Arnaud Pallotta

Ariane Boudier

Benjamin Creusot

Emilie Brun

Cécile Sicard-Roselli

Rana Bazzi

Stéphane G Roux

Igor Clarot
email: igor.clarot@univ-lorraine.fr

Quality control of gold nanoparticles as pharmaceutical ingredients

Keywords: Gold nanoparticles, Pharmaceutical ingredient, Capillary electrophoresis, Assay, Impurity profile, Stability

Introduction

Since recent years, nanoparticles (NP) development is an extensively growing research field for biomedical applications [START_REF] D'mello | The evolving landscape of drug products containing nanomaterials in the United States[END_REF] especially for cancer diagnosis and treatment [START_REF] Brigger | Nanoparticles in cancer therapy and diagnosis[END_REF][START_REF] Hardie | Simultaneous cytosolic delivery of a chemotherapeutic and siRNA using nanoparticle-stabilized nanocapsules[END_REF][START_REF] Pallotta | Nanotechnologies for Medical Devices: Potentialities and Risks[END_REF][START_REF] Rawal | Threatening cancer with nanoparticle aided combination oncotherapy[END_REF][START_REF] Silva | Current Trends in Cancer Nanotheranostics: Metallic, Polymeric, and Lipid-Based Systems[END_REF][START_REF] Yi | Targeted systemic delivery of siRNA to cervical cancer model using cyclic RGD-installed unimer polyion complex-assembled gold nanoparticles[END_REF]. Several types of NP are under investigation and among the inorganic ones, gold nanoparticles (AuNP) are widely studied and used for numerous applications (Figure 1). Their properties have led to several studies from preclinical to clinical trials.

At the moment, eight trials concerning AuNP are set up with different therapeutic targets (such as cancer treatment, type I diabetes, coronary diseases, Parkinson's disease, pulmonary hypertension), half of which implies in-vivo use (Table 1). AuNP are biocompatible, easily synthesized and can be functionalized by a wide range of molecules and active substances. Their specific optical properties make them used as biosensors [START_REF] Aldewachi | Gold nanoparticle-based colorimetric biosensors[END_REF][START_REF] Devi | Nanomaterials for early detection of cancer biomarker with special emphasis on gold nanoparticles in immunoassays/sensors[END_REF][START_REF] Nie | Applications of gold nanoparticles in optical biosensors[END_REF][START_REF] Shawky | Gold aggregating gold: A novel nanoparticle biosensor approach for the direct quantification of hepatitis C virus RNA in clinical samples[END_REF] or diagnostic agents [START_REF] Iso/Ts ; Lu | Nanotechnologies -Terminology and definitions for nano-objects, nanoparticle, nanofibre and nanoplate[END_REF]. Thus, they emerge as powerful theranostic platforms [START_REF] Guo | Gold nanoparticles enlighten the future of cancer theranostics[END_REF][START_REF] Singh | Application of gold nanoparticles for gastrointestinal cancer theranostics: A systematic review[END_REF].

The translation of AuNP from bench to bedside requires to ascertain purity and impurity profiles of the synthesized product. For classical drugs and excipients, profiles are described and reported in Pharmacopoeia (FDA for the USA, EDQM for Europe and PMDA for Japan). However, for AuNP as for all NP, no official consensus exists. Their characterization remains a challenge throughout NP synthesis. Among the numerous available characterization techniques, most focus on physicochemical parameters and allow determining:

• Their size: core size (transmission electronic microscopy (TEM)) or hydrodynamic diameter (Dh) (dynamic light scatting (DLS)).

• Their surface charge (ζ potential): DLS coupled to electrophoresis.

• Their chemical composition: atomic spectroscopies, X-Ray Photoelectron spectroscopy (XPS), Secondary Ion Mass Spectroscopy (SIMS) …

The indicated methodologies are the ones used currently to determine physicochemical parameters of NP [START_REF] Iso/Ts ; Lu | Nanotechnologies -Terminology and definitions for nano-objects, nanoparticle, nanofibre and nanoplate[END_REF]. But several drawbacks have already been reported in the literature. On the one hand, TEM is a very powerful tool to determine the morphologie and size of a batch of nano-objects (ISO/ TR-18196:2016TR-18196: , 2016;;[START_REF] Varenne | Multimodal Dispersion of Nanoparticles: A Comprehensive Evaluation of Size Distribution with 9 Size Measurement Methods[END_REF]. However, it is always performed on dry samples leading to possible modifications compared to NP in their liquid medium i.e. after synthesis or incubation in biological media (hydration and solvation layers, ligand or protein adsorption...). Cryo-TEM could be used to assess liquid samples [START_REF] Stewart | Cryo-electron microscopy and cryo-electron tomography of nanoparticles[END_REF] but is not used in routine yet and is usually difficult to access. DLS, on the other hand, allows simple measurements in liquid media. It is low-cost and easy handling (ISO-22412:2017(ISO-22412: , 2017;;ISO/TR-18196:2016[START_REF] Bhattacharjee | DLS and zeta potential -What they are and what they are not?[END_REF][START_REF] Varenne | Multimodal Dispersion of Nanoparticles: A Comprehensive Evaluation of Size Distribution with 9 Size Measurement Methods[END_REF]. DLS give accurate results as long as monodisperse samples are studied. The hydrodynamic diameter is given assimilating nano-objets to spheres. Data obtained on samples with high polydispersity or interacting with light (such as metallic NP) must be treated with caution [START_REF] Bhattacharjee | DLS and zeta potential -What they are and what they are not?[END_REF][START_REF] Varenne | Multimodal Dispersion of Nanoparticles: A Comprehensive Evaluation of Size Distribution with 9 Size Measurement Methods[END_REF]. To overcome this drawback, it is possible to associate DLS with a pre-separation step, such as Field Flow Fractionation but it can be biased by the presence of large particles [START_REF] Clogston | Polymer Nanoparticles for Nanomedicines: A Guide for their Design, Preparation and Development[END_REF].

Concerning surface charge, ζ potential is highly dependent on experimental parameters (dispersion medium, salt concentration, pH, instrument parameters …). In addition, ζ potential determination can sometimes be difficult for small particles (< 40 nm) (ISO/ TR-18196:2016TR-18196: , 2016;;[START_REF] Varenne | Standardization and validation of a protocol of zeta potential measurements by electrophoretic light scattering for nanomaterial characterization[END_REF]. Finally, the previously mentioned methods that analyze the chemical composition of NP are often used on dry samples which can be responsible of artifacts [START_REF] Muneshwar | Comparing XPS on bare and capped ZrN films grown by plasma enhanced ALD: Effect of ambient oxidation[END_REF]. Moreover, they require complex handling, are not readily accessible and thus are not used in routine.

Additionally, these different approaches do not give any information on the impurity profile of the sample.

Separative methods have been previously applied to characterize NP: liquid chromatography coupled to mass spectrometry (LC-MS) [START_REF] Mishra | TiO2 decorated functionalized halloysite nanotubes (TiO2@HNTs) and photocatalytic PVC membranes synthesis, characterization and its application in water treatment[END_REF], size exclusion chromatography (SEC) [START_REF] Duesberg | Chromatographic size separation of single-wall carbon nanotubes[END_REF]), Field Flow Fractionation (Zattoni et al., 2009). Capillary zone electrophoresis (CZE) is also suitable for this purpose [START_REF] Pallotta | Characterization and stability of gold nanoparticles depending on their surface chemistry: Contribution of capillary zone electrophoresis to a quality control[END_REF]. AuNP have been separated according to their size [START_REF] Franze | Separation and detection of gold nanoparticles with capillary electrophoresis and ICP-MS in single particle mode (CE-SP-ICP-MS)[END_REF][START_REF] Pallotta | Characterization and stability of gold nanoparticles depending on their surface chemistry: Contribution of capillary zone electrophoresis to a quality control[END_REF] and shape [START_REF] Surugau | Electrophoretic methods for separation of nanoparticles[END_REF] with a high resolution. It has also been used to evaluate the surface chemistry of AuNP and their stability. Thus CZE has been proposed as a method of choice for quality control of inorganic NP [START_REF] Pallotta | Characterization and stability of gold nanoparticles depending on their surface chemistry: Contribution of capillary zone electrophoresis to a quality control[END_REF].

International Conferences on Harmonization (ICH) is an organization regrouping Pharmacopoeias from all around the world and gives guidelines for Quality and safety of medicines. These recommendations include the definition of precise specifications so that a substance can be declared in conformity with its intended use (ICH Q6A, (ICH-Q6A)). Important specifications are related to the limits imposed for impurities (ICH Q3A(R2), (ICH-Q3A(R2))). Impurities in new substances derived from chemical synthesis may be "organic, inorganic or residual solvents". They result from the synthesis (starting materials, by-products, intermediates, reagents…)

or degradation products. For nanoparticles, these definitions must also be applied with certain adjustment: if nanoparticles other than those defined in the specifications exist in the sample, they must also be considered as impurities (populations with different sizes or charges or even aggregates for example). Their assessment and the substance content (assay percentage) are essential for the initial definition of the NP as a pharmaceutical ingredient. For a safety of use, these criteria and in particular the impurity profiles must be evaluated over time to define their stability (e. g. a maximum permissible deviation of 5% from the initial content in ICH Q1A(R2) (ICH-Q1A(R2)))

Lately, several reviews and articles have been published concerning the lack of quality control during NP manufacturing process, the complexity to switch from a lab scale to an industrial scale production [START_REF] Coty | Characterization of nanomedicines: A reflection on a field under construction needed for clinical translation success[END_REF][START_REF] Gonzalez-Fuenzalida | Miniaturized liquid chromatography coupled on-line to in-tube solid-phase microextraction for characterization of metallic nanoparticles using plasmonic measurements. A tutorial[END_REF][START_REF] Pallotta | Nanotechnologies for Medical Devices: Potentialities and Risks[END_REF][START_REF] Stavis | Nanoparticle Manufacturing -Heterogeneity through Processes to Products[END_REF] and the need for future standards for the evaluation of nano-ingredients [START_REF] Gioria | Are existing standard methods suitable for the evaluation of nanomedicines: some case studies[END_REF][START_REF] Halamoda-Kenzaoui | Mapping of the available standards against the regulatory needs for nanomedicines[END_REF].

