Bin Wang

Olivier Sandre

Kunzhou Wang

Haishan Shi

Kun Xiong

Yu-Bin Huang

Tingting Wu

Minhao Yan
email: yanminhao@swust.edu.cn

Jérémie Courtois
email: jeremiecourtois@swust.edu.cn

Auto-degradable and biocompatible superparamagnetic iron oxide nanoparticles/polypeptides colloidal polyion complexes with high density of magnetic material

Keywords:

Hypothesis: superparamagnetic iron oxide nanoparticles (SPIONs) are extensively used as building block of colloidal nanocomposites for biomedical applications. Strategies employed to embed them in a biodegradable and biocompatible polymer matrix often fail to achieve a high density of loading which would greatly benefit to applications such as imaging and hyperthermia. In this study, poly(acrylic acid) coated SPION (γ-Fe 2 O 3 -PAA) are self-assembled with hydrolysable poly(serine ester) by electrostatic complexation, leading to perfectly defined spherical particles with ultra-high density of magnetic material and an ability to auto-degrade into individual SPION and biocompatible byproducts. Experiments: self-assembly and auto-degradation of γ-Fe 2 O 3 -PAA/poly(serine ester) and γ-Fe 2 O 3 -PAA/poly(serine ester)-b-PEG colloidal particles are studied by light scattering and microscopy. Colloidal stability in bio-fluids, hyperthermia under alternating magnetic field, cellular uptake, cytotoxicity and degradation of γ-Fe 2 O 3 -PAA/poly(serine ester)-b-PEG in living cells are investigated. Findings: a remarkably slow electrostatic complexation leads to dense superparamagnetic γ-Fe 2 O 3 -PAA/poly(serine ester)-b-PEG polyion complexes (PICs) with controlled sizes (150 -500 nm) and times of degradation in aqueous solvents (700 -5000 h). The material shows good sustainability during hyperthermia, is well taken up by MC3T3 cells and non-cytotoxic. TEM images reveal a mechanism of degradation by "peeling" and fragmentation. In cells, PICs are reduced into individual SPIONs within 72 h.

Introduction

Nanomaterials have matured significantly during the last decade as they have transitioned from lab bench science to applied technology. It is reasonable to expect that their utilization will rapidly increase and their eco-and bio-toxicities are therefore major concerns. Long-term effects of nanomaterials on environment and living systems are difficult to anticipate and study. [1][2][3] The most direct approach to overcome toxicity issues is to design stable environment-friendly or biocompatible nanomaterials. However, their long-term effects will remain questioned. A smart alternative is therefore to develop nanomaterials with ability to self-degrade "by-design" into biocompatible sub-units. This way, toxicity issues are reduced to a time-scale for which exhaustive toxicological studies are possible whereas, at longer time scale, nanomaterials will no longer exist in their original form but as innocuous byproducts. In this work, focus is put on colloidal assemblies of nanoparticles. Particularly, we developed sub-micrometer polyionic complex clusters of Superparamagnetic Iron Oxide Nanoparticles (SPION) and polyelectrolytes with an ability to auto-degrade into single nanoparticles and biocompatible molecular byproducts that can be eliminated through renal system.

SPIONs are widely applied in materials science, biotechnology and nanomedicine.

