# Development of anti-EGFR targeted magnetic nanoparticles for doxorubicin delivery into triple negative breast cancer cells

Chloé Lameli, Stéphanie David, Emilie Allard-Vannier, Gaëlle Dupeyrol, Fanny Boursin, Nicolas Aubrey, Igor Chourpa, Katel Hervé-Aubert

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## ► To cite this version:

Chloé Lameli, Stéphanie David, Emilie Allard-Vannier, Gaëlle Dupeyrol, Fanny Boursin, et al. Development of anti-EGFR targeted magnetic nanoparticles for doxorubicin delivery into triple negative breast cancer cells. European Journal of Pharmaceutics and Biopharmaceutics, 2026, 222, pp.115009. 10.1016/j.ejpb.2026.115009. hal-05512417

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# Development of anti-EGFR targeted magnetic nanoparticles for doxorubicin delivery into triple negative breast cancer cells

Chloé Lameli<sup>a, *, D</sup>, Stéphanie David<sup>a</sup>, Emilie Allard-Vannier<sup>a, D</sup>, Gaëlle Dupeyrol<sup>b, D</sup>, Fanny Boursin<sup>b, D</sup>, Nicolas Aubrey<sup>b, D</sup>, Igor Chourpa<sup>a, *, D</sup>, Katel Hervé-Aubert<sup>a</sup>

## ARTICLE INFO

Keywords:

Doxorubicin
Superparamagnetic iron oxide nanoparticles
(SPIONs)
Magnetic nanovectors
Humanized scFv
EGFR targeting
Triple negative breast cancer (TNBC)

## ABSTRACT

A new generation of pH-responsive magnetic theranostic nanovectors (NV) has been developed to deliver doxorubicin (DOX) to triple negative breast cancer (TNBC) cells which overexpress epidermal growth factor receptor (EGFR). DOX was loaded onto functionalized NV using a pH-sensitive DOX-Fe²⁺ complex (hereafter called NVscFv-DOX). NVscFv-DOX consist of superparamagnetic iron oxide nanoparticles (SPIONs), labelled with Dylight™ 680 fluorophore, and coated with a layer of covalently bound polyethylene-glycol (PEG) which is partially functionalized with anti-EGFR scFvs (average ratio is ≈12 scFvs per nanovector). The physico-chemical characteristics of the new nanovectors were suitable for IV injection: hydrodynamic diameter D<sub>H</sub> below 150 nm, polydispersity index below 0.3 and slightly negative surface charge (≈ −10 mV). Thanks to the functional grafted scFvs, the NVscFv-DOX were able to recognize the EGFR antigen efficiently. Using preformed DOX-Fe²⁺ complex which binds to the SPION surface in a pH-dependent manner, about 6.5% w/w (DOX/iron oxide) of the drug was loaded onto the NVscFv-DOX. The drug loading and release in its native form at acidic pH were characterized by surface-enhanced Raman scattering (SERS) spectroscopy. The dual fluorescent response of both Dylight™680 and that of DOX was confirmed, which is promising for theranostic use of the nanovectors. Finally, the *in vitro* toxicity of NVscFv-DOX on the EGFR-overexpressing TNBC cell line MDA-MB-468 was confirmed and compared to that of free DOX and NVscFv without DOX. Together, all these properties of the NVscFv-DOX are promising for their potential use as theranostic platform for TNBC treatment.

## 1. Introduction

Triple-negative breast cancer (TNBC) represents 15 to 20% of breast cancers [1–4] and is the most aggressive type of breast cancer with a high metastasis rate and complex phenotype leading to a poor prognosis [5–7]. This breast neoplasm subtype is characterized by the absence of progesterone receptor, estrogen receptor and human epidermal growth factor receptor type 2 (HER2) expression on cells [8,9]. As a consequence, hormonal therapy and some existing molecular targeted therapies, such as Trastuzumab (an antibody targeting HER2) are not first-hand choice treatments [5] and surgery, radiotherapy and chemotherapy remain pivotal steps in the treatment of TNBC [6]. Doxorubicin (DOX), an anthracycline drug inducing DNA damage and free radicals production is an antineoplastic agent used as standard chemotherapy for the treatment of TNBC [10–12]. However, chemoresistance and important side effects such as cardiotoxicity appear quickly leading to

overall low survival [6,13,14]. Using nanoparticles for drug delivery is an effective way to overcome chemoresistance [15,16]. It increases the intracellular concentration of the drug and its blood circulation half-time, while preventing its accumulation in healthy tissues and reducing the required dosage, thereby minimizing its side effects [17,18]. Indeed, the small size of nanoparticles, usually under 200 nm, allows them to accumulate preferentially in tumors by a passive mechanism known as the enhanced permeability and retention (EPR) effect [18,19] which results from an increased vessel permeability and insufficient lymphatic drainage in tumoral tissues [20]. Consequently, CAE-LYX®, a doxorubicin pegylated liposomal drug was developed to prevent dose-dependent drug cardiotoxicity and was authorized by the European Medicines Agency (EMA) in 1996 [21]. Another promising strategy to vectorize doxorubicin is by using superparamagnetic iron oxide nanoparticles (SPIONs). These magnetic nanovectors, especially those composed of maghemite and magnetite, have theranostic

<footnote><sup>a</sup> CBM UPR 4301, CNRS, Département Nanomédicaments et Nanosondes, Université de Tours 37200 Tours, France</footnote>

<footnote><sup>b</sup> ISP UMR 1282, INRAE, équipe BioMAP, Université de Tours 37200 Tours, France</footnote>

<footnote>* Corresponding authors.
E-mail addresses: chloe.lameli@univ-tours.fr (C. Lameli), igor.chourpa@univ-tours.fr (I. Chourpa).</footnote>
properties which make them particularly interesting in the nano-
medicine field [22]. Indeed, they can be used as therapeutic agents for
delivery of chemotherapy [23] and/or gene therapy (siRNA) [24–26].
Moreover, SPIONs can induce cell death via a local increase of tem-
perature under an alternative magnetic field – this phenomenon is
called magnetic hyperthermia [27–29]. In magnetic resonance imaging
(MRI), SPIONs shorten T2 and T2* leading to tissues hypodensity thus
improving negative contrasts [30–32]. Interestingly, SPIONs have no
remanence meaning that they present magnetic properties only in the
presence of a magnetic field [22,29,33]. Finally, the surface of SPIONs is
interesting to be labelled with fluorophores [34,35], to be functionalized
with polymers such as polyethylene glycol (PEG) in order to reduce
opsonization and provide colloidal stability [22] and/or with targeting
agents (i.e. biological ligands) to obtain an active targeting of tumor
cells [23,28].

Previously, our team worked on SPIONs for the delivery of doxorubicin into TNBC cells. Indeed, DOX has been loaded onto citrated SPIONs [36] and later onto PEGylated SPIONs [37] through the use of a pre-formed doxorubicin-Fe (II) complex (DOX-Fe²⁺ complex). This complex has interesting pH-sensitive properties meaning that it will be adsorbed directly on the surface of SPIONs at neutral pH but at acidic pH, such as the one that can be encounter in the tumoral environment, only DOX will be desorbed of the complex and diffused out of SPIONs [36,37].

An important part of the scientific community working on TNBC is now focused in finding new targets to treat TNBC. As a result, epidermal growth factor receptor (EGFR) appears to be a promising candidate [4,9,38]. Indeed, EGFR is a transcriptional regulator and a protein kinase that is overexpressed in around 70% of TNBC, especially in basal-like morphology TNBC [9,39–41]. Its expression is associated with metastasis, cancer chemoresistance, and tumor progression [9,42]. EGFR tyrosine kinase inhibitors, such as erlotinib [43], and monoclonal antibodies, such as cetuximab, have previously been tested as novel methods of treating TNBC but show poor results in clinical trials [44,45]. Recently, our team worked on the development of SPIONs conjugated with anti-EGFR scFvs (single-chain variable fragments) for siRNA delivery into triple negative breast cancer cells [35]. ScFvs are the fusion of only variable domains of heavy and light chains of a whole antibody resulting in a reduced size and molecular weight (around 27 kDa versus around 150 kDa for a whole antibody) [35,46]. Due to its small size, scFv has little impact on the colloidal properties of SPIONs [35,46], while providing them with high immunotargeting potential [47,48]. Moreover, the absence of Fc domain prevents complement activation or binding with Fc receptor [49]. In order to favor the paratope binding, we optimized the geometry of scFvs grafting on the nanovectors surface using a cysteine in a hexahistidine flag engineered in its C-Terminal position [46]. In vitro assay performed by flow cytometry demonstrated that SPIONs functionalized with anti-EGFR scFvs were better internalized into MDA-MB-231 TNBC cells compared to non-targeted SPIONs thus proving the efficacy of active targeting [35,50].

The aim of this study is to load doxorubicin via a pH-sensitive DOX-Fe²⁺ complex onto SPIONs functionalized with anti-EGFR scFvs in order to develop a new generation of fluorophore and DOX decorated EGFR-targeting theranostic nanovectors. Compared to other targeted SPIONs-based DOX carriers, this novel generation is distinguished by the original combination of theranostic functionalities via MRI and dual fluorescence imaging, scFv-mediated EGFR targeting, and pH-dependent DOX release.

First of all, the surface of SPIONs was enriched with covalently
bound near infrared fluorochrome Dylight™680 to follow nanoparticles
*in vitro* and *in vivo*, and was coated with poly(ethylene glycol)₅₀₀₀ (PEG)
chains to increase stealthiness. Then, the outer terminus of PEG chains
were partially functionalized with anti-EGFR scFvs to achieve active
targeting. Finally, these nanosystems were loaded with doxorubicin in a
form of pre-formed DOX-Fe²⁺ complex to become NV<sub>scFv</sub>-DOX.

We address here a full characterization of NVscFv-DOX concerning quantification of grafted scFvs and loaded DOX, affinity for EGFR of grafted scFvs, size, zeta potential, iron concentration and fluorescent properties. Surface Enhanced-Raman scattering (SERS) was used to characterize DOX loading and its release at acidic pH. Viability assays were also performed to assess DOX cytotoxic effect when delivered by NVscFv-DOX to MDA-MB-468, a TNBC cell line overexpressing EGFR.

