-NH₂@C33s and MSN-NH₂@C33s@CDDP. In parallel, the cytotoxicity of free cisplatin was evaluated at concentrations ranging from 0 to 500 µM. Following treatment, cell culture was stopped. Cells were washed with PBS 1X, fixed in absolute ethanol and stained with 1% crystal violet. The dye was subsequently eluted using 33% acetic acid. The absorbance of the eluted dye, which correlates with cell viability, was measured spectrophotometrically at λ = 570 nm (UVM 340, BioServ). All concentrations were tested in quadruplicate for both MSNs and cisplatin, across three independent experiments. Mean values of cytotoxicity were calculated as well as their error bars that represent the standard deviation of all wells at the same concentration divided by the square root of the number of wells.

Results were expressed as concentration-response curves showing percentage cytotoxicity (calculated using Expression S3) as a function of MSNs or cisplatin concentration (using Expression 1). The half maximal inhibitory concentration (IC₅₀), representing the concentration that inhibits 50% of cell growth, was determined after linearisation of these curves.

**Expression 1.** Simplified formula used to convert the concentrations of MSN-NH₂@C33s-A@CDDP from mg MSN-NH₂ / mL to µM of cisplatin. A detailed version is provided in Expression S3.

$$y\mu\text{molCDDP/L} = 0.582 \cdot x\mu\text{gMSN} - \text{NH}_2\text{/mL}$$

## 2.10. Physicochemical characterisations

The hydrodynamic diameter of the particles was measured by dynamic light scattering (DLS) at 173° angle using a Zetasizer Nano ZSP instrument (Malvern Instruments, UK) equipped with a 4 mW He-Ne laser (λ = 633 nm). The mean particle size, Z-Ave, was obtained from fitting a single exponential to the correlation function, alongside the corresponding polydispersity index (PdI). Each measurement was performed in nonuplicate at 20 °C after 120 s of stabilisation. The reported data correspond to the mean values of the 9 independent measurements.

Zeta potential measurements were conducted on the same Zetasizer Nano ZSP instrument. Each measurement was performed in triplicate. The titration direction was selected to avoid crossing the isoelectric point (IEP) when adjusting the initial pH from the natural pH of the suspension. For every titration, 10 mL of suspension was used at the following concentrations: 1 mg MSN-NH₂ / mL, 1 mg MSN-OH / mL, 0.9 mg MSN-NH₂@C33s / mL (equivalent to 0.7 mg MSN-NH₂ / mL) and 0.9 mg MSN-NH₂/C33s / mL (equivalent to 0.7 mg MSN-NH₂ / mL). For MSN-NH₂ and MSN-OH, powdered nanoparticles were redispersed prior to analysis as described in Section 2.3, whereas MSN-NH₂@C33s and MSN-NH₂/C33s were analysed immediately after preparation and washing, without subsequent drying. No background salt was used during the titrations.

The morphology and mesostructure of the nanomaterials were characterised by transmission electron microscopy (TEM). A Hitachi HT7800 (Hitachi High Techn Corp., Tokyo, Japan) operating at 100 kV with a point-to-point resolution of 0.36 nm was used (Dimacell analysis platform, INRAe, Dijon). Additionally, a JEOL JEM 2100 microscope (JEOL (Europe) SAS, Croissy-sur-Seine, France) operating at 200 kV with a point-to-point resolution of 0.25 nm was employed (ARCEN analysis platform, Université Bourgogne Europe, Dijon, France). In all cases, samples were prepared by immersing 300-mesh carbon film-coated copper grids into the nanoparticle suspension for a few seconds, then allowing them to air dry.

Textural properties were evaluated by nitrogen physisorption at 77 K using a Micromeritics ASAP 2020 system. Powder samples were degassed at 80 °C prior to analysis. The specific surface area (S<sub>BET</sub>) was calculated using the BET method within the relative pressure range of 0.06 to 0.25. Pore size distribution and mesoporous volume (V<sub>m</sub>) were determined from the desorption branches of the isotherms using the BJH-method at P/P₀ ≤ 0.75 to minimise contributions from interparticle porosity.

Structural properties were assessed by low-angle powder X-ray diffraction (XRD) using a Bruker D8 Advance diffractometer equipped with Vantec linear PSD and CuKα radiation (35 kV, 35 mA). Diffraction patterns were recorded over the 0.3° to 10° (2θ) range, with a step size of 0.017° and a step time of 3 s. Data were normalised such that the intensity of the most intense peak (100) was consistent across samples.

Thermogravimetric analysis (TGA) was performed on a TA Instruments Discovery system (USA) to study the decomposition of organic species. The protocol involved an initial 10-minute isotherm at room temperature, followed by a temperature ramp at 5 °C/min up to 100 °C. A subsequent 1-h isotherm at 100 °C was applied to remove physisorbed water. The main analysis ramp then increased temperature from 100 to
800 °C at 5 °C/min. To avoid overestimating functionalisation rates due to water loss related to silica condensation, weight loss calculations were restricted to the 100–800 °C.

### 3. Results and discussion

#### 3.1. Preparation of amino-functionalised mesoporous silica nanoparticles (MSN-NH₂) and associated colloidal stability concerns

TEM images obtained prior to solvent evaporation revealed the faceted morphology of monodisperse and non-aggregated nanoparticles (Fig. 1A). At higher magnification, a well-defined hexagonal arrangement of the pores was observed. Since the two-dimensional cross-section of the nanoparticles can be approximated as a hexagon, size determination involved measuring two perpendicular diameters: the long diameter (D) and the short diameter (d) (Fig. 1B). Fig. 1C shows a scatter plot of D versus d for each nanoparticle measured using Image J. Most data points lie between the two lines representing the ideal geometries of a regular hexagon (red line) and a circle (blue line), with a tendency toward the latter. Points above the hexagon line correspond to elongated nanoparticles. The distribution of these two combined diameters is shown in Fig. 1D for 4 independent batches of MSN-NH₂ analysed before drying. Measurement of over 200 nanoparticles per batch yielded an average diameter of 111 nm with a standard deviation σ = 0.02.