In this study, we focused on gold nanoparticles stabilized by citrate ions (AuNP-Cit) because they are often the basic elements for the synthesis of more complex nano-objects (functionalization by ligands for example). All the batches studied in this work were analyzed using classical methodologies (i.e. UV-Vis spectrophotometry, DLS coupled or not to electrophoresis (ζ potential), TEM) and CZE coupled to a diode array detection (DAD) for contents and impurities.

Some batches synthesized from laboratories of the French network GDR Or nano (http://or-nano.insp.upmc.fr)

were monitored all along the synthesis process to evaluate purification or centrifugation processes. Eighteen batches were monitored for evaluation of their stability with time. Finally, the methodologies developed were applied to commercial AuNP batches to determine their value as pharmaceutical ingredients. The main objective of this work is to complement traditional techniques for nanoparticle analysis to ascertain their safety of use in the pharmaceutical sector. A higher quality grade can be achieved and would be a major step forward in other sectors such as cosmetics, agri-food or microelectronics. was used for the preparation of all solutions. Commercial AuNP (5 nm core diameter, citrate stabilized) samples were purchased from Nanocomposix and Sigma-Aldrich.

Citrate stabilized gold nanoparticles synthesis

Citrate stabilized AuNP (AuNP-Cit) were synthesized in aqueous solution as already described [START_REF] Tournebize | Impact of gold nanoparticle coating on redox homeostasis[END_REF]Tournebize, Boudier, Sapin-Minet, et al., 2012;[START_REF] Tournebize | Simple spectrophotocolorimetric method for quantitative determination of gold in nanoparticles[END_REF]. Briefly, 1 mL of AuCl 4 -solution (10 mg/mL) was added in 90 mL of water and 2 mL of sodium citrate 55 mM was added.

The solution was stirred for 1 min then 1 mL of NaBH 4 19.5 mM was added and the solution was stirred again for 5 min under nitrogen. The resulting suspension was immediately stored in the dark at 4 °C. The final concentration was 90 nM.

Several batches were received from other laboratories with their own labelling. The first batches (from A to D)

were not evaluated in this paper. Analyzed batches were called "E, F and G". For E batch, stock solutions of nanoparticles were prepared as proposed by Slot and Geuze [START_REF] Slot | Sizing of protein A-colloidal gold probes for immunoelectron microscopy[END_REF]. AuNP were synthesized by reducing KAuCl 4 solutions (125 nM) by a mix of tri-sodium citrate (0.04 % wt final concentration) and tannic acid (0.05 % wt final concentration), previously heated at 60 °C. Purification consisted of three successive centrifugations (195 000 × g, 40 min, 4 °C) by replacing each time the supernatant with ultra-pure water.

For F and G batches, a pre-mixing method [START_REF] Xia | Revitalizing the Frens Method To Synthesize Uniform, Quasi-Spherical Gold Nanoparticles with Deliberately Regulated Sizes from 2 to 330 nm[END_REF], was used. Two mL of HAuCl 4 (1 % wt in water) and 170 µL of AgNO 3 (0.1 % wt) were mixed. 6 mL (for F) or 2.8 mL (for G) of sodium citrate (1% wt) were added, and water for final volume of 10 mL. After 5 min (for F) or 4 min (for G), the 10 mL solution was dispersed into 190 mL of boiling water and reflux was maintained 30 min. After the partial removal of the solvent under reduced pressure and at a maximum of 40°C, the precipitate was recovered on a polymer membrane and washed thoroughly and successively with 0.01 M HCl, water, and diethylether. The resulting precipitate was dried and the recovered black powder (with a yield of up to 200 mg) was dispersed in 10 mL of NaOH with pH 8.0 (raw suspension of AuNP@DTDTPA).

Since these nanoparticles have been developed for image guided radiotherapy, a purification protocol was established in order to prepare a suspension suitable for intravenous injection. The raw suspension of AuNP@DTDTPA was purified by ultrafiltration using Vivaspin® tube (20 mL, molecular weight cut-off (MWCO) 5 kDa). The resulting suspension was diluted with an appropriate volume of a filtered aqueous solution containing NaCl (4 M) and HEPES (135 mM) in order to obtain intravenous use gold nanoparticles suspensions.

The pH was then adjusted to 7.4. Before use, the AuNP@DTDTPA suspension was filtered onto a syringe filter with a nylon membrane (pore diameter 0.22 μm). BGE (2 min). AuNP were directly injected hydrodynamically (20 mbar for 10 s). EOF was determined using a neutral marker, benzyl alcohol (migration time = 2.06 ± 0.03 min, EOF = 9.1 ± 0.1 × 10 -4 cm²V -1 s -1 (n = 5)).

Electrophoretic mobilities (µ EP ) of AuNP were calculated using equation ( 1):

μ = μ + μ (1)
where µ APP , µ EP and µ EOF correspond to apparent, electrophoretic and electroosmotic flow mobilities respectively.

Each injection was realized in triplicate and the obtained peaks were identified by their relative migration time (RMT), equation (2):

= (2)
where T m is the migration time of the peak of interest and T m neutral marker is the migration time of the neutral species.

Gold concentration measurements

Gold concentration was measured according to the method already developed in our laboratory [START_REF] Pallotta | Highly sensitive and simple liquid chromatography assay with ion-pairing extraction and visible detection for quantification of gold from nanoparticles[END_REF]. Briefly, AuNP-Cit samples (400 µL) were mixed with an HCl-NaCl-Br 2 solution (1.0 M, 0. HPLC analyses were carried out on a SpectraSystem instrument constituted by the following modules: vacuum degasser (SCM1000), quaternary pump (P1000XP), autosampler (AS300) and UV-Visible detector (UV2000).

The column was thermostated at 40 °C using a Croco-cit™ (1267) oven. Data acquisition and injection control were performed by the Azur software (version 4.6.0.0, Datalys®). The method used a Nucleosil ®(C18), 150 mm × 4.6 mm, 3 µm particles size and 100 Å porosity. The detector was set at 555 nm and the pump at 1.0 mL.min -

1

. The injection volume was 20 µL.

Results and discussion

Defining specifications for pharmaceutical raw materials can be a real challenge. Usually, for new drug substances, interlaboratory studies are a good way to define the imposed limits so that they are accepted and recognized by the manufacturing community. For eventual impurities, monographs have to be designed to cover different impurity profiles as synthesis and/or purification processes used by manufacturers could be different. Impurity specifications have also to be defined for specified impurities with individual acceptance criteria and include a limit of the total impurity accepted. Those limits derive directly from qualification data from producers and are submitted to the competent authorities. In this study, all indicated specifications are based on the numerous syntheses carried out in our laboratory and correspond to limits defined specifically for "simple" gold citrate nanoparticles of defined size (5 nm). In order to confer the grade of pharmaceutical ingredient to nanoparticles, a similar approach must be used for all type of nanoparticles (characterization of charge, size, functionalization…) that will be used for biomedical applications.

Synthesis and purification processes evaluation

As potential pharmaceutical products, AuNP require a strict control. In a previous work of our laboratory, some specifications were defined to perform quality control of day-to-day AuNP-Cit synthesis [START_REF] Pallotta | Characterization and stability of gold nanoparticles depending on their surface chemistry: Contribution of capillary zone electrophoresis to a quality control[END_REF].

As defined by ICH Q6A (ICH-Q6A): "A specification is defined as a list of tests, references to analytical procedures, and appropriate acceptance criteria, which are numerical limits, ranges, or other criteria for the tests described. It establishes the set of criteria to which a drug substance or drug product should conform to be considered acceptable for its intended use. "Conformance to specifications" means that the drug substance and / or drug product, when tested according to the listed analytical procedures, will meet the listed acceptance criteria. Specifications are critical quality standards that are proposed and justified by the manufacturer and approved by regulatory authorities as conditions of approval. The specifications imposed in our lab and coming from two years of repeated synthesis are presented in Table 2. On the one hand, classical characterization focuses on the Surface Plasmon Resonance (SPR) measurement (λ max ), the hydrodynamic diameter (Dh) and ζ potential determination. SPR is directly linked to NP size, shape, core/corona composition and suspension medium [START_REF] Vilela | Sensing colorimetric approaches based on gold and silver nanoparticles aggregation: chemical creativity behind the assay. A review[END_REF]. Dh takes into consideration the gold core size plus surface chemistry and some hydration layers. ζ potential somehow reflects ion capping AuNP surface (i.e. citrate ions).

On the other hand, CZE coupled to DAD leads to µ EP which is directly linked to the three parameters described above. As a separation method, it also allows calculating the content percentage (assay on the main product)

in each batch and is therefore highly complementary to classical approaches. Therefore, each newly synthesized AuNP-Cit batch that did not reach specifications described below was automatically discarded. µ EP (× 10 -4 cm².V -1 .s -1 ) λ max (nm) Assay (%) AuNP-Cit 514 ± 1 6.5 ± 0.9 -32.6 ± 0.7 -1.75 ± 0.44 521 ± 3 > 86.0

More than 31 independent batches with the same synthesis process were analyzed and confronted to those specifications. Four parameters (λ max , Dh, ζ potential, µ EP ) are presented in Figure 2 as an example of control card obtained through the batch-to-batch survey. Horizontal plain lines represented the specification limits of each parameter. Among the 31 batches, 9 of them were out of specifications right after synthesis.

Respectively, λ max , Dh, ζ and CZE found 16 % (5 on 31), 13 % (4 on 31), 19 % (6 on 31) and 29 % (9 on 31)

batches to be out of conformity. This indicated that 3 out of 9 non-conform batches were only detected using CZE (batches 16, 18 and 25) indicating that classical methods may not be sufficient to determine AuNP conformity or long-term stability.