## 2. Materials and methods

2.1. Materials

For nanoparticles synthesis, ferric chloride (FeCl₃), ferric nitrate nonahydrate (Fe(NO₃)₃·9H₂O) and ferrous ammonium sulfate (Fe(NH₄)₂(SO₄)₂·6H₂O) were purchased from Carlo Erba (Val-de-Reuil, France). Ferrous chloride tetrahydrate (FeCl₂·4H₂O), (3-Aminopropyl) trimethoxysilane (APS), anhydrous dimethyl sulfoxide (DMSO, 99,9%) and ethylenediaminetetraacetic acid (EDTA) disodium salt were purchased from Sigma-Aldrich (Saint-Louis, Missouri, United States). Dylight™ 680 NHS-Ester, sodium acetate and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), Premium Grade, Pierce™ were obtained from Fisher Scientific (Illkirch, France). N-Hydroxysuccinimide-Polyethylene glycol 5000-Maleimide (NHS-PEG5000-Maleimide, MW 5000 Da) was purchased from Rapp Polymere GmbH (Tübingen, Germany). Cellulose ester membrane for dialysis (molecular weight cutoff of 1000 kDa) was obtained from Biovalley (Marne-la-Vallée, France). Methanol and hydrochloric acid were purchased from Honeywell (Charlotte, North Carolina, United States). Acetone, ethanol absolute anhydrous, glacial acetic acid solution, nitric acid 65% solution and ammonia 30% solution were obtained from Carlo Erba (Val-de-Reuil, France). Doxorubicin hydrochloride was obtained from European Directorate for the Quality of Medicines & HealthCare – Council of Europe (Strasbourg, France). Finally, deionized water (18 mΩ cm) was used for experiments.

## 2.2. Synthesis of anti-EGFR scFv

The humanized scFv anti-EGFR fragment (U11K33) is the association through a (Gly₄Ser)₃ peptide link of the heavy (VH) and light (VL) variable domains based on the antibody Cetuximab (anti-EGFR) [35]. A peptide flag is also included and is composed of an hexahistidine tag, a GS spacer, and a terminal cysteine located at the C-terminus position. The chimeric antibody has been previously humanized by removing the N-glycosylation site in the VH [35]. Synthesis and cloning of the humanized scFv anti-EGFR fragment genes into pcDNA™3.4 have been realized by GeneArt (ThermoFisher, Waltham, Massachusetts, United States). ScFvs production was realized into ExpiCHO-S™ cells using the Max Titer protocol. First, the cells cultivated in ExpiCHO™ Expression Medium, must be diluted one day before transfection (D-1). ExpiCHO-S™ cells were then seeded at 3.0x10⁶ viable cells/mL and incubated overnight under agitation at 37°C with 8% of CO₂.

On the day of transfection (D0), the Max Titer transfection protocol was applied. After determining the cell concentration using CytoS-MART™ (Corning Inc., Corning, New York, United States), the cell culture was diluted to 6.0 x10⁶ viable cells/mL in fresh medium at 37°C. The cells were then transfected with 1000 ng/µL of EGFR plasmid, previously complexed with ExpiFectamine™ CHO Reagent and Opti-PRO™ medium. After 18 to 22 h of incubation post-transfection under the same cell culture conditions as on D-1, ExpiFectamine™ CHO Enhancer and ExpiCHO™ Feed were added and cells were placed under agitation at 32°C with 5% of CO₂. A second addition of ExpiCHO™ Feed medium was made five days post-transfection. Ten days after transfection, the cells were harvested and centrifuged at 10,000g for 10 min. The resulting supernatant containing the anti-EGFR scFvs was collected and stored at -20°C until further purification. On purification day, the supernatant was defrozed, re-centrifuged using the same parameters as
for the cells and passed through a 0.22 µm filter. The ÄKTApure™ liquid chromatography system, which was equipped with a HiTrap™ protein L column (Cytiva, reference number 17547815) was then used to purify and elute the anti-EGFR scFvs present in the supernatant, using a linear pH gradient in 0.1 M glycine buffer solution from pH 6 to pH 2. A desalting column was needed to replace the elution buffer with a buffered saline solution (pH 7.4 PBS, 2.7 mM KCl, 0.1 M NaCl, 2 mM KH₂PO₄, 8 mM Na₂HPO₄). The concentration of scFvs was determined by measuring the absorbance at 280 nm using a UV detector. The molecular mass and molar extinction coefficient values of the produced scFvs were calculated using the ProtParam tool (https://web.expasy.org/protparam/).

## 2.3. Nanoparticles synthesis

Synthesis of the different types of nanoparticles was realized using protocols previously published by our team. Concisely, Superparamagnetic Iron Oxide Nanoparticles (SPIONs) were obtained using a simplified Massart's protocol and were then silanized with APS [51]. Covalent grafting was performed in anhydrous DMSO to label SPIONs with Dylight™ 680 fluorochrome (Dylight™ 680 NHS-Ester) and to functionalize them with PEG (NHS-PEG5000-Maleimide) [35,52,53]. To eliminate ungrafted PEG and fluorochrome, dialysis (membrane molecular weight cutoff of 1000 kDa) was performed for 48 h and water as the acceptor volume was changed every hour. The previously synthesized anti-EGFR scFv fragments were reduced for two hours using TCEP and grafted on functionalized SPIONs by maleimide-cysteine Michael addition reaction (PBS 1X/EDTA 1 mM medium, pH 7.0) in order to obtain targeted magnetic nanovectors (NV<sub>scFv</sub>) [35,46]. Addition of an identical volume of PBS 1X instead of scFvs anti-EGFR on functionalized SPIONs allowed to synthesize control non-targeted magnetic nanovectors (NV), all other conditions were left unchanged. Size-exclusion chromatography using a Cytiva HiLoad™ 16/600 Superdex™ 200 pg column (GE Healthcare, Chicago, Illinois, United States) on an ÄKTA go™ protein purification system (Cytiva, Malboroug, Massachusets, United States) with a UV-Visible detector at 280 nm was performed to separate the components of the nanoparticles solution. Flow rate of 1 mL/min of PBS 1X as the mobile phase allowed injection of 5 mL of nanoparticles with a maximum iron content of 0.45 g/L. Purified NV<sub>scFv</sub> and NV were collected and reconcentrated using Sartorius Vivaspin® 20 centrifugal concentrators (polyethersulfone membrane, 30 kDa molecular weight cutoff, Fisher Scientific). Samples were stored at 4°C until further use.

## 2.4. Doxorubicin loading on NV and NV<sub>scFv</sub>: Formulation of NV-DOX and NV<sub>scFv</sub>-DOX

Doxorubicin loading on NV and NV<sub>scFv</sub> was performed using a DOX-Fe²⁺ complex previously developed by our team [36,37]. Briefly, aqueous solution of Mohr's salt was mixed with doxorubicin in TRIS buffer pH 7.6 using 1.5 M excess of Fe²⁺ over DOX to obtain DOX-Fe²⁺ complexes after 5 min of incubation.

The formation of complex DOX-Fe²⁺ in TRIS and PBS pH 7.6 buffers was evaluated using UV-1900i UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). The samples were analyzed in a quartz cell and the scanned spectral region was defined as 400–800 nm.

Suspensions of NV or NV<sub>scFv</sub> in TRIS buffer pH 7.6 were then incubated with DOX-Fe²⁺ complexes (10:1 w/w) for 30 min in the dark allowing the loading of DOX and the formation of NV-DOX and NV<sub>scFv</sub>-DOX respectively. PBS 10X was then added to the suspensions in order for them to be in PBS medium 1X. Unloaded DOX-Fe²⁺ complexes were eliminated by 5 to 10 min of centrifugation at 2000 rpm at 20°C (Sigma 3–16 KL, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) using Sartorius Vivaspin® 2 centrifugal concentrators (polyethersulfone membrane, 30 kDa molecular weight cutoff, Fisher Scientific). The retentate, containing NV-DOX or NV<sub>scFv</sub>-DOX was collected according to

manufacturer's instructions and its volume was quantified to later determine doxorubicin loading on nanovectors.

## 2.5. Characterization of nanovectors

a) Size, polydispersity index and zeta potential.

Hydrodynamic diameter (D<sub>H</sub>), polydispersity index (PDI) and zeta potential were measured using Malvern Nanosizer ZS (Malvern Panalytical, Malvern, United Kingdom). For NV and NV<sub>scFv</sub>, D<sub>H</sub> was determined in PBS 1X whereas it was in PBS 1X/ultrapure water (1: 1 v/v) for NV-DOX and NV<sub>scFv</sub>-DOX. All D<sub>H</sub> measurements were realized in triplicate at 25°C at an iron concentration of 50 mg/L. D<sub>H</sub> results were expressed in intensity and presented using mean values and standard deviations. Zeta potential was measured at 25°C after dilution of NV, NV<sub>scFv</sub>, NV-DOX and NV<sub>scFv</sub>-DOX at 50 mg of iron/L in ultrapure water.

b) Iron concentration determination of nanoparticles.

Iron concentration of NV, NV<sub>scFv</sub>, NV-DOX and NV<sub>scFv</sub>-DOX was determined by Atomic Absorption Spectroscopy (iCE 3000 spectrometer, Thermo Instruments, France) with measurements performed at 248.3 nm. Hydrochloric acid (6 N) was used to mineralize the samples overnight, then dilution was performed using hydrochloric acid (0.12 M) for NV and NV<sub>scFv</sub> or using ultrapure water for NV-DOX and NV<sub>scFv</sub>-DOX. Calibration curve obtained through standard solutions of iron concentrations (0.25, 0.5, 1, 2 and 5 mg/L) was used to determine the iron concentration of the different samples.

c) Qualitative characterization of Doxorubicin-loaded nanovectors.