In bio-applications, SPIONs are mainly used as MRI contrast agents, drug delivery vectors and magnetic hyperthermia tools. [4][5][6][7][8][9][10] For such applications, clustering iron oxide nanocrystals into colloidal aggregates bring several advantages such as higher macroscopic magnetic response, enhanced contrast in imaging and higher generation of heat in the case of hyperthermia treatment. [11][12][13][14][15] Various strategies have been employed to aggregate SPIONs into colloidal nano-and micro-sized objects. [11,[16][17][18][19][20][21] One of them consists in embedding SPIONs in a hydrolysable biocompatible polyester matrix, [22][23][24][25][26] but with some difficulty to reach the high content in magnetic material required by applications. [24,26,27] Another way is to densely pack SPIONs with oppositely charged polyelectrolytes into spherical or rod-like polyion complexes (PICs). [28][29][30] Entropically driven self-assembly allows synthesis route as simple as direct mixing of the oppositely charged species. [31] Dense PICs can be obtained by self-assembly of poly(acrylic acid) coated sub-10 nm maghemite nanoparticles (γ-Fe 2 O 3 -PAA) and either cationic homopolyelectrolytes, [30,31] or cationic-block-neutral copolymer. Utilization of diblock copolymers helps to obtain colloidally stable spherical and monodisperse PICs. [28] Sizes and microstructures of these PICs depend on concentration and charge ratio of the oppositely charged species during synthesis. Well-defined shapes, lower polydispersity and higher densities of the clusters are obtained when building blocks are mixed at higher concentrations and at charge stoichiometry. [29,30,32] Interaction between chemically stable γ-Fe 2 O 3 -PAA/copolymer rod-like PICs and living cells was studied in vitro. [33] Observation by TEM revealed that 24 h after incubation, the PICs were internalized by cells and, interestingly, partly degraded into smaller fragments, suggesting that byproducts will be generated over time.

These byproducts include γ-Fe 2 O 3 -PAA nanoparticles which have been recognized for their biocompatibility, [33,34,7] but also potentially stable and toxic nano-sized fragments, polycations, [35] and their various degradation products.

In the present study, issues arising from the long-term fate of γ-Fe 2 O 3 -PAA/polyelectrolyte PICs are overcome by replacing industrial cationic polyelectrolytes with biocompatible and biodegradable ones. [36] γ-Fe 2 O 3 -PAA nanoparticles are complexed with either poly(serine ester) (pSe) or poly(serine ester)-block-poly(ethylene glycol) (pSe-b-PEG), resulting in dense superparamagnetic colloidal clusters. On the one hand, positive charges of serine units allow self-assembly driven by electrostatic interaction with the negative charges of γ-Fe 2 O 3 -PAA nanoparticles, whilst hydrolysis of the ester bonds of pSe triggers its depolymerization and the complete disassembly of the nanostructured edifices in aqueous media over time on the other hand.

The first part of this article is dedicated to the physicochemical study of the synthesis of the auto-degradable PICs. Self-assembly of nanoparticles and polymers according to concentration and charge ratio are investigated. Kinetics and mechanisms of degradation are analyzed by light scattering and microscopy.

In the second part are presented results which aim to demonstrate the reliability of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs as biocompatible tools for biomedical applications.

Heat generation induced by alternating magnetic field (AMF) stimulation, PICs in cells was demonstrated by TEM analysis of cells in resin sections. Simulated Body Fluid was prepared following the procedure described by Kokubo et al. [37] SPI-Pon 812 epoxy resin Monomer (glyceringlycidether) (CAS: 90529-77-4) was purchased from SPI. All reagents and solvents were commercial grade and used as received without further purification. 18 MΩ cm resistivity MilliQ® quality (Millipore)

colloidal

Materials and methods

Synthesis and characterization

Materials

deionized water was used. Poly(acrylic acid) (PAA) coated iron oxide nanoparticles were prepared following reported protocols. In brief, magnetite (Fe 3 O 4 ) nanocrystals were synthesized by co-precipitation of iron (II) and iron (III) salts in aqueous alkaline medium at room temperature. [38] The nanocrystals were then transferred in diluted nitric acid (pH = 1.8) and oxidized by the addition of Fe(NO 3 ) 3 at boiling temperature for 30 min.

Change in colour of the dispersion from black to dark red-orange indicates formation of maghemite (γ-Fe 2 O 3 ). [39] In order to decrease their polydispersity, γ-Fe 2 O 3 nanoparticles were size-sorted by four subsequent liquid-liquid phase separations induced by the addition of nitric acid. [38,40,41] Transmission Electron stability. [42] The molecular weight of γ-Fe 2 O 3 nanoparticles was determined by Static Light Scattering (SLS) and found to be 5.82 × 10 6 g mol -1 (Supporting Information).

