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Microfluidic-assisted production of sizecontrolled SPIONs-loaded PMMA nanohybrids Shukai Ding, Mohamed F Attia, Justine Wallyn, Chiara Taddei, Christophe A Serra, Nicolas Anton, Mohamad Kassem, Marc Schmutz, Meriem Er-Rafik, Nadia Messaddeq, Alexandre Collard, Wei Yu, Michele Giordano, and Thierry F. Vandamme Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01928 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Microfluidic-assisted production of size-controlled SPIONs-loaded PMMA nanohybrids Shukai Ding1,2, Mohamed F. Attia3,4,5, Justine Wallyn3, Chiara Taddei1,6, Christophe A. Serra1*, Nicolas Anton3*, Mohamad Kassem7, Marc Schmutz1, Meriem Er-Rafik1, Nadia Messaddeq8, Alexandre Collard1, Wei Yu1, Michele Giordano6, Thierry F. Vandamme3 1

2

Université de Strasbourg, CNRS, ICS UPR 22, F-67000 Strasbourg, France

Shaanxi University of Science & Technology, Institute of Atomic and Molecular Science, CN-710021, Xi'an, Shaanxi, China 3

Université de Strasbourg, CNRS, CAMB UMR 7199, F-67000 Strasbourg, France 4

5

6

7

National Research Centre, P.O. 12622, Cairo, Egypt

Clemson University, Department of Bioengineering, Clemson, SC 29634, USA

Institute for Polymers, Composites and Biomaterials (IPCB), CNR, Portici, Italy

Université de Strasbourg, Vascular and Tissue Stress in Transplantation: microparticles and environment EA7293, F-67000 Strasbourg, France. 8

Université de Strasbourg CNRS, INSERM, Collège de France, IGBMC UMR 7104/UMR_S 964, F-67000 Strasbourg, France

* Corresponding authors: C.A. Serra, Tel: + 33 388 414 095, Email: [email protected], N. Anton, Tel: +33 368 854 251, Email: [email protected]. Mr. Attia and Mrs. Wallyn have contributed equally to this work.

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ABSTRACT In this paper, superparamagnetic iron oxide nanoparticles (SPIONs, around 6 nm) encapsulated in poly(methyl methacrylate) nanoparticles (PMMA NPs) with controlled sizes ranging from 100 nm to 200 nm have been successfully produced. The hybrid polymeric nanoparticles were prepared following two different methods: (1) nanoprecipitation and (2) nanoemulsification-evaporation. These two methods were implemented in two different microprocesses based on the use of an impact jet micromixer and an elongational-flow microemulsifier respectively. SPIONs-loaded PMMA NPs synthesized by the two methods presented completely different physicochemical properties. The polymeric nanoparticles prepared with the micromixer-assisted nanoprecipitation method showed a heterogeneous dispersion of SPIONs inside the polymer matrix, an encapsulation efficiency close to 100 wt.% and an irregular shape. In contrast, the polymeric nanoparticles prepared with the microfluidic-assisted nanoemulsification-evaporation method showed a homogeneous dispersion, almost complete encapsulation and spherical shape. The properties of the polymeric nanoparticles have been characterized by DLS, TGA and TEM. In vitro cytotoxicity assays were also performed on the nanohybrids and pure PMMA nanoparticles.

Introduction Magnetic nanoparticles (MNPs) have become one of the main scopes in nanomaterials dedicated to biomedical applications. Over the past decades researches in pharmaceutical fields have been expanding showing how such magnetic nanomaterials are versatile and possess huge potentials for enhanced diagnosis of various diseases 1, 2, 3, 4

.

Among the different types of MNPs, superparamagnetic iron oxide nanoparticles (SPIONs) have attracted great attention due to the combined merits of atomic -2ACS Paragon Plus Environment

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composition, crystal structure and nanoscale effect. Indeed they offer good magnetic properties and thus present a huge potential as magnetic nanoprobes or nanoparticulate contrast agents for magnetic resonance imaging (MRI) in non-invasive diagnoses. As regards to in vivo aspects, SPIONs already exhibit two main advantages: i) their magnetic property within body can enlarge the signal during MRI and ii) their ultrasmall size leads to their fast distribution and good diffusion in intercellular space 5. However, for in vivo applications, the design of SPIONs surface must be adapted in order to overcome a lack of stealth property which prevents them from achieving what they were introduced for. The hurdle that the unmodified SPIONs are facing is the fast opsonization (recognition and sequestration) by the reticuloendothelial system (RES) causing SPIONs metabolization and rapid excretion from body. Syntheses of SPIONs by general methods such as co-precipitation, microemulsion, and thermal decomposition, mostly provide lipophilic SPIONs due to the use of long carbon chain ligands as stabilizer agent during the synthesis process. It results in instability of SPIONs in biological environment, because they tend to aggregate to bigger particles. Efficient surface modification of SPIONs will not only prolong the circulation time in blood stream and impart bioactive function on the SPIONs surface, which will interact with the target site of cell as ligand-receptor pair, but also circumvent the aggregation of SPIONs in aqueous phase. The commonly used methods for surface modification of SPIONs have been implemented by directly inverting hydrophobic surfactant into hydrophilic ones6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. Although, the surface modification of SPIONs has been well developed, SPIONs still will face challenges in clinics. For example, SPIONs with ultra-small size may undergo fast biodegradation during in vivo assays 21. Thus, to overcome a lack of stability and long-time retention, we have investigated in this study the encapsulation of SPIONs within a polymeric matrix as an inorganicorganic nanoassembly based on magnetic seeds embedded within polymeric nanoparticles to prevent SPIONs from aggregation and impose stability in biological environment. -3ACS Paragon Plus Environment