Fluorescence of reversibly loaded DOX and covalently grafted Dylight™ 680 onto NV<sub>scFv</sub>-DOX has been studied at pH 4 using spectrofluorimetry. Two types of samples were analyzed: NV<sub>scFv</sub>-DOX diluted in sodium acetate/acetic acid in pH 4 buffer and free DOX solution mixed with chitosan solution (1: 3 v/v). Surface-enhanced Raman scattering (SERS) spectroscopy on silver colloids was used to characterize DOX loading onto NV<sub>scFv</sub>-DOX and NV-DOX. Silver colloids were previously synthesized using Lee and Meisel method [54]. Four types of samples were analyzed: free DOX solution, DOX-Fe²⁺ complex solution, purified suspensions of nanovectors (NV-DOX and NV<sub>scFv</sub>-DOX), and supernatant containing DOX released from the nanovectors (NV-DOX and NV<sub>scFv</sub>-DOX) at pH 4 (diluted in sodium acetate/acetic acid pH 4 buffer). Prior to use for SERS, the colloids were activated by 2-fold dilution in PBS pH 7.4 and mixed with 10% volume of samples (90 µL + 10 µL). Both fluorescence and SERS measurements were performed using LabRam confocal microspectrometer (Horiba Jobin-Yvon, Palaiseau, France) equipped with a 300 grooves/mm grating, an EmCCD detector and coupled to an Olympus BX-40 microscope. A non-confocal measurement mode was used (confocal hole open to 1000 mm). For measurements, a sample droplet of 40 µL was placed under a 10x microscope objective and laser beam was focused within the droplet. The experimental conditions were as follows:

**Fluorescence:** Excitation with either a 492 nm laser (6.65 µW at the sample) to induce the emission of DOX and Dylight™ 680 or with a 633 nm laser (6.35 µW at the sample) to induce Dylight™ 680 emission. Each spectrum was obtained as an average of 9 scans (0.5 s per scan) to insure an optimal signal-to-noise ratio.

**SERS:** Excitation with a 691 nm laser (20.5 mW at the sample). Each spectrum was obtained as an average of 36 scans (0.5–1 s per scan) to insure an optimal signal-to-noise ratio.

For each sample, three independent measurements were realized to obtain average spectra presented in the figures. All data were recorded and treated using LABSPEC (Horiba Scientific) software.

d) Doxorubicin loading quantification.

Previously obtained retentate containing NV-DOX or NV<sub>scFv</sub>-DOX was transferred from the centrifugal concentrator to an Eppendorf tube and 1 mL of pH 4 acetic acid/sodium acetate buffer was added. The sample was then put to sonicate in an ultrasonic bath (2800 Branson, Branson Ultrasons, Rungis, France) for 1 h at room temperature to ensure total liberation of DOX from the nanovectors following phenol
protonation leading to dissociation of DOX-Fe²⁺ complexes [36,37]. Centrifugation at 20,000 rpm for 1 h at 20°C was then performed (Sigma 3–30 K, Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany) to separate released DOX from NV-DOX or NV<sub>scFv</sub>-DOX. Intrinsic fluorescence properties of DOX were used to determine its concentration in the supernatant using FS5 spectrofluorometer (Edinburgh Instruments Ltd, Livingston, United Kingdom) and quantify doxorubicin-loading. A calibration curve was previously obtained through standard solutions of DOX in sodium acetate/acetic acid pH 4 buffer (0.5, 1, 2, 3, 4 µM). Drug loading is expressed as a ratio of doxorubicin loaded onto nanovectors mass over the mass of the iron core constituting the nanovectors.

e) Quantification of grafted anti-EGFR scFvs on NV<sub>scFv</sub>.

The quantity of anti-EGFR scFvs grafted on NV<sub>scFv</sub> was determined by a modified Bradford assay using the Coomassie Plus™ Protein Assay Reagent (Thermo Scientific, Rockford, Illinois, United States). To realize that experience, NV<sub>scFv</sub> and NV as a negative control, were incubated in a 96 well-plate at 100 mg of iron/L in PBS 1X (100 µL/well). PBS 1X or Coomassie reagent were then added to samples (100 µL/well) and after 10 min of incubation, absorbance was measured at 595 nm using an absorbance microplate reader (BioTek Instruments, Inc., United States). A calibration curve (scFv concentrations of 0, 2.5, 5, 10, 15, 20, 25 µg/mL) was used to determine the concentration of grafted scFvs (µg/mL) on NV<sub>scFv</sub>. Using scFv molar mass and the following formula, number of scFvs per NV<sub>scFv</sub> has been able to be calculated:

$$ \text{Number of scFvs per NV}_{\text{scFv}} = C_{\text{scFv}} \cdot \frac{\pi d_p^3}{6} \cdot d \cdot N_A $$

With C<sub>scFv</sub>: concentration of grafted scFvs (mol/g), d<sub>p</sub>: diameter of the core of the magnetic nanoparticle previously determined by our team by TEM at 10 x 10<sup>-9</sup> m, d: mass density of SPIONs determined at 5.2 x 10<sup>6</sup> g/m<sup>3</sup>, N<sub>A</sub>: number of Avogadro fixed at 6 × 10<sup>23</sup> mol<sup>-1</sup>.

f) Functionality of grafted anti-EGFR scFvs on NV<sub>scFv</sub> and NV<sub>scFv</sub>-DOX.

Indirect enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the functionality of anti-EGFR scFvs grafted on NV<sub>scFv</sub> and NV<sub>scFv</sub>-DOX. First, recombinant Human EGFR Protein (Sino Biological Europe GmbH, Eschborn, Germany) was coated in a high-binding 96 well-plate (Greiner Bio-One, Kremsmünster, Austria) at a concentration of 2 µg/mL (100 µL/well) in PBS 1X and incubated all night at 4°C. Saturation of wells (300 µL/well) with BSA-PBS 1X (3% m/v) was then performed for 1 h at 37°C. Subsequently, NV, NV<sub>scFv</sub>, NV-DOX, NV<sub>scFv</sub>-DOX, from concentrations of 0 mg of iron/L to 100 mg of iron/L were incubated in wells (100 µL/well) for 1 h at 37°C and PBS 1X was used as negative control. Afterwards, wells were washed 4 times (300 µL/well/washing) with Tween 20-PBS 1X (0.05% m/v) and incubated with 100 µL/well of scFv-binding Pierce™ Recombinant Protein L Peroxidase Conjugated (Fisher Scientific) diluted at 1.25 µg/mL in PBS 1X for 1 h at 37°C. After 4 more washings (300 µL/well/washing) with Tween 20-PBS 1X (0.05%; m/v), 100 µL/well of 1-Step™ Ultra TMB-ELISA (Thermo Fisher Scientific, Waltham, Massachusetts, United States) was added allowing enzymatic reaction between 3,3',5,5'-tetramethylbenzidine and protein-L peroxidase in presence of functional anti-EGFR scFvs on NV<sub>scFv</sub> and NV<sub>scFv</sub>-DOX. Reaction was then stopped by adding sulfuric acid 1 M (50 µL/well) and absorbance was measured at 450 nm using an absorbance microplate reader (BioTek Instruments, Inc., United States).

## 2.6. *In vitro* evaluation

a) Cell culture.

Overexpressing EGFR triple-negative breast cancer cell line MDA-MB-468 (American Type Culture Collection, Manassas, Virginia, United States) was cultured in Complete Cell Culture Medium (CCM) at 37°C in humid atmosphere containing 5% CO₂. CCM was composed of Minimum Essential Medium (MEM) with GlutaMAX™ (Gibco™,

Paisley, United Kingdom) supplemented with 10% of fetal bovine serum (Eurobio Scientific, Les Ulis, France), 1% of penicillin-streptomycin 10 000 U/mL (Gibco™), 1% of sodium pyruvate 100 mM (Gibco™), 1% of non-essential amino-acids (Gibco™) 100X, 1% of vitamins solution 100X for MEM and 0.008% of 50 mg/mL gentamycin (Gibco™). CCM was changed every 2–3 days and harvesting was realized using trypsin-ethylenediaminetetraacetic acid (EDTA) (0.05%) 1X solution (Gibco™) when MDA-MB-468 cells reached 80% of confluency.

b) Cell viability assessment.

RealTime-Glo™ MT Cell Viability Assay (Promega, Madison, Wisconsin, United States) was used to evaluate cell viability. In order to perform this test, MDA-MB-468 cells were seeded onto a white opaque-walled 96 well-plate with a flat bottom (Fisher Scientific) at 2.5x10³ cells/well (100 µL/well) for 36 h at 37°C/5% CO₂. Suspensions of NV, NV<sub>scFv</sub>, NV-DOX and NV<sub>scFv</sub>-DOX were prepared in CCM at 2X concentrations (iron concentration from ≈0.7 mg/L to ≈39 mg/L corresponding to doxorubicin concentration for NV<sub>scFv</sub>-DOX from 0.1 µM to 5 µM), free DOX solution was prepared in CCM at 2X concentrations (from 0.1 µM to 5 µM) and RealTime-Glo™ reagents (MT Cell Viability Substrate and NanoLuc® enzyme) were also prepared in CCM at 2X concentrations. Cells were treated with 50 µL/well of NV suspension, NV<sub>scFv</sub> suspension, NV-DOX suspension, NV<sub>scFv</sub>-DOX suspension, free DOX solution, CCM only (negative control) or 40 mM of hydrogen peroxide (positive control) and then, 50 µL of RealTime-Glo™ reagents were added per well. Consequently, the final concentration of each reagent and tested samples is 1X in each well thus studying DOX effect on cell viability from concentrations of 0.05 µM to 2.5 µM. Cell viability was monitored in real time for 72 h by measuring luminescence (gain 200) using an absorbance microplate reader (BioTek Instruments, Inc., Winooski, Vermont, United States). All the measurements were performed at least in duplicate and the assay was performed three times. The number of viables cells was correlated with the luminescence value and determined using the following formula:

$$ \text{Viable cells (\%)} = \frac{\text{Lum}_{\text{sample}}}{\text{Lum}_{\text{negative control}}} \times 100 $$

With Lum<sub>sample</sub>: luminescence value of the sample, Lum<sub>negative control</sub>: luminescence value of the negative control

The half maximal inhibitory concentration (IC₅₀) was determined for each treatment as the concentration inhibiting the cell viability by 50%.

c) Statistics.