The synthesis method demonstrated good reproducibility, as evidenced by the narrow distributions of the Z-Ave (Fig. 1E) and the PdI (Fig. 1F), both measured by DLS. The average Z-Ave across 49 synthesis batches (each Z-Ave<sub>batch</sub> calculated from 9 independent measurements) was 143 nm (σ = 3 nm). The corresponding average PdI across 49 independent PdI<sub>batch</sub> values was 0.03 (σ = 0.01), confirming the high monodispersity of the nanoparticles.

The zeta potential of MSN-NH₂ was measured as a function of pH for and the results were compared with those of MSN-OH (Fig. 2A). MSN-NH₂ exhibited an average isoelectric point (IEP) of 8.0 (σ = 0.1, n = 7 independent samples), while MSN-OH showed an IEP of 3.2 (n = 1), a characteristic value for non functionalised silica (Avadiar and Leong, 2011; Chadwick et al., 2002; Franks, 2002). The shift in IEP toward higher pH values confirmed the presence of amine groups on the external surface of MSN-NH₂ (Etienne and Walcarius, 2003; Moitra et al., 2011). Nevertheless, the presence of silanol groups on the surface could also be inferred. Given that the pKₐ of APTES is 10.4 (Mornet et al., 2005), the nanoparticles should remain positively charged at pH 8. The observation of a negative surface charge above this pH suggests the deprotonation of silanol groups.

The functionalisation rate was quantified through TGA (Fig. 2B). To avoid any overestimation, the weight loss attributed to the propylamine groups in MSN-NH₂ was calculated by subtracting the value obtained for MSN-OH. This approach assumes that water loss from silica condensation is the same for all the samples. MSN-NH₂ showed an average weight loss of 12.8 % (σ = 0.8 %, n = 30 independent samples), compared to 8.0 % for MSN-OH. Using this difference and applying Expressions S4 and S5, the experimental functionalisation rate (τ<sub>exp.</sub>) was determined to be 0.83 mmol NH₂ / g (σ = 0.14 mmol NH₂ / g, n = 30), corresponding to 0.59 NH₂ groups / nm² (σ = 0.10 NH₂ groups / nm², n = 30). The functionalisation efficiency (η), defined by Expression S6 and based on a theoretical functionalisation rate (τ<sub>theo.</sub>, Expression S7), was calculated to be 52 % (σ = 9 %, n = 30).

X-ray diffraction analysis revealed five Bragg peaks corresponding to the (100), (110), (200), (210) and (300) planes (Fig. 2C), which are characteristic of the two-dimensional hexagonal P6mm geometry typical of MCM-41-type mesoporous materials (Beck et al., 1992). Nitrogen physisorption isotherms (Fig. 2D) exhibited two capillary condensation steps and are classified as type IV isotherms according to the Brunauer, Deming, Deming and Teller (BDDT) classification (Brunauer et al., 1938; Storck et al., 1998) and IUPAC nomenclature (Thommes et al., 2015). The observed hysteresis loop is of type H4

according to the IUPAC classification. Its formation is due to nitrogen condensation in the voids between loosely agglomerated silica nanoparticles. The first condensation step, observed around P/P₀ = 0.3, corresponds to mesopores within the nanoparticles, while the second step, at P/P₀ > 0.9, is attributed to interparticle porosity. These isotherms are consistent with those typically observed for MCM-41 materials (Chen et al., 1993). The pore size distribution (Fig. 2E) was narrow. The results from these two techniques are summarised in Table 3.

In our study, the operational conditions were optimised to enable the successful redispersion of dried MSN-NH₂ powder using an ultrasonic probe. Redispersion efficiency was evaluated by comparing DLS and TEM measurements before drying and after redispersion (Fig. 3A and Fig. 3B). Additionally, the redispersed MSN-NH₂ nanoparticles remained colloidal stable for at least 7 days. Although some aggregation was observed starting on day 6, the intensity of those aggregates did not exceed 0.7 % (Fig. 3C).

Nonetheless, the colloidal stability of MSN-NH₂ was compromised under conditions A, B, C and E (see Table 2). Under condition A, MSN-NH₂ exhibited a slightly higher Z-Ave and became destabilised after 24 h. This was likely due to the suspension's pH approaching the IEP of the nanoparticles, which reduces electrostatic repulsion and promotes aggregation. Specifically, condition A had a slightly higher pH (4.6) compared to the initial suspension (3.7), resulting in a higher Z-Ave (153 nm versus 148 nm). After 24 h, the observed destabilisation in DLS correlated with a further pH increase to 6.3 (see Fig. 2A). Conditions B, C and E introduced non-negligible ionic strength. However, the degree of destabilisation under these conditions was primarily governed by the pH rather than by ionic strength alone. On day 0, condition B (0.15 M) caused less pronounced destabilisation than conditions C and E, despite the latter having lower salts concentrations (0.02 M and 0.01 M, respectively). This seemingly counterintuitive outcome was explained by differences in pH: the presence of PBS in conditions C and E resulted in pH values of 7.3 and 7.1, respectively, closer to the IEP of 8.0 and thus promoting aggregation. In contrast, condition B maintained a lower pH of 5.5, further from the IEP, leading to greater colloidal stability despite the higher ionic strength. Notably, the destabilisation under condition B after 24 h remained less severe than that observed immediately after preparation under conditions C and E. These findings highlight the critical role that pH, in relation to the IEP, plays in the colloidal stability of MSN-NH₂ for subsequent steps, such as the loading of cisplatin. Given the lack of stability of the nanoparticles under physiological conditions, surface modification of the nanoparticles was studied using a comb-like copolymer.

#### 3.2. Grafting of comb-like copolymer C33s (MSN-NH₂@C33s)

C33s is a comb-like copolymer composed of a main chain bearing two types of side chains: (i) carboxylate groups (1.78 mmol COO⁻ / g) and (ii) polyethylene glycol (PEG) chains attached to the main chain via ester linkages. The carboxylic groups of C33s react with the amine groups on MSN-NH₂ to form amide bonds, thereby grafting the copolymer onto the nanoparticle surface, as illustrated in Fig. 4. Activation of the carboxylic groups was carried out using coupling agents EDC and NHS (Thanh and Green, 2010) (Fig. S1).