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Actually, preliminary experiments have been endeavored in this respect

22

. Different

monomer were applied to encapsulate the SPIONs by hydrolysis-condensation reaction or emulsification-polymerization with silica methacrylate) (PMMA)

23

, polystyrene

24

, poly(methyl

25, 26

, or PMMA-DVB (DVB = divinylbenzene) 27. Generally,

the commonly used protocol was achieved by polymerization of monomer. However, the obtained nanohybrids showed some deficiencies regarding the coating materials and their impacts on the in vivo properties of nanohybrids, for example the molecular weight of the polymeric coat and the degradable rate or the toxicity of materials. Besides, the low entrapment efficiency of SPIONs and the poor control over the methods preclude them from being widespread use. To address these shortcomings, SPIONs-encapsulated polymeric nanoparticles have been synthesized by top-down methods, i.e. by starting from an already synthesized polymer and not from its monomer. We synthesized SPIONs-loaded PMMA NPs by nanoprecipitation and nanoemulsification-evaporation methods associated with microfluidic systems, which yielded nanohybrids with enhanced properties compared to those produced by conventional methods 28. To the best of our knowledge, it has been reported no papers in the literature about the preparation of this type of nanohybrids by such methods and none of them relied on microfluidic systems. To that extent, it will be potent methods to encapsulate SPIONs in a polymeric matrix. Basically, nanoprecipitation and nanoemulsification-evaporation methods have been prevalent strategies for the production of polymeric nanoparticles. Fessi and coll. were the pioneers in the preparation of polymeric nanoparticles by nanoprecipitation

29

.

They reported that nanoprecipitation, also known as solvent displacement process or interfacial deposition, was driven by the fast diffusion of a solvent, solubilizing the polymer, in a non-solvent of the polymer (most often an aqueous phase) in presence or absence of surfactant to provoke polymer precipitation. Ideally, the solvent and the non-solvent used in the formulation should be miscible. In contrast, during the nanoemulsification-evaporation method, the solvent and non-solvent need to be immiscible. First, those two solvents are mixed to get an emulsion (micron-or -4ACS Paragon Plus Environment

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nanosized) in which the organic solution of polymer is the dispersed phase while the non-solvent forms the continuous phase. Solvent from the emulsion droplets is then removed by evaporation to get solid polymeric nanoparticles. Compared to other methods such as salting-out 30, supercritical fluids that replace the solvent in the precipitation techniques

31

, polymerization

32

, microfluidic-assisted

methods to prepare nanoparticles offers some flexibility for the biological applications. One of the latest developments in the production of polymeric nanoparticles begun with the advent of microfluidics and its specific microfabrication procedures

33

.

Extensive microfluidic systems have been explored to enhance the property of polymeric NPs 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 due to a better control over fluid flow at small scale, even at nanoliters 50. In this current study, microfluidic-assisted nanoprecipitation and nanoemulsificationevaporation methods have been adopted for the synthesis of SPIONs-loaded PMMA NPs to present unique physicochemical properties and aiming at allowing these nanohybrids particles to be used as potential MRI contrast agent. Our strategy is to produce SPIONs by a proven organic solvent-based method and have them encapsulated in the matrix of polymer nanoparticles whose surface, coated with a PEGylated surfactant, will allow the overall nanohybrids presenting good aqueous dispersibility and stability.

Materials and procedure Materials Methyl

methacrylate,

copper

(I)

bromide,

1,1,4,7,10,10-

hexamethyltriethylenetetramine and 2-ethyl bromoisobutyrate used for the synthesis of the PMMA and iron(III) acetylacetonate (Fe(aca)3) (97%), 1,2-dodecanediol (90%), oleylamine (70%) and oleic acid (90%) used for the SPIONs synthesis were purchased from Sigma-Aldrich (Saint-Louis, USA) and used as received except 2-ethyl bromoisobutyrate initiator, which was distilled under vacuum prior to use and methyl -5ACS Paragon Plus Environment

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methacrylate monomer, which was passed through an alumina column, from Merck (Darmstadt, Germany) to remove inhibitors. Ethanol, dichloromethane (DCM), diphenyl ether, hexane, methanol, tetrahydrofuran (THF) were purchased from SigmaAldrich (Saint-Louis, USA). Nonionic surfactant (Cremophor ELP) from BASF (Ludwigshafen, Germany) was kindly provided from Laserson (Etampes, France). Cremophor ELP is a parenteral grade nonionic surfactant made by reacting ethylene oxide to castor seed oil at an ethylene oxide to oil molar ratio of 35

51

. MilliQ water

was obtained using the MilliQ filtration system (Millipore, Saint-Quentin-en-Yvelines, France).

Dimethyl

sulfoxide

(DMSO),

and

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) solutions were purchased from Sigma-Aldrich (Saint Louis, USA). Phosphate buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum were purchased from PAN Biotech (Aidenbach, Germany). The dialysis membrane (diameter 16 mm, MWCO 12-14 kDa) was purchased from Medicell Internationnal (London, UK). Synthesis and characterization of PMMA PMMA was synthesized by atom transfer radical polymerization technique (ATRP) using a modified procedure of Xu et al.