For each batch, the content percentage was calculated (Table 2). In absence of any certificate standard with known purity, in a first approximation and to compare the different batches, the assay corresponding to the main product percentage was determined by a classic normalization process (corrected area of the main peak versus the sum of all other peaks corrected area). According to ICH Q2(R1) recommendations (ICH-Q2(R1)) the limit of detection (LOD) was calculated as a Signal-to-Noise Ratio equal to 3. This LOD value represented 2.6 ± 0.5 % of the AuNP-Cit main peak. For all the 31 batches analyzed, the assay percentage was above the 86.0 % specification (data not shown). However, impurities were present. They may originate from either raw material and/or degradation products: uncapped or unfunctionalized gold core responsible for AuNP aggregation and non-homogeneous surface. Only neutral impurities were detected on the electrophoregrams. After synthesis, these impurities represented 2.7 ± 1.3 % of the synthesized AuNP-Cit (mean of the 31 different batches).

After developing quality control on our own AuNP-Cit, AuNP from other laboratories of Or Nano network (GDR-Or-Nano) were tested. The three first batches to be tested were AuNP-Cit of different sizes. All three batches (E, F and G) were received with some parameters being already measured (λ max and Dh mainly, Table 3). These parameters were compared to our own results. λ max were similar, on the contrary Dh values differed but this parameter is very dependent on the laser wavelength used (and also on other parameters such salt concentration, measurement duration, etc.). As batch E was received in very low volume from another laboratory, a complete evaluation comprising Dh and ζ potential measurements could not been performed.

A typical electrophoregram is presented in Figure 3. Detection was performed from 200 to 600 nm in order to monitor in the same experiment: possible different AuNP population (Surface Plasmon Band, around 520 nm) and organic impurity profile (at 214 nm). For batches E, F and G, only one peak was present at 214 and 520 nm.

This indicated the presence of a single AuNP population and the absence of organic impurity. CZE is an interesting tool helping to visualize the purity of newly synthetized batch and can be applied to different types of AuNP-Cit (size from 5 nm to 40 nm).

N.D.: Not determined

As synthesis of AuNP usually requires several purification steps to obtain the final product [START_REF] Alric | The biodistribution of gold nanoparticles designed for renal clearance[END_REF][START_REF] Gilles | Gold nanoparticles functionalization notably decreases radiosensitization through hydroxyl radical production under ionizing radiation[END_REF] , the impact of purification steps using CZE was monitored.

First, purification of AuNP-Cit (batch E) was investigated: electrophoregrams went from a complicated one with several peaks for raw suspension (Figure 4A) to a very pure one, showing only one AuNP population with no detected impurity in CZE after three cycles of ultracentrifugation (Figure 4B). We then evaluated our method with more complex AuNP: AuNP@DTDTPA. This synthesis implied steps of solvent removal and filtration.

AuNP@DTDTPA are designed for combining medical imaging (X-ray imaging owing to the gold core, magnetic resonance imaging (MRI) and nuclear imaging after immobilization of gadolinium ions or radioisotopes in the DTDTPA shell, respectively) and for radiosensitization [START_REF] Alric | The biodistribution of gold nanoparticles designed for renal clearance[END_REF][START_REF] Alric | Gadolinium chelate coated gold nanoparticles as contrast agents for both X-ray computed tomography and magnetic resonance imaging[END_REF][START_REF] Arifin | Trimodal gadolinium-gold microcapsules containing pancreatic islet cells restore normoglycemia in diabetic mice and can be tracked by using US, CT, and positive-contrast MR imaging[END_REF].

As demonstrated by previous in vivo experiments, AuNP@DTDTPA exhibit a high potential for MRI-guided radiotherapy [START_REF] Butterworth | Preclinical evaluation of gold-DTDTPA nanoparticles as theranostic agents in prostate cancer radiotherapy[END_REF][START_REF] Miladi | The in vivo radiosensitizing effect of gold nanoparticles based MRI contrast agents[END_REF]. Although extensive characterization of AuNP@DTDTPA has to be performed prior to in vivo administration, for this purpose, CZE could be of interest as it would provide crucial information about the quality grade of the suspension of theranostic nanoparticles before intravenous injection. Two different samples of AuNP@DTDTPA were analyzed. The first one was the raw suspension (AuNP@DTDTPA in aqueous sodium hydroxide solution at pH 8) which was prepared from a black powder obtained after filtration of the reaction mixture and rinsing the retentate successively with HCl, water and acetone. The second was prepared as for intravenous injection from the raw suspension which was used as starting material but with a final purification step. The resulting electrophoregrams are shown in Figures 4C and4D.

Before purification (Figure 4A) the sample presented 9 different peaks at 214 nm. The main one (peak n°8) was set with a relative migration time (RMT) of 1 (Table 4). Among those 9 peaks, 5 of them (peaks n°1, 3, 5, 6 and 8) were AuNP populations due to their absorbance at 520 nm. Purities were calculated by dividing the corrected area (CA) of the main peak by the sum of all other peak at the same wavelength. The sample presented a purity of 45.7 % considering the total impurity values (214 nm) or 32.6 % considering only nanoparticle populations (520 nm). Interestingly, sample purity was found lower at 520 nm than at 214 nm.

This was caused by the presence of peak n°6. Its CA was even higher than the one of the main peak, indicating that there were two major AuNP populations coexisting in the same sample. Those two populations were not detected through classical methods. DLS values of unpurified sample gave uncoherent values (Dh = 216.1 nm) certainly caused by a relatively important number of aggregates and impurities among the suspension. After purification only four peaks remained (Figure 4B). Two of them (peaks n°5 and 9) were attributed to organic impurities with no absorption at 520 nm. The other two peaks (peaks n°6 and 8) corresponded to two different AuNP populations. This led to a sample total purity of 90.4 % (normalized to the main peak at 214 nm) or a nanoparticle purity of 84.0 % (normalized to the main peak at 520 nm). Purity was respectively increased by 2.0 and 2.6 times through purification steps. CZE successfully assess the purity of a complex AuNP sample whereas classical methods give only hints of a single AuNP population. After purification steps, Dh of AuNP@DTDTPA was in accordance with expected values (9.1 nm) with a surface charge of -37.6 mV. However, these values were global results that prove neither the co-existence of several NP populations in the same sample, nor the presence of impurities.

In the end, in the presence of complex samples (in terms of populations/impurities), CZE proved its capacity to discriminate a mix of AuNP populations and showed the benefits of purification steps on two different AuNP in terms of size and chemical stabilization.

AuNP-Cit stability monitoring

Once purified and characterized, crucial questions about AuNP remains their stability and the long-term storage. A previous study has shown that AuNP-Cit remained stable for no more less than 20 days [START_REF] Pallotta | Characterization and stability of gold nanoparticles depending on their surface chemistry: Contribution of capillary zone electrophoresis to a quality control[END_REF]. This study was based only on 3 batches. Thus, this issue was more carefully addressed with 18 independent batches which were fully characterized just after synthesis (D0) and after 20 days (D20) of storage. Results are shown in Table 5. being obtained through classical methods and often a stability indication (up to 1 year in some cases). But the overall quality of AuNP is never evaluated. Indeed, as AuNP are not considered as active pharmaceutical ingredients, there is no impurity limit or stability requirements. Four commercially available AuNP-Cit batches were analyzed, under a single-blinded randomized trial, using both approaches described previously and results were compared to the provider's certificate of analysis.

Most of our results (Table 6) were in accordance with reported values on the certificate of analysis (ζ potentials for Batches #1 to #4 were not reported by suppliers). In parallel, these 4 batches were analyzed by CZE. The assay percentage, total organic impurities (at 214 nm) and nanoparticular impurities (other AuNP population at 520 nm) are presented in Table 7. Except for Batch #3 that presented a purity superior to 90 % with no other AuNP population, the other 3 batches displayed purity profiles inferior to 80 %. The low purity (76.8 %) of Batch #2 was only caused by a high organic impurity level (23.2 %). CZE electrophoregrams showed the presence of 3 distinct peaks at 214 nm. Those organic impurities probably derived the synthesis.

For the 2 batches left (#1 and #4), impurity profiles were composed of both organic and nanoparticular impurities. Batch #1 electrophoregrams were composed of 4 organic impurities and 2 other AuNP populations.

The same profile was found for Batch #4 (3 organic and 1 AuNP populations). Those AuNP populations could derive directly from a synthesis with an incomplete reaction or indicate of a loss of stability of the AuNP suspension forming aggregates. As these populations are present at low concentrations, classical methods do not detect them. Those results raised some questions concerning the quality of commercially AuNP. In the worst cases, up to 30 % of the sample was composed of AuNP different of the one expected (in terms of size, surface charge, surface chemistry …). One serious problem is the stability of suspensions. When buying AuNP, a large delay between synthesis and purchase usually occurs. In that case, characterizations made on the synthesis day can be different from the one performed at reception. If new AuNP populations are formed (as in Batches #1 and #3), their impact on experiments can affect the obtained results, leading to a lack of reproducibility or misinterpretation of results (reactivity, catalysis, functionalization, biological evaluation, …).

Conclusion

The more and more important use of nanoparticles as new therapeutic tools, considering the knowledge on their human toxicity, requires a strict control of them. As with other "conventional" drugs, all nano-objects should be submitted to a strictly control before use. In this paper, we showed the major interest of using a separation method (CZE) for evaluating AuNP quality. In the highly controlled pharmaceutical field, marketed products go through a relatively long regulatory process to secure their use and have to prove their added value in relation to what already exists on the market. For each pharmaceutical raw material (called ingredients), there is a specific monograph. This one establishes its exact definition (i.e. its identification), evaluates its quality including purity and limitation of impurities. However, to date, there is not any specific monograph for nanoparticles. Faced with their increasing use for pharmaceutical purposes, it seemed urgent and necessary to have nano-objects of utterly defined quality and meeting international standards and

procedures that yet remains to be defined.

  solvents were of analytical grade and used without further purification. Sodium borohydride, sodium citrate, dithiolated diethylenetriamine pentaacetic acid (DTDTPA), hydroxyethyl piperazineethanesulfonic acid (HEPES) , tetrachloroauric acid trihydrate (HAuCl 4 .3 H 2 O), potassium tetrachloaurate (KAuCl 4 ), HCl 1.0 M, and NaOH 1.0 M, acetonitrile, trifluoroacetic acid, tannic acid, silver nitrate, sodium borohydride (NaBH 4 ), fuming HCl, Rhodamine B and diisopropyl ether were supplied by Sigma Aldrich. Boric acid and NaOH pellets were purchased from Riedel-de-Haën. Ultrapure water (> 18.2 mΩ.cm)

2. 3 .