After washing, TEM images showed faceted, non-aggregated and monodispersed nanoparticles. The hexagonal arrangement of pores could still be observed (Fig. 5A). The zeta potential of well-dispersed MSN-NH₂@C33s was measured as a function of pH (Fig. 5B). Following the copolymer grafting, the IEP shift towards lower pH values was attributed to the neutralisation of surface amine groups upon amide bond formation and the presence of free carboxylic groups from the grafted copolymer. TGA of MSN-NH₂@C33s powder revealed a reproducible weight loss, with a mean value of 32.2 % (σ = 1.5 %, n = 14 independent samples). In most studies, the grafting ratio of organic compounds onto MSNs is calculated as the difference in weight loss before and after grafting (Cheng et al., 2019; Kim et al., 2023). However,
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** TEM images of MSN-NH₂ (A), geometric approximation of a nanoparticle to a regular hexagon (B), scatter plot of D versus d for individual nanoparticles (C), diameter distribution with error bars representing the standard deviation (D), and Z-Ave (E) and PdI (F) distributions determined by DLS for MSN-NH₂.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Zeta potential as a function of pH (A), weight loss curves determined by TGA (B), X-ray diffraction patterns (C), nitrogen physisorption isotherms (D) and pore size distribution calculated using the BJH method (E) for MSN-NH₂ (green) and MSN-OH (orange). Error bars represent the standard deviation.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Size distributions by intensity determined by DLS for MSN-NH₂ after synthesis and after redispersion (A), TEM images of MSN-NH₂ after redispersion (B), and size distributions by intensity determined by DLS for redispersed MSN-NH₂ at a concentration of 1 mg/mL (C).</figcaption></figure>

<caption>**Table 2**</caption>

Z-Ave, PdI and pH values of MSN-NH₂. Each sample under conditions A, B, C and E was prepared in duplicate, and the average values are presented. Standard deviations are shown in parentheses. For the 1 mg/mL sample, standard deviations for Z-Ave PdI are based on 9 measurements. For conditions A, B, C and E, standard deviations reflect the 2 prepared samples, with 9 measurements taken on each. Given that conditions C and E caused nanoparticles destabilisation, it was reasonable to assume that conditions D and F, which introduce an additional factor (0.15 M NaCl), would also lead to destabilisation; therefore, the values were not determined. n.d.: value not determined.

<table><thead><tr><th rowspan="2"></th><th colspan="3">Day 0</th><th colspan="3">Day 1</th></tr><tr><th>Z-Ave (nm)</th><th>PdI</th><th>pH</th><th>Z-Ave (nm)</th><th>PdI</th><th>pH</th></tr></thead><tbody><tr><td>1 mg/mL</td><td>148 (2)</td><td>0.07<br>(0.02)</td><td>3.7</td><td>146 (1)</td><td>0.08<br>(0.01)</td><td>4.1</td></tr><tr><td>A</td><td>153 (2)</td><td>0.07<br>(0.03)</td><td>4.6</td><td>1021<br>(247)</td><td>0.60<br>(0.21)</td><td>6.3<br>(0.3)</td></tr><tr><td>B</td><td>190 (4)</td><td>0.20<br>(0.04)</td><td>5.5</td><td>410 (41)</td><td>0.55<br>(0.05)</td><td>6.1<br>(0.0)</td></tr><tr><td>C</td><td>1753<br>(473)</td><td>0.21<br>(0.08)</td><td>7.3</td><td>n.d.</td><td>n.d.</td><td>n.d.</td></tr><tr><td>D</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td></tr><tr><td>E</td><td>1700<br>(421)</td><td>0.28<br>(0.11)</td><td>7.1</td><td>n.d.</td><td>n.d.</td><td>n.d.</td></tr><tr><td>F</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td></tr></tbody></table>

to improve accuracy, a new method is proposed in this work. First, it was observed that MSNs were altered by hydrolysis when stirred for several hours in water, leading to a reduction in weight loss. Therefore, nanoparticles subjected to the same grafting conditions (without the grafting entity) were characterised as a reference to avoid underestimating the

amount of grafted material. In these control samples, the weight loss of treated MSN-NH₂ was 7.4 %, compared to 12.8 % in untreated MSN-NH₂ (Fig. 2). Second, the incomplete degradation of the copolymer was taken under account, as only 94.8 % of C33s is decomposed during TGA. Assuming complete degradation would again lead to an underestimation of the grafted material. Finally, the total weight loss observed in grafted nanoparticles includes both the contribution from silica degradation (in this case, amine-modified silica) and the removal of grafted organic material. In contrast, for ungrafted nanoparticles, the weight loss arises entirely from the silica (amine-modified silica). Since grafting increases the total mass of the nanoparticles, the relative proportion of silica in the total weight becomes smaller. For example, the degradable silica fraction was 5.3 % in MSN-NH₂@C33s, compared to 7.4 % in MSN-NH₂ subjected to the same grafting conditions. Therefore, simply subtracting the weight loss percentages before and after grafting would overestimate the grafted amount. To address this, we assumed that the ratio between the degradable and non-degradable fractions of silica remains constant, regardless of the grafted material. Based on this assumption, and using Expression 2, the weight fractions of the different components in MSN-NH₂@C33s were calculated: 5.3 % for the degradable part of MSN-NH₂ in MSN-NH₂@C33s, 66.3 % for the non-degradable part of MSN-NH₂ in MSN-NH₂@C33s and 28.4 % for the grafted C33s.

Expression 2. Relationships between the weight contributions of the different components in MSN-NH₂@C33s.

% deg. fraction MSN-NH₂-graft.cond. + % deg. fraction C33s = 32.2 %.

% non-deg. fraction MSN-NH₂-graft.cond. + % non-deg. fraction C33s = 67.8 %.