24

. Reagents include methyl methacrylate as

monomer (19.91 mL), copper (I) bromide as catalysis (0.0876 g) and 1,1,4,7,10,10hexamethyltriethylenetetramine as ligand (182.75 µL). The reaction was carried out for 1.5 hours in THF (19.8 mL) at 60°C in a magnetically stirred round bottom flask under a blanket of nitrogen. Then the as-prepared polymer solution was filtrated through a neutral alumina column to remove catalyst and poured in a large volume of methanol to precipitate PMMA. Dry state polymer was finally obtained after overnight vacuum at 30°C. PMMA was analyzed by gel permeation chromatography (PL-GPC 120 platform, Polymer Laboratories, Church Stretton, UK) in THF (at a flow rate of 1 mL/min at 35°C). The number-average molecular weight of the synthesized polymer was 15,500 g.mol-1 (Ð = 1.37). Synthesis of SPIONs -6ACS Paragon Plus Environment

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Fe(acac)3 (0.71 g, 2 mmol), 1,2-dodecandiol (2.02 g, 10 mmol), oleic acid (2 mL, 6 mmol), oleylamine (2 mL, 6 mmol) and diphenyl ether (20 mL) were mixed and magnetically stirred in a 100 mL round bottom flask. The mixture was heated to reflux (200°C) for 2 hours. The black-brown oily mixture was cooled to room temperature by removing the heating source and purified by dialysis against ethanol (∼ 5 cycles). Ethanol was then removed from the product under vacuum. The resulting black product was finally suspended in THF or dichloromethane to reach a concentration of 15 mg of SPIONS per mL of organic solvent and formed black ferrofluids referred as SPIONs-THF and SPIONs-DCM respectively. SPIONs composition was investigated by infrared spectroscopy on a Nicolet 380 FT-IR Spectrometer (Thermo Electron Corporation) which revealed absorption peaks of magnetite Fe3O4 iron oxide phase (588 cm-1) with a shoulder assigned to maghemite γ-Fe2O3, the oxidized form of magnetite, due to slight oxidation naturally occurring on SPIONs surface. SPIONs size was investigated by TEM (Jeol 2100F operating at 200 kV) and ranged from 5 to 7 nm. Characterization of SPIONs-loaded PMMA NPs Diameter, size distribution and zeta potential The z-average diameter, size distribution and zeta potential of the hybrid NPs were assessed by dynamic light scattering (DLS) using a Malvern Nano ZS instrument (Malvern, Orsay, France). The helium-neon laser (4 mW) was operated at 633 nm, the scatter angle was fixed at 173° and the sample temperature was maintained at 25°C. The polydispersity index of the particle size (PDI) is a measure of the broadness of the size distribution and it is commonly admitted that PDI values below 0.2 corresponds to monomodal distributions. Analyses of nanosuspensions size and zeta potential were performed on three different samples by pouring dropwise 0.02 mL of the nanosuspensions into 1 mL MilliQ water. Five measurements were conducted for each sample. Transmission electron microscope (TEM) -7ACS Paragon Plus Environment

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To analyse the morphology and shape of the hybrid nanoparticles, TEM experiments were performed. A drop of the SPIONs-loaded PMMA NPs suspension was placed on a carbon grid (carbon type-A, 300 mesh, copper, Ted Pella Inc. Redding, PA) and dried at 40°C. Observations were carried out using a Philips Morgagni 268D (FEIEindhoven) electron microscope and images taken with a SIS Megaview camera (Olympus SIS-Munster). For cryo-TEM experiment, a 5 µL drop of the SPIONsloaded PMMA NPs suspension was deposited onto a lacey-holey carbon film (Ted Pella) freshly glow discharged (Elmo, Cordouan Technologies). The grid was rapidly frozen in liquid ethane cooled by liquid nitrogen in a home-made environment controlled machine. The grids were mounted onto a Gatan 626 cryoholder and observed in a Tecnai G2 (FEI-Eindhoven) operating at 200kV and the images were taken with an Eagle 2k2k ssCCD camera (FEI- Eindhoven) under low dose conditions. The contrast was, in that case, directly related to the atomic number of the diffusing atoms. So SPIONs were seen as black dots and the PMMA was rather invisible. Thermal gravimetric analysis (TGA) Thermogravimetric analysis (TGA) measurements were performed on a TGA 2 (Mettler Toledo). The mass losses of samples were assayed under nitrogen atmosphere from 35 to 700°C with a heating rate of 10°C/min. In vitro Cytotoxicity Assays (MTT Method) Cytotoxicity assays were performed using HeLa human

cervix epithelial

adenocarcinoma cells, and they were selected as a standard cancer cell line model. Cells were seeded in 96-well plates at a concentration of 104 cells per well in 100 µL of medium (DMEM) containing 10% FBS and 1 wt.% of commercial solution of penicillin and streptomycin. Cells were then incubated overnight at 37°C under a controlled atmosphere (5% CO2 and 80% H2O). Next, the culture medium was replaced by the same medium but containing variable concentrations of SPIONsloaded PMMA nanoparticles (see details below). After incubation for 24 h, the medium was removed and the cells were washed with PBS. Then, the wells were filled -8ACS Paragon Plus Environment