 3 Dithiolated Diethylenetriamine Pentaacetic Acid grafted AuNP (AuNP@DTDTPA) synthesis and purification For a typical preparation of AuNP@DTDTPA, 200 mg (510 µmol) of HAuCl 4 •3H 2 O, dissolved in 60 mL of methanol, were placed in a 250 mL round-bottom flask. 256 mg (500 µmol) of DTDTPA in 40 mL of water and 2 mL of acetic acid were added to the gold salt solution under stirring. The mixture turned from yellow to orange. After 5 min, 185 mg (4.89 mmol) of NaBH 4 dissolved in 14 mL of water were added to the orange mixture under vigorous stirring at room temperature. When adding NaBH 4 , the solution became dark brown then a black flocculate formed. The vigorous stirring was maintained for 1 h before adding 5 mL of 1.0 M HCl.

  FigureS1of the Supplementary Material attached file.

  3 M, 0.025 M respectively) for 20 min protected from light in LoBind Eppendorf microtubes (1.5 mL). The mixture was then partially evaporated using a Turbovap (Biotage) at 40 °C under N 2 gas for 20 min. Then, 200 µL of NH 4 Cl solution (30 % m/V), 100 µL of 6 M HCl and 100 µL Rhodamine B solution (0.084 µM) were added. The ion-pair formed between AuCl 4 -and Rhodamine B was then extracted in 200 µL of diisopropyl ether. 150 µL of the supernatant (organic phase) were withdrawn, completely evaporated (Turbovap, 40 °C, N 2 , 5 min) and the dried residues were dissolved into the mobile phase (acetonitrile/water/trifluoroacetic acid 25/75/0.1 V/V) before injection into the HPLC system.

Figure 1 Figure 2 Figure 3

 123 Figure 1

Figure 4

 4 Figure 4

Table 1 :

 1 Clinical Trials using AuNP extracted from https://clinicaltrials.gov/ using on the 2019 19 th of March with keyword AuNP. For more information on studies use the identifier number on https://clinicaltrials.gov/.

	Identifier	Study Title	Subject Conditions	Use
	NCT01436123	Plasmonic Photothermal and Stem Cell Therapy of Atherosclerosis Versus Stenting	Coronary Artery Atherosclerosis Disease	Injection
	NCT02837094	Enhanced Epidermal Antigen Specific Immunotherapy Trial -1	Type 1 Diabetes	Intradermal injection
	NCT03020017	NU-0129 in Treating Patients With Recurrent Glioblastoma or Gliosarcoma Undergoing Surgery	Gliosarcoma Recurrent Glioblastoma	Intravenous injection
			Stable Angina|Heart
	NCT01270139	Plasmonic Nanophotothermal Therapy of Atherosclerosis	Failure Multivessel Coronary Atherosclerosis	NP on patch
			Artery Disease
				Surface pre-
	NCT03669224	Effect of Nano Care Gold on Marginal Integrity of Resin Composite	Caries Class II	treatment of the resin by
				AuNP
	NCT02782026	Exhaled Breath Olfactory Signature of Pulmonary Arterial Hypertension	Pulmonary Hypertension	Sensor array with AuNP

Table 2 :

 2 Quality specifications defined for citrate stabilized AuNP, assay % refers to the main product content.

	Specifications	UV-vis/DLS/electrophoresis coupled to DLS	CZE
		λ max (nm)	Dh (nm)	ζ (mV)

Table 3 :

 3 Comparison between announced and controlled characteristics of lab-made AuNP-Cit. Announced concentrations were determined by UV-Vis spectrophotometric measurements and controlled ones using HPLC coupled to a UV-Vis detector.

			Announced Characteristics			Controlled Characteristics
		λ max
	Batch		Dh (nm)	ζ (mV)	Concentration (mM)	λ max (nm)	Dh (nm)	ζ (mV)	Assay (%)	Concentration (mM)
		(nm)
	E	524 ± 2	9.5 ± 1.8	N.D.	5.7 ± 0.5	523 ± 1	N.D.	N.D.	100.0 ± 2.5	5.3 ± 0.2
	F	516 ± 1	21.5 ± 2.7	N.D.	7.4 ± 0.7	516 ± 1	5.0 ± 1.0	-24.2 ± 3.1	100.0 ± 2.5	5.2 ± 1.1
	G	519 ± 1	34.0 ± 2.9	N.D.	10.6 ± 0.4	519 ± 2	38.8 ± 3.8	-52.3 ± 4.6	100.0 2.5	7.0 ± 0.9

Table 4 :

 4 Impurity profile of AuNP@DTDTPA by CZE analyzes after synthesis (Raw) and after purification steps (purified). The line in grey represents the main AuNP peak. RMT : Relative Migration Time; CA : corrected area

			AuNP@DTDTPA raw			AuNP@DTDTPA purified
			214 nm	520 nm		214 nm	520 nm
	Peak	RMT	CA	RMT	CA	RMT	CA	RMT	CA
	1	0.38	3490	0.38	647
	2	0.39	3103
	3	0.41	23412	0.41	5378
	4	0.59	43008
	5	0.65	6730	0.65	1013	0.63	13220
	6	0.71	95419	0.70	15647	0.73	28861	0.73	12847

Table 5 :

 5 Physicochemical characterization of AuNP-Cit using classical approaches and CZE after synthesis (Day 0) and 20 days of storage (Day 20). Values out of specifications after 20 days are marked in grey. Batch numbers correspond to the same batches than presented in Figure2. part 3.1 and show that CZE is a more powerful tool to monitor the stability of AuNP samples. Thus, if one wants to establish a gradation of the parameters in their capacity to analyze and discriminate AuNP batches according to specifications, the more powerful would be CZE, followed by ζ potential measurement, λmax and Dh being the least indicative of the AuNP colloidal state.3.3. Commercial batches analysisConsidering results obtained in part 3.2 in terms of stability or impurities, one could be skeptical about commercial AuNP-Cit. Commercially available AuNP-Cit are sold with a certificate of analysis. At best, it gives information on size (gold core or Dh), polydispersity, number of particles per mL, surface charge … all of this

	Day 0

Table 6 :

 6 Comparison of the characterization of commercially available AuNP-Cit by classical methods with the data from the provider.

	Commercial batch number	Results provenance	Core size (nm)	Dh (nm)	ζ potential (mV)	λ max (nm)
	#1	Provider This study	5 5	19 13	N.R. -34.5	521 521
	#2	Provider This study	5 5	19 7	N.R. -33.1	519 516
	#3	Provider This study	5.0 ± 0.6 5.0 ± 0.7	N.R. 7	N.R. -29.3	516 515
	#4	Provider This study	5.0 ± 0.6 5.0 ± 2.0	N.R. 7	N.R. -19.7	515 514
	N.R.: Not Reported

Table 7 :

 7 CZE analyzes of commercial AuNP-Cit.

Commercial batch number Assay (%) Organic impurities (%) Nanoparticular impurities (%)

	#1	79.8	6.1	14.1
	#2	76.8	23.2	0.0
	#3	90.5	9.5	0.0
	#4	68.3	11.6	20.1

Acknowledgments:

The authors thank Sayens (SATT French network) and Incubateur Lorrain for their financial supports and the GDR Or-Nano for its scientific support. The authors are grateful to Pr A. Le Faou for improving the English of the manuscript.

The present work has benefited from the CYTOMETRY / ELECTRONIC MICROSCOPY / LIGHT MICROSCOPY facility of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr), member of IBiSA (http://www.ibisa.net), supported by "France-BioImaging" (ANR-10-INBS-04-01), and the Labex "Saclay Plant Sciences" (ANR-10-LABX-0040-SPS).


Jonathan Bruniaux

Emilie Allard-Vannier

Nicolas Aubrey

Z Lakhrif

Ben Sanaa

Sahar Ben Djemaa

Hervé Eljack

Katel Marchais

Igor Hervé-Aubert

Stephanie Chourpa
email: stephanie.david@univ-tours.fr

David

S Eljack

H Marchais

K Hervé-Aubert

I Chourpa

S David

Magnetic nanocarriers for the specific delivery of siRNA: Contribution of breast cancer cells active targeting for down-regulation efficiency

Keywords: small interfering RNA (siRNA), superparamagnetic iron oxide nanoparticles (SPION), scFv anti-HER2, survivin, Abreviations nanoparticle, NP, single-chain antibody fragments, scFv, Stealth Fluorescent Particles, SFP, small interfering RNA, siRNA, Stealth Magnetic siRNA Nanovectors, S-MSN, superparamagnetic iron oxide nanoparticles, SPION, Targeted Stealth Fluorescent Particles, T-SFP, Targeted Stealth Magnetic siRNA Nanovectors, TS-MSN

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Breast cancer is the most common cancer and the leading cause of cancer mortality in women worldwide. In 2012, 25% of all cancers recorded worldwide are represented by new breast cancer cases [START_REF] Ferlay | Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012[END_REF]. Approximately 20% of breast cancer are caused by overexpression of the human epidermal growth factor receptor 2 (HER2) [START_REF] Anhorn | Specific targeting of HER2 overexpressing breast cancer cells with doxorubicin-loaded trastuzumab-modified human serum albumin nanoparticles[END_REF][START_REF] Slamon | Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer[END_REF][START_REF] Steinhauser | Trastuzumab-modified nanoparticles: optimisation of preparation and uptake in cancer cells[END_REF]. This HER2 overexpression is currently associated with more aggressive tumor behavior and poorer clinical outcomes [START_REF] Slamon | Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer[END_REF]. Treatment strategies for patients with HER2 positive advanced breast cancer have progressed significantly over the past few decades. The outcome of these patients has been improved with the introduction of multiple successful anti-HER2 therapies, including anti-HER2 monoclonal antibody (mAb, trastuzumab) [START_REF] Swain | Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer[END_REF][START_REF] Verma | Trastuzumab emtansine for HER2-positive advanced breast cancer[END_REF], antibody-drug conjugate (ado trastuzumab emtansine or T-DM1) and tyrosine kinase inhibitor (lapatinib). The market introduction of four trastuzumab biosimilars in 2018 underlines the great interest of this humanized monoclonal antibody for the targeting of HER2 receptors [START_REF] Santos | Biosimilar medicines used for cancer therapy in Europe: a review[END_REF]. However, many HER2-overexpressing breast cancer patients do not respond to anti-HER2 mAb treatment alone [START_REF] Marty | Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2positive metastatic breast cancer administered as first-line treatment: the M77001 study group[END_REF] and development of resistance and disease recurrence continue to be the major clinical challenges as it occurs in more than 50% of treated patients. These clinical problems underline the need of alternative therapeutic strategies combined to anti-HER2 therapies.