% non-deg. fraction MSN-NH₂-graft.cond. = $\frac{92.6 \%}{7.4 \%}$ % deg. fraction MSN-NH₂-graft.cond.

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Structure of the comb-like copolymer C33s and schematic representation of the preparation of MSN-NH₂@C33s.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** TEM images (A), zeta potential as a function of pH (B), weight loss curve determined by TGA (C), X-ray diffraction patterns (D), nitrogen physisorption isotherms (E) and pore size distributions calculated using the BJH method (F) for MSN-NH₂ (green), MSN-NH₂@C33s (red) and calcined MSN-NH₂@C33s (blue). Error bars represent the standard deviation.</figcaption></figure>
$$ \% \text{ non-deg. fraction C33s} = \frac{5.2\%}{94.8\%} \% \text{ deg. fraction C33s}. $$

The grafting rate was calculated using Expressions S8 and S9, yielding 0.36 COO⁻ groups / nm² and 0.31 mmol COO⁻ / g, respectively.

The structural analysis of MSN-NH₂@C33s by nitrogen physisorption and X-ray diffraction (Fig. 5D and E, and Fig. 5F, respectively) indicated modifications in the porous structure and volumetric properties. Only the (100) diffraction peak remained visible, this feature being characteristic of vermicular systems where pores no longer exhibit the regular hexagonal two-dimensional arrangement. The absence of the other peaks was attributed to the presence of the copolymer inside the pores rather than a change in structure. Moreover, the physisorption isotherm lacked the step at P/P₀ = 0.3 typical of MCM-41 materials. The pore size distribution shifted from 4.5 nm to less than 2 nm. Additionally, there was a significant decrease in specific surface area (–36 %) and mesoporous volume (–63 %), as shown in Table 3. The missing condensation step in the isotherms suggested that the copolymer filled the pores or blocked pore entrances, explaining the apparent loss of mesoporosity despite TEM images showing intact pores. Similar observations have been reported for polymer-grafted MSNs, suggesting both a modification of the porous structure and the incorporation of the polymer within the pores (Lin et al., 2011a). Other authors have attributed this effect solely to PEG grafted on the external surface, which blocks pore entrances to nitrogen molecules (Cheng et al., 2019), especially since XRD patterns of PEG-coated MSNs still show Bragg peaks (He et al., 2010). TEM images in these studies confirmed the preservation of the mesostructure, consistent with our observations for MSN-NH₂@C33s. To confirm the presence of copolymer inside the pores, MSN-NH₂@C33s nanoparticles were calcined in a tubular furnace (100 °C for 1 h, ramping at 1 °C/min to 500 °C, held for 1 min) to remove the organic content. The (110) and (200) diffraction peaks reappeared, along with a high specific surface area and pore size distribution similar to those of MSN-NH₂, supporting the hypothesis that the grafting occurred both on the external surface and within the pores. While calcination restores mesoporosity, the structural differences between calcined MSN-NH₂@C33s and pristine MSN-NH₂ (particularly the broader and shifted peaks observed by XRD) suggest partial degradation of the porous framework during the calcination process, likely due to the absence of CTAB as a template and to the decomposition of the amine groups.

### 3.3. Colloidal stability assays of MSN-NH₂@C33s

Colloidal stability of MSN-NH₂@C33s was evaluated over a period of 7 days under the 6 different conditions summarised in Table 1. Z-Ave and PdI values are presented in Fig. 6 (as well as in Table S1 and Table S2), while size distributions by intensity are shown in Fig. S3 and pH values are listed in Table S3. MSN-NH₂@C33s remained stable in ultrapure water (A) and in the presence of a 0.15 M NaCl concentration (B).

To study the behaviour of the nanoparticles in a medium closer to physiological conditions, two low concentrations of PBS (0.1X and 0.05X, respectively, corresponding to conditions D and F) were used to control the pH near 7.4 along with NaCl to adjust the ionic strength to

values similar to physiological media. Although the ionic strength was slightly higher than 0.15 M (0.16 M for D and 0.17 M for F), MSN-NH₂@C33s remained stable for at least 3 days (however, a slight daily increase in both Z-Ave and PdI suggested gradual destabilisation). After 7 days, the nanoparticles were no longer stable, with Z-Ave and PdI values reaching 344 nm and 0.49 for condition D, and 302 nm and 0.48 for condition F (see size distributions in Fig. S3).

The influence of PBS on the stability of MSN-NH₂@C33s was also evaluated under conditions C and E. After 7 days, 0.05X PBS (condition E) maintained nanoparticle stability, whereas a higher concentration of 0.1X (condition C) caused a significant increase in Z-Ave. The colloidal destabilisation observed under conditions C and E was attributed not to ionic strength (0.02 M in condition C compared to 0.15 M in condition B), but rather to some species present in PBS. Therefore, it was concluded that the PBS composition, rather than ionic strength, was responsible for the colloidal destabilisation observed under conditions D and F.

### 3.4. Determination of the nature of the bond between MSN-NH₂ and copolymer C33s

To determine the nature of the bond between the amine groups of MSN-NH₂ and the carboxylic groups of C33s, and to rule out the possibility that the copolymer was merely adsorbed on the nanoparticle surface, a control sample was prepared by mixing MSN-NH₂ with C33s in the absence of coupling agents (MSN-NH₂/C33s). TGA results showed similar amounts of C33s in both samples (28.1 % for MSN-NH₂/C33s and 28.4 % for MSN-NH₂@C33s). However, zeta potential measurements as a function of pH revealed notable differences between the two samples (Fig. S2). The IEP of MSN-NH₂@C33s was lower (IEP = 4.2) than that of MSN-NH₂/C33s (IEP = 5.0; σ = 0.1, n = 2 independent samples). Additionally, MSN-NH₂@C33s exhibited significantly lower zeta potential values at low pH, indicating fewer available amine groups due to efficient covalent grafting of the copolymer. Colloidal stability tests further highlighted differences between the two samples. At an equivalent concentration of 0.7 mg MSN-NH₂ / mL, MSN-NH₂/C33s suspensions were unstable in water, mainly due to a shift towards the sample's IEP (Table S4). They also showed instability under condition B, influenced by both added salt and pH. These findings contrast with MSN-NH₂@C33s, which remained stable for at least 7 days (Fig. 6). Adjusting the pH to around 7.4 with PBS at 0.1X and 0.05 x concentrations (conditions C and E, respectively) resulted in stable suspensions for both samples due to the pH being far from the IEP. The salt concentration introduced by PBS was low enough to avoid nanoparticle destabilisation for at least 24 h. Unlike previous stability tests where the mother suspension was diluted in water before PBS addition, here the required PBS volume was first mixed with the required volume of water before adding the mother suspension. This procedure ensured that the pH remained away from the IEP during nanoparticle addition to prevent aggregation. Finally, MSN-NH₂/C33s suspensions, which were moderately destabilised shortly after preparation under conditions D and F, became unstable after 24 h, whereas MSN-NH₂@C33s showed slower