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with cell culture medium containing MTT, incubated for 4 hours at 37°C, and the formed formazan crystals were dissolved by adding 100 µL of DMSO. UV absorbance was measured at 570 nm with a microplate reader (Varioskan Flash, Thermo Scientific, USA). Experiments were carried out in triplicate and expressed as a percentage of viable cells compared to the control group. Preparation of SPIONs-loaded PMMA NPs by the micromixerassisted nanoprecipitation method Formulation

of

SPIONs-loaded

PMMA

NPs

via

microfluidic-assisted

nanoprecipitation method was performed by using SPIONs-THF solution. Evaluation of the amount of SPIONs in THF was carried out by weighting the mass of SPIONs from an aliquot of SPIONs-THF after complete evaporation of solvent. The organic phase (THF) contained PMMA, non-ionic surfactant and SPIONs whereas the nonsolvent phase was only MilliQ water. The organic phase was prepared by the successive addition of purified PMMA (50 mg) and Cremophor ELP (25 mg) to 5 mL of the dispersion of SPIONs in THF adjusted to 15 mg SPIONs/mL (total amount encapsulated = 75 mg of SPIONs). Thus the resulting organic solution for the nanoprecipitation had a weight ratio of SPIONs to PMMA of 1.5. As illustrated in Figure 1, the microfluidic system consisted of two syringe pumps (PHD 2000, Harvard Apparatus) which were connected with a micromixer by two PTFE tubes (1.06 mm ID x 1.68 mm OD). The micromixer used was the K-M impact jet micromixer (Fujifilm Corporation, Kyoto, Japan) introduced by Mae and coworkers 52, 53 and was composed of 3 microchannels (150 x 150 µm square) per inlet streams and an outlet channel of 300 µm in diameter. The organic phase and MilliQ water were pumped separately at different flow rates: the organic phase flow rate was fixed at 0.2 mL/min whereas the water flow rate was varied over a range of 2, 2.4, 2.8, 3, 3.2, 3.6 and 4 mL/min. Nanoprecipitation started right away within the mixing chamber of the micromixer when both fluids were brought into contact. The resulting colloidal suspension of SPIONs-loaded PMMA NPs was then directly collected at the outlet of the

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micromixer. The suspension was left at room temperature overnight to completely remove organic solvent to be ready for the measurements and characterizations.

Figure 1. Schematic representation of the micromixer-assisted nanoprecipitation process (not at scale). Qs, Qns and Qout denote the flow rate of the polymer solution, non solvent and exit stream respectively

Preparation of SPIONs-loaded PMMA NPs by the microfluidicassisted nanoemulsification-evaporation method Formulation

of

SPIONs-loaded

PMMA

NPs

via

microfluidic-assisted

nanoemulsification-evaporation method was performed by using SPIONs-DCM solution. Similarly, to compare with the micromixer-assisted nanoprecipitation, the concentration of SPIONs in DCM was adjusted to 15 mg SPIONs/mL. PMMA (10 mg/mL DCM) and hexane (1.5 mL) were added into SPIONs-DCM (1.5 mL). Mixture was then subjected to 10 min sonication to complete PMMA solubilization. The asprepared suspension had a weight ratio of SPIONs to PMMA of 1.5 and was used as the organic phase during emulsification. The continuous phase was basically composed of Cremophor ELP (0.255 g) solubilized in MilliQ water (12.75 mL). The organic phase (2.25 mL) and aqueous phase (12.75 mL) were then vortexed for 1 min to form a premixed emulsion. The premix was then used as the raw material for the microfluidic-assisted nanoemulsification system. As illustrated in Figure 2, the emulsification system was mainly assembled with two mid-pressure syringe pumps - 10 ACS Paragon Plus Environment

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(neMESYS Mid Pressure Module, Cetoni) which can work in opposite phase (withdraw/infuse), two 25 mL stainless steel syringes (Cetoni) and one PEEK tee (Valco Vici). The system was controlled by the supplier’s software to accurately operate flow rate. The micromixer was fabricated such as to obtain three drilled cylindrical microchannels having a diameter of either 250 or 150 µm. Two microchannels were connected to the stainless steel syringes with two PTFE tubes (1.06 mm ID x 1.68 mm OD). The third microchannel was used to collect the emulsion. During the process, both pumps were operated in opposite phases at the same reciprocating flow rate so that the premixed emulsions were transferred from one syringe to the other one through the microchannels acting as a restriction to the flow. A back and forth movement of the pump counts for one cycle. The effect of different reciprocating flow rates (30 and 50 mL/min) and number of cycles (100 to 400) on the physicochemical properties of NPs was investigated 54. At the end of the operation, the samples were collected, poured in a vial and left overnight in a fume hood to let the solvent evaporated.