Data were presented as mean ± standard deviation and were plotted using GraphPad Prism 8 software. One-way ANOVA tests were performed to compare cell viability and IC₅₀ between evaluated samples. The difference was considered significant when p-value (p) < 0.05.

# 3. Results and discussion

## 3.1. Production of nanovectors

1) Synthesis of NV and NV<sub>scFv</sub>

Injectable EGFR-targeting magnetic nanovectors using anti-EGFR scFvs (NV<sub>scFv</sub>) were synthesized using optimized protocols of our team [35] (Fig. 1-A). First, superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized using Massart's method and then silanized to provide NH₂ reactive groups for functionalization. Amide reactions enabled functionalization of SPIONs with polyethylene glycol chains (NHS-PEG₅₀₀₀-Maleimide) to increase stealthiness and with Dylight™680, a near-infrared fluorochrome, to follow nanoparticles *in vitro* and *in vivo*. Anti-EGFR scFvs were then grafted onto nanovectors (NV) by cysteine-maleimide Michael Addition to obtain NV<sub>scFv</sub> according to a protocol reported previously [35].

The D<sub>H</sub> of NV and NV<sub>scFv</sub> was close to 100 nm, the PDI was below 0.3 testifying that the population is monodisperse and the zeta potential was close to neutrality (Table 1). Thus, physico-chemical characteristics of
<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

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

<caption>**Fig. 1.** Schematic representation of the synthesis of the nanoparticles (A), their loading with DOX (B) and the release of loaded DOX (C). The control nanovectors (without scFvs) were generated using the same protocols. This figure was created with some elements from Biorender.com.</caption>

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

Physico-chemical characteristics of the nanosystems, before (NV and NVscFv) and after their loading with DOX (NV-DOX and NVscFv-DOX). The data is expressed as average ± standard deviation (n = 3 for scFvs/NV, D_H, PDI, zeta potential and n = 4 for iron concentration and % DOX loaded).

<table><thead><tr><th></th><th>scFvs / NV</th><th>D<sub>H</sub> (nm)</th><th>PDI</th><th>Zeta potential (mV)</th><th>Iron concentration (mg/L)</th><th>% DOX loaded (w/w DOX/iron oxide * 100%)</th></tr></thead><tbody><tr><td>NV</td><td>0</td><td>103 ± 5</td><td>0.187 ± 0.01</td><td>-8.5 ± 1.1</td><td>826 ± 154</td><td></td></tr><tr><td>NVscFv</td><td>12 ± 2</td><td>101 ± 6</td><td>0.183 ± 0.03</td><td>-4.3 ± 4.4</td><td>1025 ± 176</td><td></td></tr><tr><td>NV-DOX</td><td>0</td><td>126 ± 3</td><td>0.198 ± 0.04</td><td>-8.3 ± 1.4</td><td>169 ± 20</td><td>6.38 ± 0.84</td></tr><tr><td>NVscFv-DOX</td><td>12 ± 2</td><td>113 ± 4</td><td>0.175 ± 0.01</td><td>-6.9 ± 0.2</td><td>157 ± 28</td><td>6.50 ± 1.08</td></tr></tbody></table>

D_H: Hydrodynamic diameter; PDI: Polydispersity index.

NVscFv were suitable for intravenous injection.

2) Formulation of NV-DOX and NVscFv-DOX.

Previous studies, conducted in our team, used pH-sensitive DOX-Fe²⁺ complex strategy to load DOX onto citrated SPIONs [36] and then onto PEGylated SPIONs [37]. At pH 7.6, the chelated iron of the pre-formed DOX-Fe²⁺ complex interacts with the OH groups on the SPIONs surface allowing the adsorption of the DOX-Fe²⁺ complex onto SPIONs. Its pH-sensitive properties strengthened our choice to use this strategy to load DOX onto the EGFR-targeting magnetic nanovectors (NVscFv), thus obtaining NVscFv-DOX and control NV-DOX. A schematic representation of the nanovectors formulation can be found in Fig. 1-B.

Firstly, DOX-Fe²⁺ complexes were obtained by mixing DOX in pH 7.6 TRIS buffer with an aqueous solution of Mohr's salt providing ferrous ions (Fe²⁺) at a molar ratio of 1.5 Fe²⁺/DOX. At this particular pH and ratio, it has been proven that the Fe²⁺ provided by Mohr's salt is chelated on the C11 phenolic group of the DOX by replacing its hydrogen [36]. The formation of the DOX-Fe²⁺ complex is an easy, quick and reproducible step. Its formation can be easily tracked through fluorescence properties, as free DOX is fluorescent, whereas the DOX-Fe²⁺ complex is not [36]. In addition, the formation of the complex is easy to control by UV-Visible spectrometry.

Results of DOX-Fe²⁺ complex formation in TRIS and PBS buffers at pH 7.6, studied by UV-Visible spectrometry, are shown in Fig. 2.

The UV-Visible spectra of the controls (light red and dark red spectra), i.e. of free DOX in PBS buffer at pH 7.6 and free DOX in TRIS

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** UV-Visible spectra of free DOX 50 µM and DOX-iron complex in TRIS and PBS buffers at pH 7.6.</figcaption></figure>

buffer at pH 7.6 are superimposed and therefore similar. The spectral changes observed upon formation of the DOX-Fe²⁺ complex (increase of the red-shifted shoulders) show that the TRIS buffer (green curve) is more favorable than PBS buffer (blue curve) to the iron chelation by DOX. Therefore, TRIS buffer at pH 7.6 will be used to pre-form DOX-Fe²⁺ complexes and to load them onto NVscFv.

The pre-formed DOX-Fe²⁺ complexes were then incubated with suspensions of NV or NVscFv prepared in pH 7.6 TRIS buffer, to obtain NV-DOX or NVscFv-DOX (Fig. 1-B). The concentration of SPIONs was adjusted to a ratio of 0.1 w/w DOX/iron of SPIONs made of iron oxides
[37]. To purify unloaded DOX from the NV-DOX or NV<sub>scFv</sub>-DOX, a centrifugation step was performed using centrifugal concentrators. It allowed to obtain a retentate with NV-DOX or NV<sub>scFv</sub>-DOX and a permeate with unloaded DOX/iron complexes (Fig. 1-B).

It is worthy to note that upon NV<sub>scFv</sub>-DOX purification, we observed that a significant fraction of the nanoparticles was adsorbed onto the membrane (Ultracel regenerated cellulose) of the centrifugal concentrators, thus inducing a bias for the DOX-loading quantification. To overcome this problem, we have tested various protocols including changing the nature of the membrane of the centrifugal concentrators, the conductivity of the sample and several alternative purification techniques such as tangential flow filtration, magnetic columns sorting and desalination. The best results have been obtained using centrifugal concentrators with polyethersulfone membrane and by adding PBS 10X to the sample before the centrifugation step to increase its conductivity.

## 3.2. Physico-chemical characterization of the nanovectors

Several batches of NV<sub>scFv</sub>-DOX were generated and characterized. Control batches without scFvs (NV-DOX) were also formulated and correspond to passive magnetic nanovectors. Physico-chemical characteristics of these nanovectors are summed up in the Table 1.

1) Colloidal properties of the nanovectors.

Around $12 \pm 2$ scFvs anti-EGFR were grafted per NV in average for NV<sub>scFv</sub>-DOX (Table 1) according to quantification by a modified Bradford assay. This method is performed directly onto NV<sub>scFv</sub> and is based on the absorption shift of Coomassie G-250 dye from 465 nm to 590 nm when it binds to proteins thus allowing quantification of scFvs grafted onto NV<sub>scFv</sub>. Nanoparticles sizes were around $113 \pm 4$ nm for NV<sub>scFv</sub>-DOX whereas the sizes were close to $126 \pm 3$ nm for NV-DOX (Table 1). Loading DOX onto NV and NV<sub>scFv</sub> results in an increase in nanoparticles sizes: $\approx+23$ nm for NV-DOX compared to NV and $\approx+12$ nm for NV<sub>scFv</sub>-DOX compared to NV<sub>scFv</sub> (Table 1). The smaller size increase observed for NV<sub>scFv</sub>-DOX compared to NV-DOX may reflect a reorganization of the PEG layer upon ligand conjugation. Although this specific effect with ligand attachment has not been directly reported in the literature, conformational changes of surface-grafted PEG chains are well documented and can alter the hydrated shell thickness and hydrodynamic diameter of the nanoparticles [55–57]. While we do not provide direct experimental evidence for this effect in the present manuscript, the observed size decrease after ligand attachment is consistent with a PEG layer compaction or reorganization. After DOX loading, the polydispersity index of both NV-DOX and NV<sub>scFv</sub>-DOX stays below 0.2 (Table 1), meaning that these nanovectors populations can be considered as monodispersed. The surface charge of both nanovectors population is slightly negative ($-8.3 \pm 1.4$ mV for NV-DOX and $-6.9 \pm 0.2$ mV for NV<sub>scFv</sub>-DOX) but close to neutrality (Table 1) meaning that grafted scFvs onto DOX-loaded nanoparticles don't affect their surface charge. Colloidal stability of the nanovectors is achieved through steric stabilization via PEGylation, which means that the zeta potential value is less predictive of colloidal stability than it is for electrostatically stabilized nanoparticles [27,57]. Therefore, the physicochemical characteristics of NV<sub>scFv</sub>-DOX batches indicate that scFv grafting and DOX loading didn't prevent NV<sub>scFv</sub>-DOX from remaining within the ranges typically reported for PEGylated nanocarriers administered intravenously [58].

2) Optical properties of the nanovectors.

Fluorescence of reversibly loaded DOX and covalently grafted Dylight™ 680 onto NV<sub>scFv</sub>-DOX has been studied at pH 4 using spectrofluorimetry.