<caption>**Table 3**</caption>

Structural characteristics of MSN-OH, MSN-NH₂ and MSN-NH₂@C33s before and after calcination, determined by X-ray diffraction and nitrogen physisorption. S<sub>BET</sub>: specific surface area determined by the Brunauer-Emmett-Teller (BET) method, calculated in the range P/P₀ = 0.06–0.25; V<sub>m</sub>: total pore volume, including both mesopore and interparticle contributions; V<sub>m</sub>: mesopore volume obtained by the Barret-Joyner-Halenda (BJH) method and calculated from the desorption branch at relative pressure P/P₀ ≤ 0.75; D<sub>p</sub>: average pore diameter derived from the pore size distribution using the BJH method; d100: correlation distance corresponding to the (100) plane of a three-dimensional hexagonal lattice; a₀ = 2d₁₀₀/√3: lattice parameter; e: silica wall thickness, calculated as a₀ – Dₚ. Standard deviations are shown in parentheses. n.d.: value not determined.

<table><thead><tr><th></th><th>S<sub>BET</sub> (m²/g)</th><th>V<sub>t</sub> (cm³/g)</th><th>V<sub>m</sub> (cm³/g)</th><th>D<sub>p</sub> (nm)</th><th>d<sub>100</sub> (nm)</th><th>a<sub>0</sub> (nm)</th><th>e (nm)</th></tr></thead><tbody><tr><td>MSN-OH</td><td>1117</td><td>1.44</td><td>0.99</td><td>2.6</td><td>3.90</td><td>4.51</td><td>1.9</td></tr><tr><td>MSN-NH<sub>2</sub></td><td>849 (13)</td><td>1.09 (0.02)</td><td>0.72 (0.01)</td><td>2.6 (0.0)</td><td>4.06 (0.06)</td><td>4.69 (0.07)</td><td>2.1 (0.1)</td></tr><tr><td>MSN-NH<sub>2</sub>@C33s</td><td>543</td><td>0.67</td><td>0.27</td><td>n.d.</td><td>3.67</td><td>4.24</td><td>n.d.</td></tr><tr><td>Calcined MSN-NH<sub>2</sub>@C33s</td><td>1180</td><td>1.50</td><td>0.86</td><td>2.7</td><td>3.52</td><td>4.07</td><td>1.4</td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Z-Ave (A) and PdI (B) of MSN-NH₂@C33s determined by DLS as a function of time. The standard deviations of Z-Ave and PdI correspond to 2 samples, with 9 measurements performed on each.</figcaption></figure>

destabilisation under the same conditions (Fig. 6). All these results confirmed that the copolymer was covalently grafted to MSN-NH₂@C33s (via EDC and NHS coupling agents), while in MSN-NH₂/C33s it was merely adsorbed.

### 3.5. Loading of CDDP in MSN-NH₂@C33s

Many methods for drug loading into MSNs involve drying the nanocarriers, either before adding a solution of the molecule to fill the pores, as in dry impregnation (Charnay et al., 2004), or after the drug has been added, to promote diffusion into the pores through slow solvent evaporation (Deng et al., 2021). The drug-loaded nanoparticles must then be redispersed, which is often a critical issue.

In our study, dried MSN-NH₂@C33s could not be properly redispersed under the same ultrasonic conditions used to redisperse MSN-NH₂ due to interactions between the copolymer chains. Additionally, the presence of cisplatin further hindered the redispersion of the nanocarriers. In fact, redispersion tests of a powder composed of MSN-NH₂@C33s and cisplatin, “loaded” via different methods, resulted in a mixture that sedimented within a few minutes.

For this reason, we studied an alternative method, which consisted of stirring a mixture of a stable nanoparticle suspension with a cisplatin solution, followed by filtration to remove the unloaded drug. This approach allowed for good dispersion of the nanoparticles at the end of the process, as will be presented below. In MSN-NH₂@C33s, although the pores are occupied by the copolymer C33s, which could *a priori* decrease the loading capacity, the free carboxylate groups in the copolymer can chelate cisplatin by substituting its chloride ligands. This complexation not only contributes to the loading process but may also enable controlled release of the therapeutic agent (Nishiyama et al., 1999; Yokoyama et al., 1996). Thus, cisplatin loading into MSN-NH₂@C33s involves both diffusion into the pores and complexation by the copolymer.

DLS measurements showed that the addition of varying amounts of cisplatin to a stable suspension of MSN-NH₂@C33s led to aggregate formation. This was explained by a decrease in pH towards the IEP (from pH 6 to 5), which resulted in a lower absolute value of the zeta potential. This reduction in zeta potential is also due to the decreased availability of free carboxylate groups after cisplatin complexation. To confirm these hypotheses and optimise the conditions that ensure good dispersion of the nanocarriers during and after drug loading, nanoparticle stability was studied by varying the concentrations of both cisplatin and PBS. The results are reported in Table 4.

To prevent aggregation, the pH of the medium should be maintained away from the IEP, which necessitates the use of a buffer solution. As previously, PBS was selected at relatively low concentrations to avoid

destabilising the nanoparticles. A higher PBS concentration could induce a competitive effect between the chloride ions provided by the buffer and those from cisplatin, potentially affecting the complexation of cisplatin with the carboxylate groups of the copolymer mentioned earlier.