Figure 2. Schematic drawing of the microfluidic-assisted nanoemulsification process (not at scale)

Results and discussion Preparation and properties of SPIONs-loaded PMMA NPs - 11 ACS Paragon Plus Environment

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Micromixer-assisted nanoprecipitation method This method has already been applied for the synthesis of drug-loaded PMMA NPs

35

in our previous work. The main component of the system is the micromixer, which can split each inlet phase into three sub-streams flowing in microchannels (150 x 150 µm square) and then collides the six flows to support the fast mixing of organic phase and aqueous phase by kinetic energy. The resulting suspension is collected in the outlet microchannel (300 µm diameter) which concentrated the collided six flows to improve molecular diffusion. The two key parameters, kinetic energy and molecular diffusion, are thus required to achieve the nanoprecipitation of polymeric nanoparticles. So far, this micromixer has been reported as the most suitable device for the preparation of polymeric nanoparticles with enhanced properties

28

As for the mixing process, it has

also been proved that to ensure good physicochemical features to suspensions formulated via microfluidic-assisted nanoprecipitation, fast mixing remained one of the most important conditions to apply 55. The mixing performance can be impacted by the difference of water and polymer solvent flow rates ratio. Indeed, a higher continuous phase flow rate compared to a low organic phase flow rate would support better performance of mixing due to the collided flows and the fast molecular diffusion in the outlet microchannels. We consequently selected in our study a constant flow rate (0.2 mL/min) for the polymer solution and a range of higher flow rates for the water phase to change the kinetic energy during the process. The sizes of the nanoparticles that were obtained are displayed in Figure 3(a). Influence of kinetic energy on the size of PMMA NPs and SPIONs-loaded PMMA NPs has been assessed. First it is observed for PMMA NPs that the PDI values tend to decrease when the water flow rate increases and stabilize around 0.16 when the flow rate is higher than 2.4 mL/min indicating a monomodal distribution of the z-average diameter. Then size has apparently been greatly influenced by the water flow rate, decreasing sharply when the flow rate was increased. This phenomenon has been observed in different studies 34, 42, 56, 57, 58 about the precipitation of a polymer or a pure - 12 ACS Paragon Plus Environment

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drug and results from two reasons: 1) a higher water phase to polymer solution flow rate ratio induced a higher supersaturation of the polymer or drug, which initially resulted in smaller nuclei formation during the nanoprecipitation

59

; small nuclei

formation is a key parameter to obtain nanoparticles whatever the mechanism they follow next to get bigger, i.e. growth or aggregation; 2) higher flow rates (organic phase or water phase) induce a higher kinetic energy resulting in higher Reynolds numbers, as such, the streamlines of the two phases intermesh rapidly decreasing the diffusion path of the polymer solvent into the non-solvent phase and thus speeding up the mixing 42. For the SPIONs-loaded PMMA NPs, the PDI values also decrease when the water flow rate increases and stay around 0.24 pass 0.2 mL.min-1. Thus the nanohybrids’ size distribution could be considered as nearly monomodal. The size of the nanoparticles has been surprisingly slightly influenced by the ratio of water phase to polymer solution flow rate compared to pure PMMA NPs. This observation is unquestionably linked to the presence of SPIONs in the formulation. One can hypothesize that the presence of inorganic nanoparticles in the polymer solution alters somehow the formation of nuclei or most probably their growth because the size of a nuclei is expected to be of the same order as SPIONs size. Therefore, due to the nuclei-growth mechanism, it is likely that SPIONs would be mostly present at the surface of the polymeric nanoparticles as TEM micrographs may suggest (Figure 8(a)). In such situation, the presence of SPIONs may favor the adsorption of further macromolecules and thus sustains the polymeric nanoparticles growth. When the water flow rate increases, the mixing is more intense and the SPIONs deposition on the surface of growing polymeric nanoparticles may be faster allowing them to grow even further. As a consequence the hybrid nanoparticles will have a bigger size than the pure PMMA NPs as stressed out by size distributions of unloaded and SPIONs-loaded PMMA NPs (Figure 3(b)).

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Figure 3. Size variations of SPIONs-loaded PMMA NPs and PMMA NPs obtained by the micromixer-assisted nanoprecipitation method with respect to the water flow rate (a); figures above the bars indicate the PDI values. Size distribution of two samples, SPIONs-loaded PMMA NPs and PMMA NPs, obtained for a water and polymer solution flow rates of 4 mL/min and 0.2 mL/min respectively (b).

Microfluidic-assisted nanoemulsification-evaporation method In contrast to the conventional shear flow emulsifiers (rotor-stator or high pressure homogenizer) or ultrasonicators, elongational-flow emulsifiers were recently proved to be more efficient in controlling the size and size distribution of a nanoemulsion in the range 50-300 nm 54. Furthermore, this kind of emulsifiers operates at low pressure (ca. 2.5 bars) and can accommodate high dispersed phase viscosities. However until now, we used a labscale or a microfluidic elongational-flow emulsifier to produce nanoemulsions composed of a pure monomer solution (MMA) as the dispersed phase and an aqueous continuous phase. Then the monomer nanodroplets were polymerized either by UV irradiation or heating 45, 60. In this study a microfluidic elongational-flow emulsifier

was

employed

to

emulsify

a

SPIONs/MMA

solution

or

a

SPIONS/PMMA/solvent solution instead of a simple monomer solution (Table 1). Once the nanodroplets were formed, the solvent was removed by heating to let the polymer to form solid hybrid nanoparticles. Table 1 Composition of the coarse emulsions used for the microfluidic-assisted nanoemulsification-evaporation method Formulation

Organic Phase (0.75 mL)


Water Phase (4.25 mL)

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1

*

Hexane

MMA

SPIONs-DCM

PMMA

Cremophor ELP

×

0.75 mL

0.75 mL*

-

2 wt.%

2

×

×

0.75 mL

10 mg/mL DCM

2 wt.%

3

0.375 mL

×

0.375 mL

10 mg/mL DCM

2 wt.%

after mixing with MMA, DCM was evaporated by heating the mixture at 70°C for 30 min