As shown in Fig. 3, the dual-mode fluorescence of the nanovectors has been confirmed, by using two excitation wavelengths. With 633 nm excitation, only Dylight™ 680 was fluorescent, with the emission maximum at 703 nm, since DOX has no absorbance at this spectral range. In contrast, Dylight™ 680 absorbs also at 492 nm and can emit its fluorescence band at 703 nm, at both pH 4.0 and 7.4. With 492 nm

<figure><img src="image_7.png" /><figcaption>Fig. 3. Fluorescence spectra of the nanovectors NV<sub>scFv</sub>-DOX (492 nm excitation wavelength or 633 nm excitation wavelength) at pH 4 and free DOX at pH 4 (492 nm excitation wavelength) showing dual-mode emission of NV<sub>scFv</sub>-DOX at pH 4 originating from covalently bound Dylight™ 680 and reversibly loaded DOX.</figcaption></figure>

excitation, free DOX emits its fluorescence with a maximum at 596 nm, while the drug-iron complex is non-fluorescent. The DOX-Fe<sup>2+</sup> complex is favored at pH 7.4 and is dissociated at pH 4, as the phenol group of the drug is re-protonated [36]. As a result, at pH 4, with 492 nm excitation, both DOX dissociated from Fe<sup>2+</sup> and Dylight™ 680 covalently bound to the nanovectors were producing their emission, with respective positions of their maxima at 596 nm and 703 nm. Such a dual-mode fluorescence is of potential use for co-detection of both nanovectors and of their payload, especially in acidic environments, thus allowing to observe the drug release from the nanocarriers in targeted cells and tissues. This advantage will be used in the future biological evaluation which will be a subject of an independent study.

3) Qualitative and quantitative assessment of loaded DOX.

Further qualitative characterization of the DOX loading onto NV-DOX and NV<sub>scFv</sub>-DOX was performed by Surface-Enhanced Raman Scattering (SERS) spectroscopy. This sensitive, semi-quantitative and non-destructive technique allows to increase the Raman structure-specific signature of DOX molecules (as free DOX and/or as the DOX-Fe<sup>2+</sup> complex). SERS assays were first performed on the solutions of free DOX, then those of DOX-Fe<sup>2+</sup> complex in TRIS buffer at pH 7.6, in order to have the respective reference spectra.

As shown in Fig. 4, the SERS spectra of free DOX at pH 7.6 present very strong doublet at 465 and 443 cm<sup>-1</sup> assigned to deformational modes d(C=O) and d(C-O) respectively [59]. The weak band at 1082 cm<sup>-1</sup> was assigned to skeletal deformations. The additional bands between 1210 and 1460 cm<sup>-1</sup> were due to ring stretching coupled with other vibration modes. The two stronger bands at 1244 and 1210 cm<sup>-1</sup> were from ring stretching coupled with deformational modes of O-H. However, the band at 1296 cm<sup>-1</sup> and those at 1434–1456 cm<sup>-1</sup> were coupled with C-O stretching and with CCOCH bending. As already described by our group [59], DOX-Fe<sup>2+</sup> complex formation induces several changes compared to the SERS spectra of the free drug: the shift of the strong band from 1224 to 1244 cm<sup>-1</sup> and the increase of the intensity ratio for the bands at 1434/1413, 1244/1224, 1210/1224 and 465/443 cm<sup>-1</sup>. Consequently, these spectral differences will ease the SERS recognition of free and bound forms of doxorubicin.

In order to assess if there was residual drug, free and/or in complex with Fe<sup>2+</sup>, the aqueous suspensions of the DOX-loaded nanovectors (NV-DOX and NV<sub>scFv</sub>-DOX) purified by centrifugation were analyzed by SERS, after being incubated with pre-aggregated silver nanoparticles. Due to the short-range nature of SERS effect, only DOX or DOX-Fe<sup>2+</sup> complexes located at a distance below 2 nm from the silver
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** SERS spectra of various samples: free DOX solution (pH 7.6); DOX-Fe²⁺ complex solution (pH 7.6); residual drug in suspensions of purified NV-DOX or NV<sub>scFv</sub>-DOX (pH 7.6); released drug in supernatants of NV-DOX and NV<sub>scFv</sub>-DOX (pH 4).</figcaption></figure>

nanoparticles surface can give SERS signal. Therefore, almost no DOX signal can be detected from the drug burried in the PEG shell of the nanovectors that is supposed to be of about 1–2 nm thick. The SERS spectra (Fig. 4) from the nanovectors suspensions (both the NV-DOX and the NV<sub>scFv</sub>-DOX batches) were of very low intensity, thus indicating that there was only minor residual/released drug in the outer layers of PEG shell and/or in the bulk medium surrounding the nanovectors, thus remaining accessible to the silver nanoparticles surface, which gives the SERS effect. The shape of those spectra was closer to that of the free DOX than to that of the DOX-iron complex.

As we reported before [36,37], at pH 4, re-protonation of phenolic groups of DOX leads to dissociation of DOX-Fe²⁺ complex and favors the free drug release from the nanovectors. Thus, we assessed the free DOX released from NV<sub>scFv</sub>-DOX and NV-DOX transferred to pH 4 (diluted in acetic acid/sodium acetate buffer pH 4) and sonicated. After centrifugation step, the supernatants were incubated with pre-aggregated silver nanoparticles. As expected, for all NV-DOX and NV<sub>scFv</sub>-DOX supernatants at pH 4, the SERS spectra (Fig. 4) correspond to free DOX released from the nanovectors. The observation of the strong SERS signal of free DOX in those supernatants is concomitant with the pH-dependent DOX desorption we previously described [37,59].

To quantify doxorubicin-loading and determine doxorubicin loading rate, several steps were performed as reported in materials and methods. First, pH 4 acetic acid/sodium acetate buffer was added to the retentate containing NV-DOX or NV<sub>scFv</sub>-DOX (Fig. 1-C). At an acidic pH, the C11 phenol and the amino group of the sugar moiety of the drug are reproxtonated, thus the DOX-iron complex dissociates leading to desorption of DOX and its diffusion out of the SPIONs [36,37]. A sonication step was carried out followed by a centrifugation step to ensure, respectively, a total desorption of loaded-DOX and separate released DOX from NV-DOX or NV<sub>scFv</sub>-DOX (Fig. 1-C). The supernatant in pH 4 acetic acid/sodium acetate buffer with DOX released from NV-DOX or NV<sub>scFv</sub>-DOX was then used to quantify DOX-loading by spectrofluorimetry. Iron concentration was determined to calculate DOX loading rate (Table 1). The DOX loading rate is 6.38 ± 0.84% for NV-DOX and 6.50 ± 1.08 for NV<sub>scFv</sub>-DOX (Table 1). Therefore, the two are rather similar. Consequently, the presence of grafted scFvs on NV<sub>scFv</sub>-DOX does not impede the diffusion of DOX-Fe²⁺ complexes within the PEG shell to reach the surface of SPIONs surface. The reported DOX loading rate (≈ 6.5% w/w (DOX/iron oxide)) is lower than that of some other drug delivery

systems in which DOX has been encapsulated [60]. When compared nanocarriers with drugs adsorbed onto the surface of a core, such as with DOX electrostatically bound to SPIONs, our results are close to those reported in the literature [61]. Considering similar physico-chemical characteristics and DOX-loading for NV-DOX and NV<sub>scFv</sub>-DOX, these nanoparticles would be later used for *in vitro* and *in vivo* testing.

### 3.3. Biological properties of the nanovectors

1) Functionality of the scFvs assessed by indirect ELISA.

Following DOX loading, the functionality of the scFvs grafted onto NV<sub>scFv</sub>-DOX was qualitatively verified using an indirect enzyme-linked immunosorbent assay (ELISA) (Fig. 5). Increasing concentrations of SPIONs (NV, NV<sub>scFv</sub>, NV-DOX, NV<sub>scFv</sub>-DOX) were incubated in presence of EGFR antigen. Detection of scFvs was realized using Protein L that binds to kappa light chain variable domains while preserving antigen-binding site.

The results obtained after indirect ELISA assay (Fig. 5) show that for the batches without scFvs (NV and NV-DOX), the optical density at 450 nm does not increase with the iron concentration of SPIONs. However, for the batches with scFvs, whether there has been DOX loaded (NV<sub>scFv</sub>-DOX) or not (NV<sub>scFv</sub>), the optical density at 450 nm increases with the iron concentration of SPIONs. Similar profiles can be observed for NV<sub>scFv</sub> and NV<sub>scFv</sub>-DOX, as the slight shift observed is less than one log₁₀ unit. This increase in optical density at 450 nm indicates the recognition of EGFR antigen by NV<sub>scFv</sub> and NV<sub>scFv</sub>-DOX and demonstrates

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Results of indirect ELISA assay for NV, NV-DOX, NV<sub>scFv</sub> and NV<sub>scFv</sub>-DOX batches.</figcaption></figure>
regioselectivity during the grafting of the scFvs onto nanovectors. As a result, the presence of DOX-Fe²⁺ complexes on the surface of NV<sub>scFv</sub> (NV<sub>scFv</sub>-DOX) does not impair the functionality of the grafted antibody fragments.

2) Impact of NV<sub>scFv</sub>-DOX on TNBC cells viability.

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** *In vitro* cell viability profiles of MDA-MB-468 after 24 h of treatment (A), 48 h (B) or 72 h (C) with NV, NV<sub>scFv</sub>, NV-DOX, NV<sub>scFv</sub>-DOX or free DOX (DOX in solution). The data is expressed as average ± standard deviation (n = 3). One-way ANOVA test was used to perform statistical analysis. No significant difference (ns, p > 0.05) for NV-DOX vs free DOX, NV<sub>scFv</sub>-DOX vs free DOX, and NV-DOX vs NV<sub>scFv</sub>-DOX. Significant difference (****, p < 0.0001) for NV-DOX vs NV, NV<sub>scFv</sub>-DOX vs NV<sub>scFv</sub>, free DOX vs NV, and free DOX vs NV<sub>scFv</sub>.</figcaption></figure>

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

IC<sub>50</sub> of NV, NV<sub>scFv</sub>, NV-DOX, NV<sub>scFv</sub>-DOX and free DOX (DOX in solution) after 24 h, 48 h or 72 h of treatment on MDA-MB-468 cells. The data is expressed as average ± standard deviation (n = 3). One-way ANOVA test was used to perform statistical analysis (ns p > 0.05, *** p < 0.001).