RNA interference strategy using small interfering RNA (siRNA) is an attractive and innovative strategy to inhibit targeted proteins involved in treatment resistance.

The first RNA interference drug based on siRNA, Patisiran, was approved by the US Food and Drug Administration in 2018 and other siRNA drugs are already used in clinical trials [START_REF] Kaczmarek | Advances in the delivery of RNA therapeutics: from concept to clinical reality[END_REF][START_REF] Ledford | Gene-silencing technology gets first drug approval after 20-year wait[END_REF]. However, their development as therapeutics is limited by several hurdles including (i) weak biostability as a consequence to nucleases digestion, (ii) poor uptake in cancer cells and, (iii) nonspecific biodistribution. Therefore, the effective, targeted and safe delivery of siRNAs across the cell membrane to the cytoplasm remains a main obstacle that could be overcome by the complexation of siRNA with a nanosized cargo called nanoparticle (NP) (Wang et al., 2017, Ben Djemaa et al., this issue).

Passive targeting is known as accumulation of NP at the tumor site at high concentrations due to the pathophysiological differences between normal tissues and tumor tissues (Enhanced Permeability and Retention (EPR) effect) [START_REF] Jee | Cancer targeting strategies in nanomedicine: Design and application of chitosan nanoparticles[END_REF].

In addition, active targeting is investigated through molecular recognition by conjugation of targeting moieties onto NP to obtain targeted delivery to specific cells, tissues or organs [START_REF] Chattopadhyay | Role of antibody-mediated tumor targeting and route of administration in nanoparticle tumor accumulation in vivo[END_REF][START_REF] Slavoff | Discovering ligand-receptor interactions[END_REF].

Cancer cells express different molecular targets (antigens and/or receptors) than do normal cells and tissues. While some of these molecules are down-regulated, others are either newly expressed or significantly up-regulated on the surface of target cells, thus offering possibility of targeting strategies [START_REF] Allen | Ligand-targeted therapeutics in anticancer therapy[END_REF]. Therefore, biological ligands such as antibodies are associated to nanovectors [START_REF] Tatiparti | siRNA Delivery Strategies: A Comprehensive Review of Recent Developments[END_REF]. For the NP targeting function, a single-chain variable fragment (scFv) of trastuzumab offers several advantages over the whole antibody, mainly lower immunogenicity due to its reduced size (~27 kDa compared to 150kDa for the whole mAb) [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF].

The purpose of this study was to develop Targeted Stealth Magnetic siRNA Nanovectors (TS-MSN) as a novel approach in breast cancer diagnosis and treatment to achieve maximal therapeutic benefit of anti-cancer siRNA. To combine targeted cancer imaging and therapy, we used superparamagnetic iron oxide nanoparticles (SPION) that can provide imaging ability through MRI. SPION were covalently conjugated to a near-infrared fluorophore, sulfocyanine 5 (sCy5), for optical imaging and to polyethylene glycol (PEG) for improved immune stealthiness (prolonged lifetime in blood). These Stealth Fluorescent Particles (SFP) were conjugated with anti-HER2 scFv of trastuzumab in order to target HER2overexpressing cells, such as SK-BR3 and BT-474 human breast cancer cells [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF]. Finally, these targeted SFP (T-SFP) were loaded with siRNA polyplexes (complexes with polycations such as chitosan and poly-L-arginine) using a protocol previously developed by our lab [START_REF] Bruniaux | Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics[END_REF]. In addition to enabling siRNA complexation, the polycations favore endosomal escape of siRNA in order to reach its cytoplasmic targets [START_REF] Bruniaux | Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics[END_REF]. The therapeutic target aimed in this study was survivin, a chemotherapy-induced anti-apoptotic gene. Survivin is highly and selectively expressed in a majority of human cancers, including breast cancer, representing a potential biomarker [START_REF] Jha | Survivin expression and targeting in breast cancer[END_REF][START_REF] Lv | The role of survivin in diagnosis, prognosis and treatment of breast cancer[END_REF][START_REF] Nassar | Survivin expression in breast carcinoma: correlation with apoptosis and prognosis[END_REF]. In addition, high survivin expression is correlated with chemo and radioresistance in multiple tumor types whereas low expression enhances cell death [START_REF] Guan | Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity[END_REF][START_REF] Kunze | siRNA-mediated inhibition of antiapoptotic genes enhances chemotherapy efficacy in bladder cancer cells[END_REF]. In this way, survivin down-regulation through siRNA specific delivery could be considered as a therapeutic approach.

The final Targeted Stealth Magnetic siRNA Nanovectors (hereafter called TS-MSN) and the control non-targeted Stealth Magnetic siRNA Nanovectors (S-MSN) were both characterized and evaluated in vitro, in terms of their ability to down regulate survivin in HER2+ (SK-BR3 and BT-474 cell lines) and HER2-(MDA-MB-231 cell line) breast cancer models. In particular, we investigated the HER2 protein binding, their cellular uptake in HER2+/HER2-cells the siRNA transfection potential.

The latter was determined using anti-GFP siRNA delivery to the GFP-producing cells or anti-survivin siRNA delivery to the non-fluorescent cancer cells.

Materials and methods

Materials

NHS-PEG-Maleimide (NHS-PEG-Mal, Mw 5000 Da) and sulfocyanine 5 NHS ester were obtained from Rapp Polymer GmbH (Tübingen, Germany) and Lumiprobe (Hannover, Germany) respectively.

Poly-L-arginine (MW 15.000-70.000) and high purity chitosan (MW 110.000-150.000), used for S-MSN and TS-MSN formulation, were provided by Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany).

For physico-chemical characterization, model siRNA (targeted against PCSK9, sense sequence GGAAGAUCAUAAUGGACAGdTdT with lower case letters representing deoxyribonucleotides) were provided by Eurogentec (Angers, France). Poly-Larginine (MW 15.000-70.000) and high purity chitosan (MW 110.000-150.000; degree of acetylation: ≤ 40 mol. %), used for MSN and S-MSN formulation, were from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). For transfection assay, commercial transfection reagent Oligofectamine ® and siRNA control were purchased from Life Technologies (Paisley, UK). siRNA against survivin was purchased from Sigma Aldrich Chemie GmbH (St. Quentin Fallavier, France, sense sequence GUCUGGACCUCAUGUUGUUdTdT with lower case letters representing deoxyribonucleotides). For gel retardation assays, loading buffer, agarose and ethidium bromide were purchased from Fisher Bioreagents® (Illkirch, France). All the culture media and supplements for cell culture were supplied by life technologies (Paisley, UK).

Nanocarriers preparation

Stealth fluorescent nanoparticle (SFP) and Targeted SFP (T-SFP) synthesis

T-SFP were synthesized using previously developed protocols. Briefly, SPIONs obtained using the Massart protocol were first silanized [START_REF] Hervé | The development of stable aqueous suspensions of PEGylated SPIONs for biomedical applications[END_REF]. Then, silanized SPION were labelled with the fluorochrome sulfocyanine 5 NHS and functionalized with PEG leading to SFP [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF][START_REF] Perillo | Synthesis and in vitro evaluation of fluorescent and magnetic nanoparticles functionalized with a cell penetrating peptide for cancer theranosis[END_REF]. In parallel, scFv anti HER2 were produced in E.coli and purified using affinity chromatography [START_REF] Alric | Covalent conjugation of cysteine-engineered scFv to PEGylated magnetic nanoprobes for immunotargeting of breast cancer cells[END_REF]. Finally, SFP were functionalized with purified scFv leading to T-SFP [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF][START_REF] Alric | Covalent conjugation of cysteine-engineered scFv to PEGylated magnetic nanoprobes for immunotargeting of breast cancer cells[END_REF]. SFP as control were treated in the same conditions (reaction and purification conditions) as T-SFP.

Stealth magnetic siRNA nanovector (S-MSN) and targeted S-MSN (TS-MSN)

preparation TS-MSN were prepared based on a protocol previously described by Bruniaux et al. [START_REF] Bruniaux | Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics[END_REF]. Briefly, siRNA were pre-complexed with poly-L-arginine (PLR), then added to a suspension containing T-SFP and chitosan. The amount of T-SFP (quantified by its iron content) was defined as iron/siRNA mass ratio and fixed at 10. Chitosan and PLR content were expressed by the charge ratio (CR) of positive polymer charges to negative siRNA charges. CR of chitosan/siRNA (CRCS) and CR of PLR/siRNA (CRPS) were respectively set to 30 and 10. S-MSN, as control, were prepared in the same conditions using SFP instead of T-SFP.