The bare maghemite nanocrystals were then coated with poly(acrylic acid) (PAA) oligomers (M w = 2000 g mol -1 ) using the precipitation-redispersion process. [43,44] D H and polydispersity of γ-Fe 2 O 3 -PAA dispersion at pH = 7 are 34 ± 1 nm and 0.25 ± 0.05, respectively. This D H is 5 nm larger than that of the bare particles, indicating the presence of PAA layer surrounding the particles which has been measured by Small Angle Neutron Scattering to be 1.25 nm thick. [45] Colloidal stability of γ-Fe 2 O 3 -PAA is function of the negative charges density of the PAA layer, which depends on the pH of the dispersion. [43] Surface charge density of γ-Fe 2 O 3 -PAA depending on pH was determined by acid-base titration according to the method developed by M. Szekeres (Figure S3, Supporting Information). [46] At pH = 7, the amount of surface carboxylate groups is 0.577 mmol per g of iron oxide, corresponding to 3347 ± 50 negative elementary charges per nanoparticle and correlated to a ξ p of -48 mV which is negative enough to prevent aggregation. [47] The superparamagnetic behaviour of γ-Fe (Figure S4, Supporting Information).

In this work, the nanoparticle concentration is defined by the percentage by weight of iron oxide in the dispersion and is determined from the whole UV-Vis spectrum measurement between 220 and 800 nm using a priory made master curve obtained by independent iron titration by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) (Figure S5, Supporting Information). The method is both very sensitive and non-disruptive. [48] 2 pSe homopolyelectrolytes and pSe-b-PEG diblock copolymer were synthesized according to previously reported protocol. [36] The M w were determined by gel permeation chromatography (GPC). Briefly, poly(N-trityl serine lactone) with M w (pTSL) = 33050 g mol -1 (pTSL 33K ), and poly(N-trityl serine lactone)-b-PEG with M w (pTSL) = 33050 g mol - 1 and M w (PEG) = 10000 g mol -1 (pTSL 33K -b-PEG 10K ), were synthesized via ring-opening polymerization of N-trityl serine lactone with diethyl zinc as catalyst. Trityl groups were further removed by trifluoroacetic acid and methanol in dichloromethane at room temperature. This de-protection procedure induces a polyelectrolyte backbone degradation about 20 % whereas the PEG 10K block did not degrade. [36,49] The obtained pSe and pSe-b-PEG with acetate counter-ions are characterized by a M w (pSe, Acetate) = 11840 g mol -1 , corresponding to an average degree of polymerization DP(pSe) = 80. S1 andS2). Then, they were postfixed in 1% osmium-phosphate buffer 0.1 M for 2 h at 20 °C in dark conditions. After 0.1 M PBS washes, the samples were dehydrated in increasing concentrations of ethanol/acetone mixtures and embedded in SPI-Pon 812 epoxy resin. 70 nm-thick sections were cut with an ultramicrotome (LEICA, UC7), stained in uranyl acetate and lead citrate, and analyzed with a Hitachi HT7700 electron microscope.

Preparation of γ-Fe

Experimental methods

Dynamic

Electron microscopy

Magnetic hyperthermia

The experimental setup used for magnetic field hyperthermia is described in previous publication. [50] In brief, a radiofrequency power generator delivers current to a 4-turn coil of 55 mm outer diameter. In our experiment, quasi-sinusoidal alternating magnetic field at f = 755 kHz and B 0 = 13 mT (field strength H 0 = 10 kA m -1 ) was generated. γ-Fe

Determination of the amount of iron oxide adsorbed and internalized by cells:

Immediately after cell viability assessment, cells were extensively washed with PBS and detached by 0.25% trypsin/EDTA mixture (Gibco, USA) (50 μL per well). After 5 min, the trypsinized cells were mixed with PBS (150 μL per well) and collected. Then, the collected samples (cells with adsorbed or internalized γ- = 1 M, which proves the electrostatic nature of the association. [31,52] Thus, among the attractive intermolecular interactions, long-range van der Waals forces are not sufficient to trigger co-assembly ; short-range Coulombic forces are required. [31] An interesting phenomenon is the delayed aggregation of nanoparticles and pSe chains at c = 6 × 10 -4 wt% (Figure 2a, dark red symbols). Indeed, the D H of the mixture remains at the constant value of single nanoparticles during 45 h before it starts to increase. A possible explanation is that at this concentration, 𝐼𝐼 𝑠𝑠 is at a critical value for which electrostatic interactions are almost screened.