To elucidate the mechanisms behind nanoparticle destabilisation, both pH and zeta potential were measured. These tests aimed to determine the optimal loading conditions that would maximise the amount of cisplatin loaded while ensuring good dispersion of the nanoparticles after 72 h. The obtained results revealed general trends. First, colloidal stability decreased over time, as evidenced by a daily increase in Z-Ave and PdI, alongside a decrease in the absolute value of the zeta potential. Constant agitation of the nanoparticles contributed to their destabilisation (as observed for MSN-NH₂@C33s when stirred during the colloidal stability tests; results not shown). For a given cisplatin concentration, increasing the PBS concentration shifted the pH further from the IEP, resulting in an increase in the absolute value of the zeta potential and, consequently, enhanced nanoparticle stability. For instance, at cisplatin concentrations of 1 and 1.5 mg/mL, nanoparticles that showed poor dispersion after 24 h in 0.05X PBS exhibited improved stability up to 48 h in 0.1X PBS and up to 72 h in 0.2X PBS. Similarly, at 2 mg CDDP / mL, colloidal stability increased with higher PBS concentrations. Conversely, for a fixed PBS concentration, increasing the cisplatin concentration led to reduced colloidal stability. This effect is attributed to a decrease in the absolute value of the zeta potential, caused either by pH reduction or cisplatin complexation on the nanoparticle surface. For example, in 0.2X PBS, nanoparticles remained stable for up to 72 h at cisplatin concentrations of 1 and 1.5 mg/mL, but stability dropped to 24 h at 2 mg/mL and was entirely lost at 2.5 and 3 mg CDDP / mL, where proper dispersion could not be achieved.

Since a high PBS concentration may limit the complexation of cisplatin with copolymer's carboxylate groups, concentrations above 0.2X were not tested. The 0.2X PBS condition was sufficient to maintain nanoparticle dispersion for 3 days at the tested cisplatin concentrations. Based on these findings, a cisplatin concentration of 2 mg/mL was selected as optimal, being the highest concentration that preserved colloidal stability. While lower cisplatin concentrations might have enabled the use of a less concentrated PBS solution, they would also have reduced the drug loading efficiency.

Once the nanoparticle stability was maintained for 3 days, the removal of free (unbound) cisplatin became essential. It was equally important to preserve colloidal stability during this removal process so that the final suspension remained stable after filtration, without requiring ultrasonication for redispersion. Ultrasonication could potentially cause the premature release of the loaded cisplatin. Following the removal of free cisplatin by filtration, the suspension was
<caption>**Table 4**</caption>

Loading of cisplatin in MSN-NH₂@C33s under different conditions. For each sample, the Z-Ave and PdI were obtained from 9 measurements, while zeta potential (ζ) was determined from 3 measurements. When n > 1, both the average value and the standard deviation are reported. The most favourable conditions, those ensuring nanoparticle stability for at least 3 days, are highlighted in yellow. n.d.: value not determined.

<table><tbody><tr><td>[MSN-NH₂@C33s]<br>(mg/mL)</td><td colspan="7">0.9</td></tr><tr><td>[MSN-NH₂] (mg/mL)</td><td colspan="7">0.7</td></tr><tr><td>CDDP (mg)</td><td>8</td><td></td><td>12</td><td></td><td>16</td><td>20</td><td>24</td></tr><tr><td>[CDDP] (mg/mL)</td><td>1</td><td></td><td>1.5</td><td></td><td>2</td><td>2.5</td><td>3</td></tr><tr><td>[PBS] added</td><td>0.1X</td><td>0.2X</td><td>0.4X</td><td>0.1X</td><td>0.2X</td><td>0.4X</td><td>0.1X</td><td>0.2X</td><td>0.4X</td><td>0.4X</td></tr><tr><td>[PBS] final</td><td>0.05X</td><td>0.1X</td><td>0.2X</td><td>0.05X</td><td>0.1X</td><td>0.2X</td><td>0.05X</td><td>0.1X</td><td>0.2X</td><td>0.2X</td><td>0.2X</td></tr><tr><td colspan="11" style="text-align: center; font-weight: bold;">After 24 h</td></tr><tr><td>Z-Ave (nm)</td><td>381</td><td>147</td><td>152<br>σ=6<br>n=3</td><td>217</td><td>143</td><td>151<br>σ=7<br>n=12</td><td>3926</td><td>745</td><td>145</td><td>276</td><td>495</td></tr><tr><td>PdI</td><td>0.41</td><td>0.10</td><td>0.13<br>σ=0.03<br>n=3</td><td>0.25</td><td>0.10</td><td>0.12<br>σ=0.03<br>n=12</td><td>0.50</td><td>0.36</td><td>0.11</td><td>0.38</td><td>0.50</td></tr><tr><td>pH</td><td>6.5</td><td>6.5</td><td>6.7<br>σ=0.2<br>n=2</td><td>5.9</td><td>6.1</td><td>6.6<br>σ=0.1<br>n=12</td><td>5.7</td><td>6.1</td><td>6.3</td><td>6.4</td><td>6.1</td></tr><tr><td>ζ (mV)</td><td>-4.9</td><td>-8.6</td><td>-8.2<br>σ=0.3<br>n=2</td><td>-1.1</td><td>-5.6</td><td>-7.4<br>σ=0.2<br>n=12</td><td>1.0</td><td>-2.9</td><td>-5.5</td><td>-5.9</td><td>-4.3</td></tr><tr><td colspan="11" style="text-align: center; font-weight: bold;">After 48 h</td></tr><tr><td>Z-Ave (nm)</td><td>n.d.</td><td>164</td><td>162<br>σ=3<br>n=3</td><td>n.d</td><td>159</td><td>160<br>σ=8<br>n=12</td><td>n.d</td><td>n.d</td><td>602</td><td>n.d</td><td>n.d</td></tr><tr><td>PdI</td><td>n.d.</td><td>0.17</td><td>0.17<br>σ=0.02<br>n=3</td><td>n.d</td><td>0.15</td><td>0.14<br>σ=0.02<br>n=12</td><td>n.d</td><td>n.d</td><td>0.38</td><td>n.d</td><td>n.d</td></tr><tr><td>pH</td><td>n.d.</td><td>6.5</td><td>6.8<br>σ=0.0<br>n=2</td><td>n.d</td><td>6.3</td><td>6.6<br>σ=0.0<br>n=12</td><td>n.d</td><td>n.d</td><td>6.5</td><td>n.d</td><td>n.d</td></tr><tr><td>ζ (mV)</td><td>n.d.</td><td>-5.7</td><td>-7.4<br>σ=0.4<br>n=2</td><td>n.d</td><td>-3.7</td><td>-5.6<br>σ=0.2<br>n=12</td><td>n.d</td><td>n.d</td><td>-3.9</td><td>n.d</td><td>n.d</td></tr><tr><td colspan="11" style="text-align: center; font-weight: bold;">After 72 h</td></tr><tr><td>Z-Ave (nm)</td><td>n.d</td><td>214<br>σ=55<br>n=2</td><td>187<br>σ=5<br>n=2</td><td>n.d</td><td>264</td><td>175<br>σ=11<br>n=12</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td></tr><tr><td>PdI</td><td>n.d</td><td>0.26<br>σ=0.09<br>n=2</td><td>0.23<br>σ=0.01<br>n=2</td><td>n.d</td><td>0.33</td><td>0.19<br>σ=0.03<br>n=12</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td></tr><tr><td>pH</td><td>n.d</td><td>6.3<br>σ=0.1<br>n=2</td><td>6.7<br>σ=0.1<br>n=3</td><td>n.d</td><td>6.3</td><td>6.5<br>σ=0.2<br>n=3</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td></tr><tr><td>ζ (mV)</td><td>n.d</td><td>-3.1<br>σ=1.5<br>n=2</td><td>-6.6<br>σ=0.3<br>n=3</td><td>n.d</td><td>-3.0</td><td>-5.0<br>σ=0.5<br>n=2</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td><td>n.d</td></tr></tbody></table>