<table><thead><tr><th></th><th>NV</th><th>NV<sub>scFv</sub></th><th>NV-DOX</th><th>NV<sub>scFv</sub>-DOX</th><th>Free DOX</th></tr></thead><tbody><tr><td>24 h of treatment IC<sub>50</sub> (µM)</td><td>>100</td><td>>100</td><td>0.518 ± 0.166</td><td>0.495 ± 0.192</td><td>0.493 ± 0.076</td></tr><tr><td>48 h of treatment IC<sub>50</sub> (µM)</td><td>>100</td><td>>100</td><td>0.185 ± 0.074</td><td>0.189 ± 0.076</td><td>0.129 ± 0.027</td></tr><tr><td>72 h of treatment IC<sub>50</sub> (µM)</td><td>>100</td><td>>100</td><td>0.100 ± 0.039</td><td>0.106 ± 0.040</td><td>0.067 ± 0.006</td></tr></tbody></table>

>100 µM indicated no measurable IC<sub>50</sub> up to 100 µM.

*In vitro* evaluation of NV<sub>scFv</sub>-DOX was performed on TNBC cells MDA-MB-468 by performing a RealTime-Glo™ MT Cell Viability assay. This nonlytic bioluminescent test, based on reducing potential of metabolically active cells, allows to assess cell viability in real time. Indeed, the produced luminescent signal will be correlated to the number of viable MDA-MB-468 cells. NV<sub>scFv</sub>-DOX and NV<sub>scFv</sub> were tested on cells at the same iron concentrations. NV<sub>scFv</sub>-DOX and free DOX (DOX in solution) were tested on cells at the same DOX concentrations. Similarly, untargeted NV-DOX and NV were tested on MDA-MB-468 cells at the same iron concentrations, whereas NV-DOX and free DOX were tested on cells at the same DOX concentrations. Biological activity of NV<sub>scFv</sub>-DOX on TNBC cells viability was compared to NV<sub>scFv</sub>, free DOX and untargeted NV-DOX. Viability profiles according to iron concentration of SPIONs (for NV-DOX, NV<sub>scFv</sub>-DOX, NV and NV<sub>scFv</sub>) and to DOX concentration (for NV-DOX, NV<sub>scFv</sub>-DOX and free DOX) are shown in Fig. 6.

For 24 h, 48 h or 72 h of treatment, the biological activity of NV<sub>scFv</sub>-DOX on MDA-MB-468 cell viability does not differ significantly from the activity of free DOX. Cell viability decreases with the DOX concentration, whether vectored by NV<sub>scFv</sub>-DOX or not, making it DOX dose-dependent. However, NV<sub>scFv</sub> effect on TNBC cell viability is expressed according to its iron concentration because no DOX is loaded onto these nanovectors. Treatment with NV<sub>scFv</sub> results in TNBC cell viability of over 75% at all tested iron concentrations and for each treatment time. This is significantly different from the effect of NV<sub>scFv</sub>-DOX and free DOX demonstrating that the cytotoxic effect on MDA-MB-468 cell is indeed caused by DOX and not by the nanovectors. The IC<sub>50</sub> values for NV<sub>scFv</sub>, as shown in table 2, are above 100 µM after 24, 48 or 72 h of treatment underlining its non-cytotoxic effect on MDA-MB-468 TNBC cells at the tested iron concentrations. They are significantly different from the IC<sub>50</sub> values of NV<sub>scFv</sub>-DOX and free DOX (p < 0.001) for all treatment times. Similar observations can be made for control untargeted nanovectors, NV and NV-DOX, underlining the non-cytotoxic effect of NV on MDA-MB-468 cells (IC<sub>50</sub> > 100 µM for all treatment times) and the DOX dose-dependent cytotoxic effect of NV-DOX on MDA-MB-468 cells. Finally, for NV<sub>scFv</sub>-DOX, NV-DOX and free DOX, DOX cytotoxicity is time-dependent as cell viability decreases with treatment time. Indeed, after 72 h of treatment with NV<sub>scFv</sub>-DOX, NV-DOX or free DOX, the viability profile of TNBC cells reaches almost 0% as can be seen in Fig. 6-C. Furthermore, the decrease in IC<sub>50</sub> values, as shown in table 2, for NV<sub>scFv</sub>-DOX, NV-DOX and free DOX with time underlines the time-dependent cytotoxicity of DOX.

Indeed, IC<sub>50</sub> value for NV<sub>scFv</sub>-DOX after 24 h of TNBC cells treatment is 0.495 ± 0.192 µM whereas it is 0.189 ± 0.076 µM after 48 h of treatment and 0.106 ± 0.040 µM after 72 h of treatment (Table 2). IC<sub>50</sub> value for NV-DOX after 24 h of TNBC cells treatment is 0.518 ± 0.166 µM whereas it is 0.185 ± 0.074 µM after 48 h of treatment and 0.100 ± 0.039 µM after 72 h of treatment (Table 2). IC<sub>50</sub> value for free DOX after 24 h of TNBC cells treatment is 0.493 ± 0.076 µM whereas it is 0.129 ± 0.027 µM after 48 h of treatment and 0.067 ± 0.006 µM after 72 h of treatment (Table 2). Values of IC<sub>50</sub> for NV<sub>scFv</sub>-DOX and free DOX are not significantly different (p > 0.05) for all treatment times. Consequently, vectorization of DOX onto NV<sub>scFv</sub>-DOX using the DOX-Fe²⁺ complex strategy did not prevent the release of DOX and maintain its biological cytotoxic effect on MDA-MB-468 TNBC cells. Furthermore, values of IC<sub>50</sub> for NV-DOX and free DOX are not significantly different (p > 0.05) for all treatment times. Thus, under continuous-treatment conditions,
the DOX payload associated with NV<sub>scFv</sub>-DOX and NV-DOX is sufficient to achieve *in vitro* cytotoxicity that is comparable to that of free DOX on the studied TNBC cell line. Notably, cell viability profiles (Fig. 6) and IC<sub>50</sub> of NV<sub>scFv</sub>-DOX and untargeted NV-DOX (Table 2) do not differ significantly (p > 0.05) at any considered treatment time. This similarity likely reflects the high intrinsic sensitivity of the MDA-MB-468 cell line to DOX, which masks any potential enhancement from the scFv targeting, resulting in comparable cytotoxicity between NV<sub>scFv</sub>-DOX and NV-DOX. As anti-EGFR scFvs grafted onto NV<sub>scFv</sub>-DOX remain functional (Fig. 5), this suggests that the targeting feature could confer additional cytotoxic benefits under more drastic conditions where cells' intrinsic sensitivity to DOX is lower, or in *in vivo* conditions where the active targeting is more relevant.

## 4. Conclusion

A new generation of anti-EGFR targeted theranostic nanovectors was developed in this study. First of all, the covalently-assembled nano-platforms were generated, composed of fluorescently labelled SPIONS coated with PEG and decorated with anti-EGFR scFvs (nearly 12 scFvs per SPION). The nanoplatforms were then successfully loaded with fluorescent anticancer drug DOX (ca 6.5% w/w of drug/iron oxide). The resulting nanovectors NV<sub>scFv</sub>-DOX possessed a list of physico-chemical characteristics (D<sub>H</sub> < 150 nm, PDI < 0.3, zeta potential ca -10 mV) making them potentially suitable for IV injection. In spite of the complexity of these nanovectors, the functionality of each component has been confirmed: dual-mode fluorescence, pH-dependent release of DOX, EGFR recognition, and drug cytotoxicity against MBA-MB-468 cancer cells after 24 h, 48 h and 72 h of treatment. Mechanisms of the anticancer action involving aspects such as internalization of NV<sub>scFv</sub>-DOX into TNBC cells and evaluation of the drug release from the nanovectors within cells will be evaluated in further studies.

## CRediT authorship contribution statement

**Chloé Lameli:** Writing – review & editing, Writing – original draft, Methodology, Investigation.
**Stéphanie David:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Funding acquisition.
**Emilie Allard-Vannier:** Writing – review & editing, Writing – original draft, Validation, Supervision.
**Gaëlle Dupeyrol:** Resources.
**Fanny Boursin:** Resources.
**Nicolas Aubrey:** Resources, Methodology.
**Igor Chourpa:** Writing – review & editing, Writing – original draft, Conceptualization.
**Katel Hervé-Aubert:** Writing – review & editing, Writing – original draft, Validation, Supervision, Funding acquisition.

## 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 was supported by the Institut National du Cancer (MAG-COT INCa_16718) and the Région Centre-Val de Loire for the PhD funding of Chloé Lameli. This work was also supported by the French National Research Agency under the program "Investissements d'avenir" Grant Agreement LabEx MAbImprove (ANR-10-LABX-53-01).

## Data availability

The data that has been used is confidential.

## References

[1] J. Lee, Current treatment landscape for early triple-negative breast cancer (TNBC), J. Clin. Med. 12 (2023) 1524, https://doi.org/10.3390/jcm12041524.

[2] H. Yao, G. He, S. Yan, C. Chen, L. Song, T.J. Rosol, X. Deng, Triple-negative breast cancer: is there a treatment on the horizon? Oncotarget 8 (2016) 1913–1924, https://doi.org/10.18632/oncotarget.12284.

[3] C.K. Anders, V. Abramson, T. Tan, R. Dent, The evolution of triple-negative breast cancer: from biology to novel therapeutics, Am. Soc. Clin. Oncol. Educ. Book (2016) 34–42, https://doi.org/10.1200/EDBK.159135.

[4] D. Liu, J. He, Z. Yuan, S. Wang, R. Peng, Y. Shi, X. Teng, T. Qin, EGFR expression correlates with decreased disease-free survival in triple-negative breast cancer: a retrospective analysis based on a tissue microarray, Med. Oncol. 29 (2012) 401–405, https://doi.org/10.1007/s12032-011-9827-x.