Nanocarriers' characterization

Size and zeta potential measurements

The mean hydrodynamic diameter and zeta potential of S-MSN and TS-MSN in suspension were determined using a Malvern Nanosizer ZS (Malvern Instruments, Malvern, UK). Before measurement, the S-MSN and TS-MSN preparations were diluted in NaNO3 0.01M at a ratio 1:25 to obtain a constant ionic strength (pH=5.6) (n=3).

siRNA protection assay

To analyze the siRNA protection, S-MSN and TS-MSN (at an initial siRNA concentration of 2.5 µM) were incubated with an aqueous ribonuclease A solution (1.2µg/ml, Sigma-Aldrich, Chemie GmbH, Schelldorf, Germany) at a ratio of 2:1 for 8 to 24 hours at 37°C. Afterwards, ribonuclease A was inactivated by heating the suspensions at 70°C for 30 min. An equivalent amount of free siRNA was incubated for 30 minutes and used as a positive control to check the ribonuclease A activity.

To analyze the amount of free siRNA, samples were diluted in water (Milli-Q system, Millipore, Paris, France) or aqueous heparin sodium solution (10mg/ml, Sigma-Aldrich, Chemie GmbH, Schnelldorf, Germany) and mixed with loading buffer (Agarose gel loading dye 6X) in order to deposit 20mol of siRNA per well on 1% agarose gel containing ethidium bromide. With its strong negative charge, heparin is used as control to displace complexed siRNA from S-MSN or TS-MSN. A 100 V voltage was applied for about 15 min in a Tris/acetate/EDTA buffer (TAE 1X, 40 mM acetate, EDTA 1 mM, pH 7.6). Gels were visualized and analyzed with EvolutionCapt software on a Fusion-Solo.65.WL imager (Vilbert Lourmat, Marne-la-Vallée, France).

Antigen-binding analysis through ELISA assay

The functionality of SFP in S-MSN formulation, and functionalized SPION (T-SFP) in TS-MSN formulation, were checked by indirect enzyme-linked immunosorbent assays (ELISA) using the HER2 protein (Sino Biologicals, Beijing, P. R. China.) as a target. scFv fragments were detected by protein L-peroxidase in the presence of chromatic substrate, 3, 30, 5, 50-tetrame-thylbenzidine (TMB), through the high affinity of protein L to the κ light chain of scFv. Briefly, HER2 protein was coated in a 96-well plate at 1 µg/mL and incubated at 37°C during 1 h. The wells were then saturated with 3% BSA-PBS for 1 h at 37°C and washed with PBS prior to incubation with an increasing concentrations of S-MSN and TS-MSN (ranging from 0 to 50 mg/L iron, 0 to 376 nM siRNA) during 1 h at 37°C. Wells were then washed and incubated with 100 µL/wells of protein L-peroxidase (Life Technologies) for 1 h at 37°C before addition to TMB substrate (Sigma-Aldrich). Enzymatic reactions were stopped with the addition of 1M H2SO4 and the absorbance was measured at 450 nm using a microplate reader (Biotek). Wells coloration correlated to the presence of scFv and the absorbance at 450 nm was then proportional to scFv content.

Immunofluorescence imaging

SK-BR3 cells, overexpressing HER2, grown on cover glasses were fixed in 4% paraformaldehyde solution for 20 min at room temperature. The cover glasses surface was saturated with a 20% fetal calf serum solution in PBS for 1 h at 37°C. The fixed cells were then incubated with 40 µL of PBS, S-MSN or TS-MSN (at an iron concentration of 267 mg/l which is equivalent to a siRNA concentration of 2000nM) all day night at 4°C in a humidified chamber box. The cover glasses were washed three times with PBS and incubated for 1h at 37 °C with protein-L-FITC (ACROBiosystems, Newark, USA) at 5 µg.mL -1 for 1h at 37 °C. Cells were finally washed with PBS and placed between slide and slip cover with 10µL of Fluoromount G ® mounting medium. Observations were then made with a fluorescent inverted microscope (Olympus, IX51).

In vitro evaluation

Cell culture

BT-474 and SK-BR3 human breast carcinoma cell lines with HER2 overexpression were purchased from Cell Lines Service (CLS Eppelheim, Germany). BT-4747 cells grow in compact, slowly growing multilayered colonies which rarely become confluent and are tumorigenic in mice. In contrast, SK-BR3 cells form monolayer colonies but are not tumorigenic in mice. MDA-MB231-GFP (Euromedex, Souffelweyersheim, France), MDA-MB231 (ECACC, Salisbury, UK) cell lines were used as negative controls as they express a low level of HER2 receptors [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF]. SK-BR3 cells were grown at 37°C/5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. BT-474 cells were grown (37°C/5% CO2) in DMEM:Ham's F12 (1:1 mixture) supplemented with insulin 1X, 10% fetal bovine serum and 1% penicillin/streptomycin. MDA-MB231 cells expressing or not GFP were routinely cultured in DMEM supplemented with 10% fetal calf serum, non-essential amino acid 1X and 1% penicillin/streptomycin. Culture medium was changed every 48 hours and the cells were harvested using trypsin as soon as 80% confluency was reached.

Internalization studies

2.4.2.1. Flow cytometry 24h before transfection, 25.10 3 BT-474 cells (over-expressing HER2 receptor)/wells were seeded in a 12-well plate. Transfections of 20 nM siRNA were carried out by adding a suspension of (T)S-MSN diluted in DPBS into 12-well plate containing equal parts OptiMEM serum-free medium and culture medium conventionally used for this cell line. Cells were treated with the prepared suspensions and maintained in normal growth conditions for different time points from 2 to 48h.Cells were analyzed using a FACSCalibur flow cytometer (BD Bioscience, Franklin Lakes, NJ). At least 10 4 events were collected and analyzed using the WinMDI 2.9 software.

Confocal spectral imaging (CSI)

For multispectral confocal imaging, the analysis of nanocarrier distribution was performed on cell-adherent cover slips. Cover glasses treated with poly-D-lysine were placed in 24-well plates. They were seeded with BT-474 cells and placed for 48 h in culture medium. The cells were then incubated with S-MSN and TS-MSN at an iron concentration of 2.66 mg/L (= 20nM siRNA concentration) for 24 h and washed three times with PBS. The cover glasses were then placed between a microscope slide and a cover slip to be scanned for CSI using a LabRAM laser scanning confocal microspectrometer (Horiba SA, Villeneuve d'Ascq, France), equipped with a 300 '/mm diffraction grating and a CCD detector air-cooled by Peltier effect. The sulfocyanine fluorescence was excited using a 633 nm line of a built-in He-Ne laser, under a 50 ᵡ long focal microscope objective. The laser light power at the sample was approximately 0.1 mW and the acquisition time was 20 ms per spectrum. For the analysis of adherent cells, an optical section (x-y plane) situated at half-thickness of the cell was scanned with a step of 0.8 μm that provided maps containing typically 2500 spectra. Both acquisition and treatment of multispectral maps were performed with LabSpec software version 5.

Co-culture experiment

For co-culture experiment 30.10 3 cells/wells and 10 4 cells/wells were respectively seeded for BT-474 and MDA-MB231/GFP considering their growth difference.

Transfections of 20 nM siRNA were carried out by adding a suspension of (T)S-MSN diluted in PBS into 12-well plate containing equal parts OptiMEM serum-free medium and culture medium conventionally used for these cell lines. GFP expression of MDA-MB231 promote their discrimination with flow cytometry (FL-1 canal) in order to differentiate (T)S-MSN interaction with each cell line. Cells were treated with the prepared suspensions and maintained in normal growth conditions for different time points from 2 to 48h. Cells were analyzed using flow cytometry as described above (n = 5).

Efficacy studies

siRNA transfection

24h before transfection, 25.10 3 cells/well were seeded in a 12-well plate. The day of transfection, TS-MSN (at an initial siRNA concentration of 0.25 µM) were prepared and diluted in a mix of OptiMEM serum-free medium and the culture medium conventionally used for the studied cell line (1:1 v/v) in order to obtain a final siRNA concentration of 20nM. S-MSN and Oligofectamine™ (Invitrogen, Thermo Fisher Scientific, Paisley, UK) were used as controls and prepared according to the protocol described above and the manufacture recommendation, respectively. Cells were treated with the prepared suspensions and maintained in normal growth conditions for 72 h.

To validate the down-regulation functionality of siRNA formulated in TS-MSN, transfection was performed on MDA-MB231-GFP cells with anti-GFP siRNA which specifically down-regulate GFP protein expression. For statistical analysis, the level of GFP expression was determined by flow cytometry analysis as described above.

To determine the down-regulation efficacy of our target protein survivin, BT-474 cells (overexpressing HER2 receptor) and MDA-MB231 (control) cells were transfected with TS-MSN containing anti-survivin siRNA as described above. The level of survivin protein expression was determined by Western Blotting as described below.

Western Blot (WB) -

After transfection of siRNA down-regulating survivin with the different nanocarriers for 72 h, transfected cells were washed with cold PBS and total proteins were extracted on ice by RIPA supplemented with anti-protease (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany). After 15 min centrifugation (10 000 G, 4°C) to collect supernatant, protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Bio-rad, Hercules, CA). The cell lysate (30 µL protein for each sample) was boiled for 5 min in SDS sample buffer and subjected to SDSpolyacrylamide gel electrophoresis (PAGE). The proteins are transferred to nitrocellulose membrane by a transfer using iBlot system. After blocking with 5 % nonfat milk at room temperature for 1 h, the membrane was incubated with the primary antibody against survivin (anti-rabbit, 1:1000, Life technologies), or glyceraldehyde-3-phosphate dehydrogenase GAPDH (anti-rabbit, 1:1000, Life technologies) at 4°C overnight. After incubating with the peroxidase conjugated secondary antibody (HRP goat to rabbit, 1:1000, Life technologies), the protein was visualized using an enhanced chemiluminescence (ECL) kit (Thermo Pierce) on a Fusion-Solo.65.WL imager (Vilbert Lourmat, Marne-la-Vallée, France) using EvolutionCapt software.