analysed by DLS. A Z-Ave of 187 nm (σ = 6 nm, n = 5) and a PdI of 0.23 (σ = 0.03, n = 5) were recorded, which are within acceptable limits. ICP-AES analysis determined a platinum concentration of 79.53 mg/L (σ = 6.71 mg/L, n = 3 independent samples), corresponding to a cisplatin weight percentage of 12.0 % in MSN-NH₂@C33s@CDDP (calculated with Expression S10).

### 3.6. CDDP release kinetics

The release kinetics of cisplatin from colloidal stable MSN-NH₂@C33s@CDDP were evaluated in DMEM High Glucose medium

supplemented with 10 % FBS (Fig. 7). A rapid burst release was observed within the first few hours of incubation, with 66.7 % of the total cisplatin released after 1 h and 83.2 % after 4 h. Following this initial phase, the release rate gradually decreased, and complete release of the drug was achieved after 48 h. These results indicated that the entire cisplatin cargo was successfully released from the MSN-NH₂@C33s@CDDP nanocarriers, allowing the drug to be effectively utilised, in contrast to other systems in which a substantial portion of the loaded drug remains confined within the nanoparticle.

While not optimal, a burst release of the drug during the initial hours is commonly observed in the literature. In this context, numerous studies
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Cisplatin release profile from MSN-NH₂@C33s@CDDP in DMEM High Glucose medium containing 10 % FBS. Error bars indicate the standard deviation for n = 2.</figcaption></figure>

have focused on the design of sophisticated delivery systems capable of triggering cargo release only in response to specific stimuli, which may be either internal or external. However, despite the success of these strategies in achieving more controlled drug release, the colloidal stability of the nanoparticles, which is a critical parameter for biomedical applications, is often overlooked. One of the main advantages of our system is that the colloidal stability of drug-loaded nanoparticles is preserved.

The drug release profiles were fitted to five kinetic models: zero-order, first-order, Higuchi, Hixson-Crowell and Korsmeyer-Peppas. The corresponding parameters for each model are summarised in Table S5. Among these models, the Korsmeyer-Peppas model provided the highest coefficient of determination (R² = 0.969), and an exponent value of n = 0.33. Since n < 0.45, this suggests that the cisplatin release follows Fickian diffusion. This finding is consistent with previous studies reporting similar diffusion-driven release kinetics from mesoporous silica nanoparticles (Shariatinia and Pourzadi, 2021; Peng et al., 2022).

### 3.7. Cytotoxicity evaluation

The cytotoxicity of cisplatin-loaded nanoparticles was assessed using two human cancer cell lines: SW480 (colorectal cancer) and A549 (lung adenocarcinoma), as platinum-based anticancer drugs are commonly used to treat these cancers. The results were compared with those obtained for empty nanovectors and free cisplatin.

Empty nanovectors (MSN-NH₂ and MSN-NH₂@C33s) exhibited negligible cytotoxic in both A549 and SW480 cell lines (Fig. 8, left), with cell viability reductions ranging from 0 to 3 % at 3.125 µg/mL, and 0 to 11 % at 200 µg/mL.

Cisplatin-loaded nanoparticles (MSN-NH₂@C33s@CDDP) exhibited a concentration-dependent cytotoxic response, reaching approximately 90 % cytotoxicity in A549 and 77 % in SW480 cancer cell lines at 200 µg/mL (Fig. 8, left). Considering that the cytotoxicity assay was performed on subconfluent cells, the IC₅₀ values of cisplatin-loaded MSNs were relatively low, with the A549 cell line showing higher sensitivity (IC₅₀ = 23 ± 2 µg MSN-NH₂ / mL) compared to the SW480 cell line (IC₅₀ = 40 ± 4 µg MSN-NH₂ / mL).