[5] L.A. Hoeferlin, C.E. Chalfant, M.A. Park, Challenges in the treatment of triple negative and HER2-overexpressing breast cancer, J. Surg. Sci. 1 (2013) 3–7.

[6] O. Obidiro, G. Battogtokh, E.O. Akala, Triple negative breast cancer treatment options and limitations: future outlook, Pharmaceutics 15 (2023) 1796, https://doi.org/10.3390/pharmaceutics15071796.

[7] R.Q. Li, L. Yan, L. Zhang, H.X. Ma, H.W. Wang, P. Bu, Y.F. Xi, J. Lian, Genomic characterization reveals distinct mutational landscapes and therapeutic implications between different molecular subtypes of triple-negative breast cancer, Sci. Rep. 14 (2024) 12386, https://doi.org/10.1038/s41598-024-62991-3.

[8] J.D. Marotti, F.B. de Abreu, W.A. Wells, G.J. Tsongalis, Triple-negative breast cancer: next-generation sequencing for target identification, Am. J. Pathol. 187 (2017) 2133–2138, https://doi.org/10.1016/j.ajpath.2017.05.018.

[9] R. Costa, A.N. Shah, C.A. Santa-Maria, M.R. Cruz, D. Mahalingam, B.A. Carneiro, Y. K. Chae, M. Cristofanilli, W.J. Gradishar, F.J. Giles, Targeting epidermal growth factor receptor in triple negative breast cancer: new discoveries and practical insights for drug development, Cancer Treat. Rev. 53 (2017) 111–119, https://doi.org/10.1016/j.ctrv.2016.12.010.

[10] S.G. Smoots, A.R. Schreiber, M.M. Jackson, S.M. Bagby, A.T.A. Dominguez, E. D. Dus, C.A. Binns, M. MacBeth, P.A. Whitty, J.R. Diamond, T.M. Pitts, Overcoming doxorubicin resistance in triple-negative breast cancer using the class I-targeting HDAC inhibitor bocedepsin/OKI-179 to promote apoptosis, Breast Cancer Res. 26 (2024) 35, https://doi.org/10.1186/s13058-024-01799-5.

[11] Y. Shi, L. Cao, W. Zhao, S. Lai, Z. Chen, Z. Ye, M. Guo, J. He, Y. Zhou, G. Li, L. Lv, Enhancing chemo-immunotherapy in triple-negative breast cancer: Co-delivery of doxorubicin and berberine using nanoparticles to downregulate PD-L1 and eliminate cancer stem cells, Int. J. Pharm. 670 (2025) 125134, https://doi.org/10.1016/j.ijpharm.2024.125134.

[12] A. Mandapati, K.E. Lukong, Triple negative breast cancer: approved treatment options and their mechanisms of action, J. Cancer Res. Clin. Oncol. 149 (2023) 3701–3719, https://doi.org/10.1007/s00432-022-04189-6.

[13] A.N. Linders, I.B. Dias, T. López Fernández, C.G. Tocchetti, N. Bomer, P., Van der Meer, A review of the pathophysiological mechanisms of doxorubicin-induced cardiotoxicity and aging, Npj, Aging 10 (2024) 1–9, https://doi.org/10.1038/s41514-024-00135-7.

[14] A.R. Palvia, A.R. Damera, A.R. Nandi, S. Magar, S. Patidar, S. Kasarla, V. Ghantasala, M.K. Shah, M. Goyal, A.R. Palvia, A.R. Damera, A.R. Nandi, S. Magar, S. Patidar, S. Kasarla, V. Ghantasala, M.K. Shah, M. Goyal, Cardio-oncology's modern approaches to prevent doxorubicin-induced cardiotoxicity: a systematic review, Cureus 16 (2024), https://doi.org/10.7759/cureus.66215.

[15] V.C. Deivayanai, P. Thamarai, S. Karishma, A. Saravanan, P.R. Yaashikaa, A. S. Vickram, R.V. Hemavathy, R.R. Kumar, S. Rishikesavan, S. Shruthi, A comprehensive review on advances in nanoparticle-mediated cancer therapeutics: current research and future perspectives, Cancer Pathog. Ther. (2024), https://doi.org/10.1016/j.cpt.2024.11.002.

[16] B. Wang, S. Hu, Y. Teng, J. Chen, H. Wang, Y. Xu, K. Wang, J. Xu, Y. Cheng, X. Gao, Current advance of nanotechnology in diagnosis and treatment for malignant tumors, Signal Transduct. Target. Ther. 9 (2024) 1–65, https://doi.org/10.1038/s41392-024-01889-y.

[17] W. Zeng, Y. Luo, D. Gan, Y. Zhang, H. Deng, G. Liu, Advances in Doxorubicin-based nano-drug delivery system in triple negative breast cancer, Front. Bioeng. Biotechnol. 11 (2023) 1271420, https://doi.org/10.3389/fbioe.2023.1271420.

[18] H. Yoo, Y. Kim, J. Kim, H. Cho, K. Kim, Overcoming cancer drug resistance with nanoparticle strategies for key protein inhibition, Molecules 29 (2024) 3994, https://doi.org/10.3390/molecules29173994.

[19] J. Wu, The enhanced permeability and retention (EPR) effect: the significance of the concept and methods to enhance its application, J. Pers. Med. 11 (2021) 771, https://doi.org/10.3390/jpm11080771.

[20] Y. Nakamura, A. Mochida, P.L. Choyke, H. Kobayashi, Nanodrug delivery: is the enhanced permeability and retention effect sufficient for curing cancer? Bioconjug. Chem. 27 (2016) 2225–2238, https://doi.org/10.1021/acs.bioconjchem.6b00437.

[21] Y. (Chezy) Barenholz, Doxil® – the first FDA-approved nano-drug: from Basics via CMC, Cell Culture and Animal Studies to Clinical Use (2016), https://doi.org/10.1039/9781782622536-00315.

[22] G. Kandasamy, D. Maity, Recent advances in superparamagnetic iron oxide nanoparticles (SPIONS) for *in vitro* and *in vivo* cancer nanotheranostics, Int. J. Pharm. 496 (2015) 191–218, https://doi.org/10.1016/j.ijpharm.2015.10.058.

[23] F.S. Abdulwahid, A.J. Haider, S. Al-Musawi, Iron oxide nanoparticles (IONPs): synthesis, surface functionalization, and targeting drug delivery strategies: mini-review, Nano 17 (2022) 2230007, https://doi.org/10.1142/S1793292022300079.

[24] U.M. Mahajan, S. Teller, M. Sendler, R. Palankar, C. van den Brandt, T. Schwaiger, J.-P. Kühn, S. Ribback, G. Glöckl, M. Evert, W. Weitschies, N. Hosten, F. Dombrowski, M. Delcea, F.-U. Weiss, M.M. Lerch, J. Mayerle, Tumour-specific
delivery of siRNA-coupled superparamagnetic iron oxide nanoparticles, targeted against PLK1, stops progression of pancreatic cancer, Gut 65 (2016) 1838-1849, https://doi.org/10.1136/gutjnl-2016-311393.

[25] Z. Yang, J. Duan, J. Wang, Q. Liu, R. Shang, X. Yang, P. Lu, C. Xia, L. Wang, K. Dou, Superparamagnetic iron oxide nanoparticles modified with polyethylenimine and galactose for siRNA targeted delivery in hepatocellular carcinoma therapy, Int. J. Nanomedicine 13 (2018) 1851-1865, https://doi.org/10.2147/IJN.S155537.

[26] K. Karpinska, L. Li, T. Wang, Dual conjugation of magnetic nanoparticles with antibodies and siRNA for cell-specific gene silencing in vascular cells, Front. Drug Deliv. 4 (2024), https://doi.org/10.3389/fdev.2024.1416737.

[27] T. Vangijzegem, V. Lecomte, I. Ternad, L. Van Leuven, R.N. Muller, D. Stanicki, S. Laurent, Superparamagnetic iron oxide nanoparticles (SPION): from fundamentals to state-of-the-art innovative applications for cancer therapy, Pharmaceutics 15 (2023) 236, https://doi.org/10.3390/pharmaceutics15010236.

[28] C. Pucci, A. Degl'Innocenti, M.B. Gümüş, G. Ciofani, Superparamagnetic iron oxide nanoparticles for magnetic hyperthermia: recent advancements, molecular effects, and future directions in the omics era, Biomater. Sci. 10 (2022) 2103-2121, https://doi.org/10.1039/D1BM01963E.

[29] C. Martins, C. Rolo, V.R.G. Cacho, L.C.J. Pereira, J. Paulo Borges, J. Carvalho Silva, T. Vieira, P.I.P. Soares, Enhancing the magnetic properties of superparamagnetic iron oxide nanoparticles using hydrothermal treatment for magnetic hyperthermia application, Mater. Adv. 6 (2025) 1726-1743, https://doi.org/10.1039/D4MA011120A.

[30] C.R. Cogle, G.J. Madlambayan, G. Hubsher, C. Beckman, R. Speisman, R. Tran-Son-Tay, C.J. Pepine, Narrow cell therapies for cardiovascular diseases, Exp. Hematol. 36 (2008) 687-694, https://doi.org/10.1016/j.exphem.2008.01.012.

[31] Y.-X.-J. Wang, Superparamagnetic iron oxide based MRI contrast agents: current status of clinical application, Quant. Imaging Med. Surg. 1 (2011) 35, https://doi.org/10.3978/j.issn.2223-4292.2011.08.03.

[32] D. Lee, J. Sohn, A. Kirichenko, Quantifying liver heterogeneity via R2*-MRI with super-paramagnetic iron oxide nanoparticles (SPION) to characterize liver function and tumor, Cancers 14 (2022) 5269, https://doi.org/10.3390/cancers14215269.

[33] J. Dulińska-Litewka, A. Łazarczyk, P. Halubiec, O. Szafrański, K. Karnas, A. Karewicz, Superparamagnetic iron oxide nanoparticles—current and prospective medical applications, Materials 12 (2019) 617, https://doi.org/10.3390/ma12040617.