At first a solution of pure MMA was used to encapsulate SPIONs (Entry 1 of Table 1). However, the nanoemulsion produced with the microfluidic elongational-flow emulsifier was not stable. The emulsion fast separated into two layers after few minutes. Aggregation of SPIONs was observed in the upper water layer and also in the organic layer (Figure 4(1)). Afterwards, the SPIONs-DCM suspension was used as the organic phase in which PMMA was dissolved (Entry 2 of Table 1). The resulting coarse nanoemulsion was turbid and SPIONs aggregates were clearly visible (Figure 4(2)). It is assumed that the density of DCM (1.33 g/cm3) was probably too high to promote a fine emulsion. Therefore we prepared a third formulation (Entry 3 of Table 1) by mixing the SPIONs-DCM/PMMA solution with 50 vol.% hexane (density 0.65 g/cm3) to lower down the density of the dispersed phase accordingly to Seifriz recommendations

61

. After the emulsification, a fine nanoemulsion was obtained

without evidence of SPIONs aggregation (Figure 4(3)). In the following, the results will be presented for nanoemulsions prepared with the formulation 3 of Table 1.

Figure 4. Optical micrographs of the samples 1, 2 and 3 of Table 1 after microfluidic-assisted nanoemulsification-evaporation. Nanoemulsification was performed with a bore size of 150 µm, a reciprocating flow rate of 50 mL/min and a number of cycles of 100.


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The influence of the operating parameters, namely the number of cycles, the reciprocating flow rate (FR) and the bore size of the elongational-flow emulsifier, on the size of the SPIONs-loaded PMMA NPs produced is presented in Figure 5(a). Conversely to the previous method, the PDI values do not seem to be greatly affected by the operating parameters and stay close to 0.23 whatever the bore size and the number of cycles highlighting the production of nearly monomodal nanohybrids. A combination of a large bore size (250 µm) along with a low reciprocating flow rate (30 mL/min) induced the formation of the biggest nanoparticles whose size tended to decrease with an increase in the number of cycles. When the bore size was reduced down to 150 µm and the reciprocating flow rate increased up to 50 mL/min, the size stayed pretty much constant. If one assumes that during the evaporation of hexane, there is no droplet fusion, this result complies with a previous study conducted on the nanoemulsification of a MMA solution

60

. In brief the smaller the bore size and the

higher the reciprocating flow rate, the higher is the elongational strain rate ( ε& ) which is the driving force of the rupture of a big mother droplet into smaller daughter ones and thus the smaller are the nanodroplets. However, the value of the strain rate is not the same in the whole volume of the restriction being higher close to wall and smaller in the center. Therefore some time is required to decrease the size of the nanodroplets and ultimately reach a plateau value corresponding to the lowest size that can be ruptured for the highest value of ε& imposed by the operating parameters used. In other words, the size should decrease with the number of cycle and reach a constant value especially for the combination of a large bore size and a low reciprocation flow rate. This is what one can observe in Figure 5(a). Figure 5(b) displays the two size distributions returned by the DLS measurements for unloaded and SPIONs-loaded PMMA NPs produced under same operating conditions. It observed that both distributions almost superimposed perfectly. It is known from our previous work, that a higher dispersed phase viscosity will generate bigger nanodroplets

60

. This result

may thus be interpreted by the assumption that the addition of the SPIONs into the Hexane/PMMA dispersed phase did not alter too much its viscosity. However, given - 16 ACS Paragon Plus Environment

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the concentration of SPIONs in the dispersed phase (5 mg/mL) and from the numerous SPIONs to be seen in the TEM micrographs (Figure 8(b)), this is unlikely. It is most probable that the high density of SPIONs would have significantly increased the density of the PMMA NPs to the point that DLS measurements had retuned much higher values than they should have been. This will be corroborated by the cryo-TEM micrographs that will exhibit plenty of loaded SPIONs-loaded PMMA NPs smaller than 100 nm. In summary, the microfluidic-assisted nanoemulsification-evaporation method allowed producing SPIONs-loaded PMMA NPs which size was up to 100 nm.

Figure 5. Effect of the number of cycles in the microfluidic-assisted nanoemulsification-evaporation method on the size of SPIONs-loaded PMMA NPs for two different micromixer bore sizes (250 and 150 µm) and two different reciprocating flow rates (30 and 50 mL/min) (a); figures above the bars indicate the PDI values. Size distribution of two samples, SPIONs-loaded PMMA NPs and PMMA NPs, obtained for a number of cycles of 400, a micromixer bore size of 150 µm and a reciprocating flow rate of 50 mL/min (b).