Statistical analysis

Data are expressed as mean ± SD of the variables and are compared among groups by using one-way ANOVA followed by Fisher's protected Least Significance Difference test calculated with GraphPad Prism7 software.

Results and Discussion

Formulation and physico-chemical characterization of targeted stealth magnetic siRNA nanovectors

Targeted Stealth Magnetic siRNA Nanovectors (called TS-MSN) were formulated combining two previously developed protocols: the synthesis protocol of Targeted Fluorescent Particles (T-SFP, [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF]) and the formulation protocol of Stealth Magnetic siRNA Nanovectors (S-MSN, [START_REF] Bruniaux | Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics[END_REF]). Briefly, T-SFP, siRNA and the cationic polymers (chitosan and poly-L-arginine) were mixed together in well-defined ratio to self-assembly via electrostatic interactions and form TS-MSN.

HER2-targeting scFv incorporation into S-MSN formulation using T-SFP led to an

increase in physico-chemical characteristics: the hydrodynamic diameter of formulated TS-MSN doubled almost compared to S-MSN to attain about 160nm for TS-MSN and the zeta potential value of the formulation in aqueous buffer tripled almost to attain about +17mV for TS-MSN (Table 1). Both have an acceptable polydispersity index for self-assembled nanocarriers, i.e. below or around 0.3. Even if the formulation process is easy, the formulation parameters have to be carefully chosen and optimized to obtain reproducible physico-chemical parameters. Results

showed that the modification of SFP with HER2 antibody fragment did not affect the formulation parameters and led to reproducible sizes and zeta potential. Despite the increase in size and zeta potential for TS-MSN compared to S-MSN, they are still acceptable considering a future intravenous administration as the size is below 200nm and the zeta potential below 20mV in aqueous buffer (pH5.6).

To verify the protection of siRNA by our nanocarriers against enzymatic degradation, a gel electrophoresis experiment using RNAse A was performed. An aqueous siRNA solution (control) and samples of S-MSN or TS-MSN were incubated in presence and absence of RNAse A and/or heparin for 30min, 8h or 24h (Fig. 1). Heparin was added as control to liberate siRNA of the nanocarriers and to visualize complexed siRNA. Unprotected siRNA is completely degraded within 30 minutes by RNAse A as no fluorescence band is visible. In absence of RNAse A and heparin, the fluorescence intensity of free siRNA is similar in the lanes containing the control solution and in the lanes containing S-MSN (Fig. 1A) and TS-MSN (Fig. 1B). In contrast, in absence of RNAse A and heparin, no fluorescence intensity is visible for S-MSN and TS-MSN indicating complete retention of siRNA in the nanocarriers. After 8 and 24h incubation, in presence and absence of RNAse A, samples with heparin show the same fluorescence intensity of free siRNA for S-MSN and TS-MSN indicating no siRNA degradation up to 24h. These results are consistent with previous published work [START_REF] Abdelrahman | siRNA delivery system based on magnetic nanovectors: Characterization and stability evaluation[END_REF][START_REF] Bruniaux | Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics[END_REF] indicating that the modification of the SPION core with HER2-targeting scFv antibodies did not alter the siRNA protection.

To validate the specific HER2 receptor recognition by TS-MSN, an ELISA assay was performed on an immobilized recombinant protein. Results are presented in Fig. 2A.

With S-MSN, there is no absorbance measured for iron concentrations up to 8 mg/L. For iron concentrations greater than 8 mg/L the absorbance increase slightly (absorbance of 0.38 for 50 mg/L iron). This increase is related to the absorbance of the SPION and not to the association with HER2. In contrast, with TS-MSN, the absorbance start to increase for iron concentrations about 0.8 mg/L. At iron concentration of 2mg/L the absorbance is about 1 indicating a high receptor recognition. The absorbance increase in correlation with iron concentration up to a maximum absorbance for iron concentration of 50 mg/L (absorbance about 1.7), displaying the specific recognition of HER2 receptor with formulations containing HER2 antibody fragments. These results show that TS-MSN containing HER2targeting scFv were efficiently bound to immobilized HER2 receptor while S-MSN demonstrate no association with HER2 receptor In parallel, immunofluorescence images were realized after the incubation of S-MSN and TS-MSN with SK-BR3 cells overexpressing HER2 receptors located at the plasmic membrane [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF]. HER2-targeting scFv were recognized by the specific interaction between κ light chain of scFv and FITC-labeled protein L (PpL-FITC) and are represented by a green fluorescence in the images (Fig. 2B). Samples treated with TS-MSN showed an intense green fluorescence distribution on the plasma membrane demonstrating the presence of antibody fragments interacting with the HER2 proteins. In contrast, samples treated with S-MSN showed only low green fluorescence on the plasma membrane. These results are in accordance with previous results obtained with T-SFP [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF]. Thus, the immunofluorescence images proved that the anti-HER2 targeting with TS-MSN is still efficient even if the presence of siRNA and cationic polymers added to T-SFP These results confirmed that TS-MSN have appropriated physico-chemical characteristics for systemic administration and that the combination of the two protocols did not affect the characteristics of the initial properties: siRNA protection and HER2 receptor recognition.

The improved internalization through active targeting

The specific recognition of HER2 receptor with TS-MSN should improve the specific distribution inside cancer cells overexpressing this receptor. The results of internalization experiments on BT-474 cell line over-expressing HER2 receptor using flow cytometry are represented in Fig. 3A. Results showed that TS-MSN did not induce more interaction with cells for incubation time below 4 hours compared to S-MSN. Nevertheless, whereas S-MSN fluorescence with cells remained constant above 24 hours incubation, TS-MSN fluorescence continued to increase displaying a better interaction with this cell line until 48h. After 24 and 48 hours incubation, the amount of TS-MSN found inside the BT-474 cells is respectively 1.4 and 2.2 times higher compared to the amount of S-MSN in the same cell line. This result was confirmed and completed with multispectral confocal imaging. After 24 hours incubation, TS-MSN had a massive internalization into cytoplasmic compartment with a homogenous distribution (Fig. 3B). T-SFP (red) and siRNA-alexa488 (green) were represented with high intensity signal suggesting that the nanovectors allow the delivery of siRNA inside the cells with high efficacy. In the same conditions with S-MSN, despite an accumulation into cytoplasmic compartment, the intensity signal remained weaker confirming the contribution of active targeting with TS-MSN.

To ensure the privileged TS-MSN distribution into cell lines overexpressing HER2 receptor, internalization studies on cellular co-culture were performed (Fig. 4). S-MSN and TS-MSN were incubated for increasing incubation times into a mixed culture of MDA-MB231/GFP (HER2-) and BT-474 (HER2+) cells. The different cell lines were discriminated through the green fluorescent protein (GFP) expression of MDA-MB231/GFP in order to compare nanocarrier interactions with cells depending on their HER2 receptor expression (Fig. 4A).

For TS-MSN, the fluorescence signal increased significantly over time in MDA-MB231/GFP cells up to 4h (p < 0.001) with a threshold between 4 and 24h and a slight increase between 24 and 48h (p< 0.01). In BT-474 cells, the fluorescence signal increased continuously between 2 and 48h but without any threshold (p < 0.001 up to 24h and p < 0.01 between 24 and 48h) (Fig. 4B). These results displayed more interaction between cells overexpressing HER2 receptors and nanocarriers with active targeting (TS-MSN). In contrast, with S-MSN the fluorescence signal increased significantly in both cell lines between 2 and 24h with a threshold after 24h (p < 0.001 for all points except at 24h for MDA-MB231/ GFP cells where p < 0.01), indicating that the entry of S-MSN inside both cell lines is similar.

However, at all time points the fluorescence signal intensity measured on cells incubated with TS-MSN is significantly higher than that measured on cells incubated with S-MSN indicating more interaction between TS-MSN with cells. The difference between TS-MSN and S-MSN was the same in both cell lines after 2h incubation (3.4 times). This phenomenon can be explained by the highly positive zeta potential value of TS-MSN compared to S-MSN allowing stronger interaction with the plasmic membrane (Table 1). However, this difference appeared to be higher after longer incubation: a fluorescence intensity 4 times higher for 4h incubation, and respectively 3.2 and 3.7 times higher for 24h et 48h indicating a more pronounced internalization of TS-MSN in BT-474 thanks to the overexpression of the HER2 receptor.

In summary, TS-MSN containing HER2-targeting scFv showed (i) the specific HER2 receptor recognition in BT-474 cells while S-MSN demonstrated no visible distinction between both cell lines, (ii) an enhanced internalization in BT-474 (HER2+) cells compared to MDA-MB231/GFP cells for long term incubation (> 24 hours).

These results are in accordance with our previous study performed on T-SFP [START_REF] Alric | Targeting HER2-breast tumors with scFv-decorated bimodal nanoprobes[END_REF] and with other studies that have previously reported the improved delivery of nanocarriers to the tumor sites by conjugation of HER2 antibody to the delivery carriers. Kievit et al. developed multifunctional superparamagnetic iron oxide nanoparticles containing trastuzumab antibody for active targeting and displayed the efficient targeting effect on HER2 expressing mouse mammary carcinoma (MMC) cells in vitro and in vivo in a transgenic mouse model [START_REF] Kievit | Targeting of primary breast cancers and metastases in a transgenic mouse model using rationally designed multifunctional SPIONs[END_REF]. In this study on HER2 expressing MMC, nanocarriers (50 µg/mL nanoparticles) containing trastuzumab antibodies led to an internalization two times higher after 2 hours of treatments compared to non-functionalized nanoparticles [START_REF] Kievit | Targeting of primary breast cancers and metastases in a transgenic mouse model using rationally designed multifunctional SPIONs[END_REF]. In the same way, Choi et al. described that the extent of cellular uptake of anti-HER2 antibody-conjugated iron oxide nanoparticles (150 µg/mL iron) was approximately 5 times higher in HER2-positive cells than in HER2-negative cells at all time points [START_REF] Choi | Targeted antitumor efficacy and imaging via multifunctional nano-carrier conjugated with anti-HER2 trastuzumab[END_REF]. Despite a considerably lower iron concentration (ca. 2.67 µg/mL), our study reports an impact of active targeting on HER2-overexpressed BT-474 cells after comparison with MDA-MB231/GFP cells.