[34] S.K. Yen, P. Padmanabhan, S.T. Selvan, Multifunctional iron oxide nanoparticles for diagnostics, therapy and macromolecule delivery, Theranostics 3 (2013) 986-1003, https://doi.org/10.7501/thno.4827.

[35] P.V. Nguyen, K. Hervé-Aubert, S. David, N. Lautram, C. Passirani, I. Chourpa, N. Aubrey, E. Allard-Vannier, Targeted nanomedicine with anti-EGFR scFv for siRNA delivery into triple negative breast cancer cells, Eur. J. Pharm. Biopharm. 157 (2020) 74-84, https://doi.org/10.1016/j.ejpb.2020.10.004.

[36] E. Munnier, S. Cohen-Jonathan, C. Linassier, L. Douziech-Eyrolles, H. Marchais, M. Soucé, K. Hervé, P. Dubois, I. Chourpa, Novel method of doxorubicin-SPION reversible association for magnetic drug targeting, Int. J. Pharm. 363 (2008) 170-176, https://doi.org/10.1016/j.ijpharm.2008.07.006.

[37] J. Gautier, E. Munnier, A. Paillard, K. Hervé, L. Douziech-Eyrolles, M. Soucé, P. Dubois, I. Chourpa, A pharmaceutical study of doxorubicin-loaded PEGylated nanoparticles for magnetic drug targeting, Int. J. Pharm. 423 (2012) 16-25, https://doi.org/10.1016/j.ijpharm.2011.06.010.

[38] P.-P. Than, S.-J. Yao, E. Althagafi, K. Kaur, A conjugate of an EGFR-binding peptide and doxorubicin shows selective toxicity to triple-negative breast cancer cells, ACS Med. Chem. Lett. 16 (2025) 109-115, https://doi.org/10.1021/acsmedchemlett.4c00480.

[39] C. Mamot, A. Wicki, U. Hasler-Strub, S. Riniker, Q. Li, L. Holer, D. Bärtschi, K. Zaman, R. von Moos, K.J. Dedes, L.A. Boos, U. Novak, A. Bodmer, R. Ritschard, E.C. Obermann, A. Tzankov, C. Ackermann, V. Membrez-Antonioli, U. Zürrer-Härdi, C.B. Caspar, S. Deuster, M. Senn, R. Winterhalder, C. Rochlitz, A multicenter phase II trial of anti-EGFR-immunoliposomes loaded with doxorubicin in patients with advanced triple negative breast cancer, Sci. Rep. 13 (2023) 3705, https://doi.org/10.1038/s41598-023-30950-z.

[40] L.C. Collins, A. Martyniak, M.J. Kandel, Z.K. Stadler, S. Masciari, A. Miron, A. L. Richardson, S.J. Schnitt, J.E. Garber, Basal cytokeratin and epidermal growth factor receptor expression are not predictive of BRCA1 mutation status in women with triple-negative breast cancers, Am. J. Surg. Pathol. 33 (2009) 1093-1097, https://doi.org/10.1097/PAS.0b013e31819c1c93.

[41] C. Rao, J. Shetty, K.H. Prasad, Immunohistochemical profile and morphology in triple-negative breast cancers, J. Clin. Diagn. Res. JCDR 7 (2013) 1361-1365, https://doi.org/10.7860/JCDR.2013/5823.3129.

[42] Y. Si, Y. Zhang, H.G. Ngo, J.-S. Guan, K. Chen, Q. Wang, A.P. Singh, Y. Xu, L. Zhou, E.S. Yang, X. (Margaret) Liu, Targeted liposomal chemotherapies to treat triple-negative breast cancer, Cancers 13 (2021) 3749. 10.3390/cancers13153749.

[43] N.T. Ueno, D. Zhang, Targeting EGFR in triple negative breast cancer, J. Cancer 2 (2011) 324-328, https://doi.org/10.7150/jca.2324.

[44] A.E. Guerrab, M. Bamdad, F. Kwiatkowski, Y.-J. Bignon, F. Penault-Llorca, C. Aubel, Anti-EGFR monoclonal antibodies and EGFR tyrosine kinase inhibitors as combination therapy for triple-negative breast cancer, Oncotarget 73618-73637 (2016) 10.18632/oncotarget.12037.

[45] K. Nakai, M.-C. Hung, H. Yamaguchi, A perspective on anti-EGFR therapies targeting triple-negative breast cancer, Am. J. Cancer Res. 6 (2016) 1609-1623.

[46] C. Alric, N. Aubrey, É. Allard-Vannier, A. Di Tommaso, T. Blondy, I. Dimier-Poisson, I. Chourpa, K. Hervé-Aubert, Covalent conjugation of cysteine-engineered scFv to PEGylated magnetic nanoprobes for immunotargeting of breast cancer cells, RSC Adv. 6 (2016) 37099-37109, https://doi.org/10.1039/C6RA06076E.

[47] C. Alric, K. Hervé-Aubert, N. Aubrey, S. Melouk, L. Lajoie, W. Même, S. Même, Y. Courbebaisse, A.A. Ignatova, A.V. Feofanov, I. Chourpa, E. Allard-Vannier, Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes, J. Nanobiotechnology 16 (2018) 18, https://doi.org/10.1186/s12951-018-0341-6.

[48] X. Liu, L. Zhang, S. Lu, D. Liu, Y. Huang, J. Zhu, W. Zhou, X. Yu, R. Liu, Superparamagnetic iron oxide nanoparticles conjugated with Aβ oligomer-specific scFv antibody and class a scavenger receptor activator show therapeutic potentials for Alzheimer's Disease, J. Nanobiotechnology 18 (2020) 160, https://doi.org/10.1186/s12951-020-00723-1.

[49] K.L. Vigor, P.G. Kyrtatos, S. Minogue, K.T. Al-Jamal, H. Kogelberg, B. Tolner, K. Kostarelos, R.H. Begent, Q.A. Pankhurst, M.F. Lythgoe, K.A. Chester, Nanoparticles functionalised with recombinant single chain Fv antibody fragments (scFv) for the magnetic resonance imaging of cancer cells, Biomaterials 31 (2010) 1307-1315, https://doi.org/10.1016/j.biomaterials.2009.10.036.

[50] H. Chen, L. Wang, Q. Yu, W. Qian, D. Tiwari, H. Yi, A.Y. Wang, J. Huang, L. Yang, H. Mao, Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer, Int. J. Nanomedicine 8 (2013) 3781-3794, https://doi.org/10.2147/IJN.S49069.

[51] K. Hervé, L. Douziech-Eyrolles, E. Munnier, S. Cohen-Jonathan, M. Soucé, H. Marchais, P. Limelette, F. Warmont, M.L. Saboungi, P. Dubois, I. Chourpa, The development of stable aqueous suspensions of PEGylated SPIONs for biomedical applications, Nanotechnology 19 (2008) 465608, https://doi.org/10.1088/0957-4484/19/46/465608.

[52] E. Perillo, K. Hervé-Aubert, E. Allard-Vannier, A. Falanga, S. Galdiero, I. Chourpa, Synthesis and *in vitro* evaluation of fluorescent and magnetic nanoparticles functionalized with a cell penetrating peptide for cancer theranosis, J. Colloid Interface Sci. 499 (2017) 209-217, https://doi.org/10.1016/j.jcis.2017.03.006.

[53] J. Bruniaux, S.B. Djemaa, K. Hervé-Aubert, H. Marchais, I. Chourpa, S. David, Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics, Int. J. Pharm. 532 (2017) 660-668, https://doi.org/10.1016/j.ijpharm.2017.05.022.

[54] P.C. Lee, D. Meisel, Adsorption and surface-enhanced Raman of dyes on silver and gold sols, J. Phys. Chem. 86 (1982) 3391-3395, https://doi.org/10.1021/j100214a025.

[55] O. Garbuzenko, Y. Barenholz, A. Priev, Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer, Chem. Phys. Lipids 135 (2005) 117-129, https://doi.org/10.1016/j.chemphylipid.2005.02.003.

[56] M. Li, S. Jiang, J. Simon, D. Paßlick, M.-L. Frey, M. Wagner, V. Mailänder, D. Crespy, K. Landfester, Brush conformation of polyethylene glycol determines the stealth effect of nanocarriers in the low protein adsorption regime, Nano Lett. 21 (2021) 1591-1598, https://doi.org/10.1021/acs.nanolett.0c03756.

[57] L. Shi, J. Zhang, M. Zhao, S. Tang, X. Cheng, W. Zhang, W. Li, X. Liu, H. Peng, Q. Wang, Effects of polyethylene glycol on the surface of nanoparticles for targeted drug delivery, Nanoscale 13 (2021) 10748-10764, https://doi.org/10.1039/D1NR02065J.

[58] P.-V. Nguyen, E. Allard-Vannier, N. Aubrey, C. Labrugère-Sarroste, I. Chourpa, J. Sobilo, A. Le Pape, K. Hervé-Aubert, Radiolabeling, quality control and in vivo imaging of multimodal targeted nanomedicines, Pharmaceutics 14 (2022) 2679, https://doi.org/10.3390/pharmaceutics14122679.

[59] J. Gautier, E. Munnier, L. Douziech-Eyrolles, A. Paillard, P. Dubois, I. Chourpa, SERS spectroscopic approach to study doxorubicin complexes with Fe2+ ions and drug release from SPION-based nanocarriers, Analyst 138 (2013) 7354-7361, https://doi.org/10.1039/C3AN00787A.

[60] Z.A. Mohammadi, S.F. Aghamiri, A. Zarrabi, M.R. Talaie, Liposomal Doxorubicin delivery systems: effects of formulation and processing parameters on drug loading and release behavior, Curr. Drug Deliv. 13 (2016) 1065-1070, https://doi.org/10.2174/1567201813666151228104643.

[61] S. Mohapatra, M. Asfer, M. Anwar, K. Sharma, M. Akhter, F.J. Ahmad, A. A. Siddiqui, Doxorubicin loaded carboxymethyl Assam bora rice starch coated superparamagnetic iron oxide nanoparticles as potential antitumor cargo, Heliyon 5 (2019) e01955, https://doi.org/10.1016/j.heliyon.2019.e01955.