Characterization of SPIONs-loaded PMMA NPs To prove the presence of SPIONS inside PMMA NPs and to quantify the difference between the nanohybrid particles produced by the two methods used, thermal gravimetric analyses were performed on the dried SPIONs, SPIONs-loaded PMMA NPs either produced with the nanoprecipitation or nanoemulsification method and unloaded PMMA NPs. SPIONs thermogram (Figure 6(a)) exhibited a rapid loss of weight till 450°C, then a plateau at 65% up to 650°C and finally another loss passed this temperature. The first weight loss is attributed to the removal of all organic - 17 ACS Paragon Plus Environment

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compounds (like the oleic acid) used in the synthesis of the inorganic nanoparticles and the plateau to bare SPIONs. The second weight loss is attributed to the oxidation of magnetite

62, 63

. PMMA thermogram (Figure 6(d)) indicates that past 450°C all the

polymer has been destroyed. Whatever methods used for their production, SPIONsloaded PMMA NPs thermograms (Figure 6(b,c)) present a plateau between 450°C and 650°C which thus proves the presence of the inorganic NPs in the PMMA nanoparticles. However the plateau for those produced with the nanoemulsification methods is higher (20%) than that for the nanoprecipitation method (10%). This seems to indicate that the former method allows encapsulating twice as much SPIONS than the latter. To check that SPIONS-loaded PMMA NPS had some magnetic properties, two nanosuspensions prepared accordingly to the two aforementioned methods were placed in two separated vials and observed in a magnetic field created by the proximity of a simple magnet. Before adding the magnetic field, the two samples were left side by side to rest for 30 min (Figure 7(a)). One clearly observes a difference in color between the two nanosuspensions. This cannot be attributed to a difference in the weight ratios of SPIONs to PMMA since both nanosuspensions were produced so that this ratio was kept constant and equal to 1.5. However the operating conditions used for the two methods implied different volume ratios between the oil and water phases. Thus the mass concentration of SPIONs in the micromixer outlet suspension (nanoprecipitation) was 0.7 mg/mL; while upon complete evaporation of DCM and hexane, the nanoemulsification-evaporation method produced an aqueous solution containing 1.3 mg of SPIONs per milliliter (1.8 times higher than the former method). Therefore the nanosuspension prepared with the nanoemulsification-evaporation method appears much darker than its nanoprecipitation counterpart due to the presence of a much higher concentration of SPIONs. Then the magnet was placed in between the two samples overnight. In the morning of next day, a concentrated layer of SPIONs-loaded PMMA NPs was observed on one side of the vials close to the magnet (Figure 7(b)). The magnetic attraction seems to be more pronounced for the sample - 18 ACS Paragon Plus Environment

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prepared by the nanoemulsification-evaporation method, but can be likely due to the difference of SPIONs concentration that affects the visual aspect of the samples. This experiment sufficiently proved that the hybrid nanoparticles that were produced can be actuated by an external magnetic field. This ability is an important property which makes them suitable as MRI contrast agents.

Figure 6. TGA curves of (a) SPIONs nanoparticles (b), SPIONs-loaded PMMA nanoparticles produced with the microfluidic-assisted nanoemulsification-evaporation method, (c) SPIONs-loaded PMMA nanoparticles synthesized by the micromixer-assisted nanoprecipitation method and (d) PMMA nanoparticles.

Figure 7. Optical micrographs of the two SPIONs-loaded PMMA NPs suspensions obtained with the nanoemulsification (left) and nanoprecipitation (right) methods without magnetic field (a) and in presence of a simple magnet (b).

The morphology of the above two samples has been analyzed by TEM. SPIONsloaded PMMA NPs prepared according to the micromixer-assisted nanoprecipitation - 19 ACS Paragon Plus Environment

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method is shown in Figure 8(a). The micrograph indicates that the whole amount of SPIONs has been encapsulated in the matrix of PMMA NPs; there are indeed little isolated inorganic nanoparticles in the background. However, the SPIONs are unevenly distributed in or at the surface of the PMMA NPs (see inset of Figure 8(a)). Furthermore, the shape of these hybrid nanoparticles looks irregular and some hybrid nanoparticles seem to have been aggregated (Figure 8(a)). Morphology of SPIONsloaded PMMA NPs prepared by the microfluidic-assisted nanoemulsificationevaporation method is displayed in Figure 8(b). One can clearly see round shape structures (spheres) containing a high amount of SPIONs. The size of the hybrid nanoparticles varies from 20 nm up to 250 nm with a majority around 40 nm. The long arrows point to the PMMA nanoparticles containing the SPIONs and the short arrow points to the few free SPIONs. The difference in morphology and distribution of SPIONs for the two types of loaded PMMA NPs produced may be explained considering the inherent mechanism of organic nanoparticles formation of the two methods. For the nanoprecipitation method, the polymer particles are formed upon the diffusion of the polymer solvent into the aqueous phase. This diffusion is so fast that it generates turbulences which are at the origin of the spinodal decomposition of the organic phase.64,

65, 66

In such condition one can hypothesize that this rapid flow of

polymer solvent convects SPIONs towards the periphery of the forming organic nanoparticles. In contrast, the nanoemulsification-evaporation method proceeds with a slow evaporation of the polymer solvent resulting in the formation of spherical PMMA NPs with a homogeneous distribution of SPIONs inside the particles. To confirm the difference in NPs morphologies induced by the two methods employed, the zeta potential of SPIONs-loaded PMMA NPs were measured and reported in Table 2. Unfortunately, since the SPIONs were synthesized in an organic solvent (diphenyl ether), they were not dispersible in an aqueous phase making impossible the measurement of their own zeta potential. However, the significant difference in zeta potential values reported in Table 2 (-42.3 vs. -1.5 mV) confirms that the SPIONs distributions at the surface of the nanoparticles are not the same and - 20 ACS Paragon Plus Environment

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thus supports the difference in morphologies observed in the micrographs of Figure 8. Table 2 presents also the z-average diameters of the SPIONs-loaded PMMA NPs freshly prepared and after 12 months. An increase of about 50 nm is observed for each sample after one year highlighting that some aggregation occurs with time.