Furthermore, MSN-NH₂@C33s@CDDP displayed cytotoxicity comparable to that of free cisplatin in both cell lines. As observed with the

empty vectors, toxicity was consistently higher in A549 cells than in SW480 cells, with maximum cytotoxicity levels of approximately 90 % and 80 %, respectively. Accordingly, the IC₅₀ values for MSN-NH₂@C33s@CDDP were slightly lower in A549 (13 ± 1 µM CDDP) than in SW480 cells (23 ± 2 µM CDDP) (Table 5). Notably, the IC₅₀ of cisplatin-loaded nanoparticles were comparable to those of free cisplatin, indicating similar efficacy when cisplatin is delivered via the nanocarrier.

Although the cytotoxic effect of cisplatin-loaded nanoparticles was similar to that of free cisplatin *in vitro*, the potential advantage of this nanoplatform lies in its preferential accumulation in tumour tissues *in vivo* according to the Enhanced Permeation Retention (EPR) effect. Drug-loaded MSNs could deliver a higher quantity of the drug in tumour cells compared with the free drug, whose non-specific distribution in healthy tissues results in a lower concentration at the tumour site. These nanocarriers could enhance tumoral drug concentration, thereby increasing therapeutic efficacy, ultimately leading to a reduction in acquired drug resistance and improved patient survival.

### 4. Conclusions

In this study, MCM-41 mesoporous silica nanoparticles functionalised with amine groups (MSN-NH₂) were synthesised with high reproducibility, as confirmed by size, zeta potential, functionalisation rate and mesoporous structure across independent batches. An efficient redispersion method was developed for powdered MSN-NH₂, leading to a particle dispersion similar to that obtained before drying. The redispersed nanoparticles remained colloidally stable for at least 7 days; however, they aggregated under physiological conditions. To enhance their colloidal stability, a comb-like copolymer (C33s) was grafted onto the surface of MSN-NH₂ via amide bond formation between the surface amine groups and the carboxylate groups of the copolymer, using EDC and NHS as coupling agents. The copolymer was found to be grafted on the external surface and within the pores of the nanoparticles. The resulting MSN-NH₂@C33s nanoparticles exhibited colloidal stability for at least 7 days in ultrapure water and in the presence of 0.15 M NaCl, and for at least 3 days under near-physiological ionic strength and pH. The observed destabilisation was attributed to the PBS components. To investigate the nature of the interactions between the amine groups of the nanoparticles and the carboxylate groups of the copolymer, the same preparation conditions were tested in the absence of coupling agents to prevent the formation of covalent bonds. The zeta potential curve as a function of pH displayed a markedly different profile and, more importantly, the colloidal stability of these nanoparticles was significantly reduced, confirming that covalent grafting was responsible for copolymer attachment in MSN-NH₂@C33s, rather than simple adsorption. An aqueous-based cisplatin loading method was optimised by varying the concentrations of cisplatin and PBS concentrations to identify conditions that preserved nanoparticle colloidal stability throughout the process, achieving a drug loading of 12 wt%. Drug release kinetics revealed an initial burst release during the first hour, followed by sustained release governed by Fickian diffusion of cisplatin, with complete drug discharge observed after 48 h. Finally, the biological performance of the system was validated by showing the innocuousness of the unloaded nanoparticles and a cytotoxicity of the loaded nanoparticles similar to that of free cisplatin on human lung adenocarcinoma and colorectal cancer cell lines.

To the best of our knowledge, this work represents the first systematic investigation of the conditions required to develop stable colloidal suspensions of monodispersed mesoporous silica nanoparticles loaded with cisplatin, with high reproducibility. Furthermore, optimising cisplatin loading into MSNs was a key step, given the drug's poor solubility and the need to synthesise nanoobjects exhibiting at least comparable cytotoxic potential to the free drug. These findings provide a foundation of the rational design and synthesis of a new generation of CDDP@MCM-41-MSN nanocarriers with promising therapeutic
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Cytotoxicity of cisplatin-loaded nanoparticles (blue curves) in SW480 (A) and A549 (B) cell lines after 72 h of treatment. In the left panels, cytotoxicity is compared to that of empty nanovectors, MSN-NH₂ (green) and MSN-NH₂@C33s (red), and is expressed as a function of MSN-NH₂ concentration in mg/mL. In the right panels, cytotoxicity is compared to that of free cisplatin (grey) and is expressed according to cisplatin content in µM.</figcaption></figure>

<table><caption>**Table 5**
IC₅₀ values of MSN-NH₂@C33s@CDDP and free cisplatin, expressed in µM of cisplatin.</caption><thead><tr><th rowspan="2"></th><th colspan="2">IC<sub>50</sub> (µM CDDP)</th></tr><tr><th>SW480</th><th>A549</th></tr></thead><tbody><tr><th>MSN-NH<sub>2</sub>@C33s@CDDP</th><td>23 ± 2</td><td>13 ± 1</td></tr><tr><th>CDDP</th><td>24 ± 0</td><td>11 ± 4</td></tr></tbody></table>

potential.

### CRediT authorship contribution statement

**Eduardo Hernando Abad:** Writing – review & editing, Writing – original draft, Visualization, Investigation.
**Florence Bouyer:** Writing – review & editing, Visualization, Investigation.
**Igor Bezverkhyy:** Writing – review & editing, Visualization, Investigation.
**Rémi Chassagnon:** Writing – review & editing, Visualization, Investigation.

**Nicolas Geoffroy:** Writing – review & editing, Visualization, Investigation.
**Lucien Saviot:** Writing – review & editing, Visualization, Validation, Supervision, Formal analysis.
**Frédéric Bouyer:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

### Declaration of competing interest

The authors 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 has been supported by the EIPHI Graduate School (Contract No. ANR-17-EURE-0002) operated by the French National Research Agency (ANR) and the European Union through the PO
FEDER-FSE Bourgogne 2021/2027 program.

We thank DImaCell Platform (AgroSup Dijon, INRAe, Univ. Burgundy Franche-Comté, F-21000 Dijon, France) and the technological platform ARCEN Carnot (funded by the Région de Bourgogne Franche-Comté, the CNRS and the French RENATECH + network) for allowing access to the TEMs.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2025.126395.

## Data availability

Data will be made available on request.

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