The impact of active targeting on down-regulation efficiency after siRNA transfection

To verify the down-regulation efficiency of TS-MSN, flow cytometry experiments were performed on a MDA-MB231/GFP model using nanocarriers formulated with siRNA against GFP (Fig. 5A). Results demonstrated GFP down-regulation efficiency about 60% after siRNA transfection with TS-MSN, at the same level than S-MSN or commercial lipoplex formulation, Oligofectamine ® . These results are in accordance with previous obtained results for S-MSN [START_REF] Bruniaux | Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics[END_REF].

To check the down-regulation efficiency of survivin by TS-MSN, Western Blot experiments were performed on MDA-MB231 cells using nanocarriers formulated with siRNA against survivin (Fig. 5B). Results demonstrated survivin down-regulation efficiency about 70% after siRNA transfection with TS-MSN, at a similar level as S-MSN. Surprisingly, commercial lipoplex formulation, Oligofectamine ® inhibited the survivin expression only about 15%.

These results indicate that modification of SFP by antibodies fragments grafting to obtain T-SFP, in order to upgrade S-MSN to TS-MSN, did not alter the downregulation efficiency on this HER2 negative model.

To verify the impact of the observed active targeting of TS-MSN on the specific down-regulation, transfection on BT-474 (HER2+) cells was performed in order to down-regulate survivin expression. After 72 hours transfection of siSurvivin with Oligofectamine ® , S-MSN and TS-MSN, survivin protein level was analyzed through western-blot and compared to transfection with siControl (Fig. 5C). As expected, transfection with siControl did not provide any survivin protein down-regulation. As in the previous experiment on MDA-MB231 cells, siSurvivin transfection with Oligofectamine ® enabled only about 15% survivin down-regulation whereas S-MSN induced about 70% down-regulation in the same conditions. With active targeting allowed by TS-MSN, survivin down-regulation was improved to about 90% demonstrating a direct contribution of HER2-targeting scFv integration in the formulation. Thus TS-MSN provide efficient down-regulation efficiency of survivin on BT-474 cells in vitro.

The study was performed with siRNA targeting GFP and survivin showing significant protein down-regulation and indicating the adaptability of the delivery system to many target genes. Additionally, results show that the functionalization of T-SFP with scFv did not disturb the optimized formulation protocol of S-MSN indicating the adaptability of the delivery system to other cell types.

The correlation between selective targeting and down-regulation efficiency was also demonstrated by Jiang et al.: siRNA transfection (from 10 to 40 nM) with e23sFv-9R protein, allowing specific HER2 recognition, induced significant CXCR4 expression decrease in BT-474 cells (HER2+) whereas no obvious change in MDA-MB231 cells (HER2-) was observed [START_REF] Jiang | Targeted delivery of CXCR4-siRNA by scFv for HER2(+) breast cancer therapy[END_REF]. Despite the difference in both delivery system and siRNA target, the same behavior were observed with the specific antibody fragment grafting. Such a correlation was also observed with other targeting ligands on other cell types using nanovectors more similar to ours. For example, Veiseh et al. used SPION coated with PEG-grafted chitosan and PEI which were functionalized with siRNA and the tumor-targeting ligand, chlorotoxin (CTX). They showed that CTX-targeted nanovectors were internalized by C6 tumor cells 2-fold more than untargeted nanovectors and that the gene knock-down was correlated (35% reduction in GFP expression with non-targeted nanovectors vs 62% with CTXtargetd nanovectors) [START_REF] Veiseh | Chlorotoxin bound magnetic nanovector tailored for cancer cell targeting, imaging, and siRNA delivery[END_REF]. However, to achieve this down-regulation efficiency, they have to use 7.5 times more siRNA, compared to the present study.

Another example is the publication of Yang et al. who functionalized their SPION via electrostatic absorption of PEI and Gal-PEG-NH2 (using galactose as targeting ligand) and loaded the obtained Gal-PEI-SPION with siRNA via electrostatic interactions. They emphasized the importance of nanoparticles protecting cargo siRNA from nuclease degradation during in vivo siRNA delivery. Their serum stability study showed no siRNA degradation up to 48h. Furthermore, cy5-siRNA loaded Gal-PEI-SPIO nanoparticles were still observed with strong fluorescence intensity 24h after intravenous injection into C57BL/6 tumor-bearing mice showing the successful complexation of the siRNA into Gal-PEI-SPIO nanoparticles with high protection efficiency. They showed that the tumor volume, the liver/body weight ratio and the mRNA levels were significantly reduced using siRNA against c-Met compared to a negative control siRNA after repeated intravenous administration in an orthotopic hepatocellular carcinoma mouse model [START_REF] Yang | Superparamagnetic iron oxide nanoparticles modified with polyethylenimine and galactose for siRNA targeted delivery in hepatocellular carcinoma therapy[END_REF]. However, they did not compare their formulation with a non-targeted formulation.

The main advantage of using SPION as delivery system is related to their theranostic properties, i.e. their possible monitoring using imaging techniques such as MRI thus allowing to combine their diagnostic and their therapeutic functions. The above cited examples and the promising results presented here (physico-chemical characteristics of the nanocarriers compatible with an intravenous administration, siRNA protection against nuclease degradation up to 24h, active targeting and efficient downregulation in vitro) encourage us to continue towards in vivo preclinical assays. Moreover, in vivo, active cancer targeting through HER2 recognition, combined to an enhanced permeability and retention (EPR) effect in tumor environment, should promote specific biodistribution and improved down-regulation efficiency compared to control non-targeted nanovectors.

Conclusion

In this study, Targeted Stealth Magnetic siRNA Nanovectors (TS-MSN), were developed as alternative to existing HER2 cancer therapies in the objective to achieve a maximal therapeutic benefit. Therefore T-SFP functionalized with trastuzumab scFv (targeting HER2) were combined with S-MSN (for efficient siRNA delivery) and loaded with siRNA targeting survivin (highly expressed in cancers such as HER2 breast cancer). The main conclusions of our developed delivery system are that: 1) the formulation is rapid and simple 2) this delivery system can be adapted to many target genes, 3) the transfection efficacy is very high even if the nanovector is not targeted (S-MSN), 4) the active targeting with anti-HER2 antibody fragment increased the uptake of TS-MSN into the cells and especially in HER2 overexpressing cells, 5) the gene silencing effect of anti-survivin siRNA is even stronger for TS-MSN on cells that overexpressed the HER2 receptor.

Fig. 1 .

 1 Figure captions:

Fig. 2 .

 2 Fig. 2. HER2 protein recognition of S-MSN and TS-MSN. A: Indirect ELISA test of the immunoreactivity of TS-MSN (red curve) and S-MSN (green curve). B: Immunofluorescence images of SK-BR3 breast cancer cells incubated with TS-MSN and S-MSN (detection with PpL-FITC) and white light images to visualize the cells.

Fig. 3 .

 3 Fig. 3. Uptake of TS-MSN and S-MSN in BT-474 (HER2+) cells . A: Uptake kinetics of TS-MSN (red curve) and S-MSN (green curve). B: Confocal spectral imaging data showing the fluorescence of the sulfocyanine labelled nanosystems (red) and the Alexa-488 labelled siRNA (green) after 24h of incubation. Representative spectra of both fluorochromes (emission maximum: 525 for Alexa-488 and 710 nm for Sulfocyanine-5) are shown below.

Fig. 4 :

 4 Fig. 4 : Uptake kinetics of TS-MSN and S-MSN in a co-culture of BT-474 and MDA-MB231-GFP cells. A. Schematic representation of the co-culture and cytometry separation of the two cell lines according to green fluorescence intensity. B. Representation of the sulfocyanine fluorescence ratio found in BT-474 versus MDA-MB231-GFP cell lines after incubation of TS-MSN (left) and S-MSN (right) according time (2 -48h). *: p<0.05; **: p<0.01; ***: p< 0.001 correspond to the comparison between MDA-MB231/GFP and BT-474 for a same incubation time. ##: p<0.01; ###: p<0.001 correspond to the comparison with the previous incubation time for the same cell line.

Fig. 5 .

 5 Fig. 5. Down-regulation efficiency of S-MSN and TS-MSN. A. GFP fluorescence expression in MDA-MB-231/GFP cells either untreated (Cell MDA-MB-231/GFP) or treated with oligofectamine ® , S-MSN or TS-MSN formulated with anti GFP siRNA. B. Western Blot membranes of survivin expression in MDA-MB-231 cells either untreated (Cell MDA-MB-231) or treated with oligofectamine ® , S-MSN or TS-MSN formulated with anti-survivin siRNA. C. Survivin down-regulation in BT474 cells. Western Blot membranes of survivin expression in BT474 cells either untreated (Cell

Table 1 .

 1 Physico-chemical characteristics of S-MSN and TS-MSN

		Hydrodynamic	Polydispersity	Zeta potential
		diameter		mV
		nm
	TS-MSN	157 ± 22	0.30 ± 0.04	+17 ± 4
	S-MSN	73 ± 6	0.17 ± 0.03	+5 ± 2

Acknowledgments

This work was supported by the "Institut National du Cancer (INCa)", the "Fondation ARC" and the "Ligue Nationale Contre le Cancer (LNCC)" (ARC_INCa_LNCC_7636), the "Région Centre-Val de Loire" (NCIS Project) and the "Cancéropole Grand Ouest". "This work has been funded with support from the French Higher Education and Research ministry under the program "Investissements d'avenir" Grant Agreement: LabEx MAbImprove ANR-10-LABX-53-01."The authors would like to thank Isabelle Dimier-Poisson and Nathalie Moire (UMR INRA 1282, team of "Infectiologie et Santé Publique", University François Rabelais of Tours) for their help with flow cytometry experiments and Lizzy Angevin for skillful technical support with cells.