Figure 8. TEM micrographs of unstained SPIONs-loaded PMMA NPs obtained with the micromixer-assisted nanoprecipitation method (a). Inset in a) shows that the SPIONs are attached mainly outside of the PMMA nanoparticles. Cryo-TEM of SPIONs-loaded PMMA NPs obtained with the microfluidic-assisted nanoemulsification-evaporation method (b). The white short arrows point to some individual SPIONs and the long white arrow to PMMA nanoparticles fully loaded with SPIONs. The inset in b) shows at a higher magnification the high density of SPIONs inside the PMMA nanoparticles.

Table 2 Zeta potential and z-average diameter of the different SPIONs-loaded PMMA NPs prepared Method

Zeta potential (mV)

Z-average diameter right after preparation (nm)

Z-average diameter after 12 months (nm)

Nanoprecipitation*

-42.3±0.3

199±3

246±5


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Emulsificationevaporation**

-1.5±0.2

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113±2

179±5

*: data collected for the sample reported in Fig. 3b; **: data collected for the sample reported in Fig. 5b

Cytotoxicity assay of SPIONs-loaded PMMA NPs

In vitro toxicity of SPIONs-loaded PMMA NPs suspensions was assessed by MTT test after incubation of the NPs for 24 hours with HeLa cells (cancer cell line model) at different SPIONs and PMMA concentrations (Figure 9). The toxicity was globally similar whatever method was used to produce the hybrid nanoparticles. The LD50 is around 5 mg/mL for SPIONs-loaded PMMA NPs and 1 wt.% for pure PMMA NPs. This means that the toxicity profiles are very low if compared with the SPIONs-based products reported in the literature. These experiments have been performed in direct contact with cells which thus were exposed to harsh conditions, but the cell viability remains high. This negligible toxicity could be attributed to the PMMA polymer and the PEGylated surfactant coating of the nanohybrids, giving rise to biocompatible and stealth NPs which can be promising in in vivo experiments.


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Figure 9. Cell viability measurements for different concentrations of SPIONS-loaded PMMA NPs obtained with the nanoprecipitation (a) and nanoemulsification (b) method. Cell viability measurements for different concentrations of pure PMMA NPs obtained by both methods (c). Experiments have been performed in triplicates.

Conclusion Two different methods, namely nanoprecipitation and nanoemulsification-evaporation, were carried out in two different microfluidic devices (micromixer and elongationalflow microemulsifier respectively) to produce SPIONs-loaded PMMA NPs whose size could be tuned from 100 to 200 nm. However, the results shown that the morphologies of the hybrid nanoparticles and the distribution of SPIONs in the organic nanoparticles were different. The microfluidic-assisted nanoemulsification-evaporation method - 23 ACS Paragon Plus Environment

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allowed producing spherical nanoparticles with a homogeneous distribution of SPIONs while the nanoprecipitation method produced irregular nanoparticles with an unevenly distribution of inorganic nanoparticles at the surface or in the PMMA NPs. These observations result from the inherent difference in the mechanism of particles formation induced by the two methods. In the nanoprecipitation method, the fast diffusion of the polymer solvent into the non-solvent expulsed the SPIONs towards the periphery of the forming PMMA NPs; while for the nanoemulsification-evaporation method the slow evaporation of the polymer solvent allowed encapsulating the SPIONs inside the PMMA chains network ultimately leading to the formation of spherical nanoparticles and homogeneous distribution of SPIONs. Nevertheless, the two methods allowed producing SPIONs-loaded PMMA NPs that exhibit low cell toxicity. The step beyond would consist in i) assessing the effect of Fe3O4 synthesis operating parameters, mainly the reaction time, on the SPIONs properties (size, size distribution, shape, iron and ligands contents, relaxivity parameters (r2,r1) and r2/r1, dispersibility in an organic solvent) by employing a complete set of characterization techniques (FT-IR, XRD, TEM, DLS, iron titration, relaxometry) in order to select the best formulation for producing the nanohybrids and ii) conducting in vitro/vivo assays to assess the potential of these nanoparticulate systems as effective MRI contrast agents.

Acknowledgments SD would like to acknowledge the China Scholarship Council for his Ph.D fellowship. CS is grateful to P. Allgayer for the mechanical engineering support. ICS electronic microscopy and polymer characterization platforms are acknowledged for the use of their instruments. The company Fujifilm is also acknowledged for providing the KM micromixer and Prof. Kazuhiro Mae for helpful discussion.


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Graphical abstract


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Fig 1 47x44mm (300 x 300 DPI)

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Fig 2 50x38mm (300 x 300 DPI)


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Fig 3 101x55mm (300 x 300 DPI)


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Fig 4 35x36mm (300 x 300 DPI)


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Fig 5 508x287mm (300 x 300 DPI)


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Fig 6 104x73mm (300 x 300 DPI)


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Fig 7 39x21mm (300 x 300 DPI)


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Fig 8 79x106mm (300 x 300 DPI)


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Fig 9 82x82mm (300 x 300 DPI)


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Graphical abstract 2sd eds. 1175x779mm (90 x 90 DPI)


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