Consequently, oppositely charged species only aggregate slowly and particle growth occurs in a reaction-controlled manner rather than diffusion-controlled.

That allows the formation of dense colloidal clusters instead of formation of a fractal macroscopic precipitate. [31] As concentration and therefore 𝐼𝐼 𝑠𝑠 decrease, larger clusters are formed and with faster kinetics. Once aggregation starts, increases of D H according to the time are almost linear with values of 1.42 nm h -1 , 1.73 nm h -1 and 2.80 nm h -1 for c = 6 × 10 -4 wt%, 6 × 10 -5 wt% and 6 × 10 -6 wt%, respectively. The highest reached values of D H are 220 nm, 640 nm and 1150 nm for c = 6 × 10 -4 wt% at t = 210 h, c = 6 × 10 -5 wt% at t = 307 h and c = 6 × 10 -6 wt% at t = 337 h, respectively. A scanning electron micrograph of a γ-Fe 2 O 3 -PAA/pSe PIC obtained at c = 6 × 10 -5 wt% 300 h after mixing is presented in Figure 2b. Its irregular shape and nano-structure are interpreted as resulting from an inhomogeneous assembly of small and dense aggregates which suggest a diffusion-limited growth mechanism. [31] The image also shows numerous single nanoparticles which indicate a low degree of interaction.

After the γ-Fe First, a polyelectrolyte-b-neutral double hydrophilic block copolymer helps to obtain spherical clusters with controlled size and polydispersity. [28] Second, PEG surface blocks generate steric repulsion which participate to colloidal stability. [53,54] Third, hydrophilic PEG coating of colloidal objects increases their stealth capability toward immune system, thereby permitting longer circulation in blood stream. [53,[55][56][57] Growth and degradation of γ-Fe S10c and S10d). With growth periods as long as ≈ 300 h, diffusion limited aggregation leading to inhomogeneous assembly (such as the one of the γ-Fe 2 O 3 -PAA/pSe PIC pictured in Figure 2b) or fractal clusters would have been expected. [31] Instead, spherical morphology and dense nanostructure of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs made at Z ≥ 1 indicate that they were grown by nucleation and growth process, [28] during which diblock copolymers were regulating nanoparticle organisation at the surface of the growing clusters while surface PEG blocks forbade their precipitation. Complete degradation into single nanoparticles was achieved in about 1400 h, 960 h and 620 h for samples at Z = 1, 10 and 100 respectively (Figure 3). DLS study demonstrates that, for Z ≥ 1, size and time of studies report the possibility of inducing significant cell death with internalized magnetic nanoparticles without observing a macroscopic temperature increase during AMF exposure. [58] The Specific Absorption Rate (SAR) corresponds to the generated thermal power per unit of mass of iron oxide. SAR was calculated using calorimetric method as described in previous publication. [59] Because in such diluted conditions, heat loss toward external environment is comparable to heat generated by the material, the heating rate was compensated by the cooling rate at same temperature in the SAR calculation. [60] SAR value of 31.0 ± 1.5 W g -1 was estimated for these conditions. γ-Fe measured on individual γ-Fe 2 O 3 -PAA nanoparticles in accordance to the well known effect on r 2 by aggregation of iron oxide nanoparticles. [61] These results tend to demonstrate that γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs can endure multiple AMF hyperthermia cycles without dramatic modification of their structure and magnetic properties. 6, closed green symbols). In DMEM supplemented with 3.7 g L -1 of sodium bicarbonate, PICs were stable for only 5 h before macroscopic precipitation occurred (Figure 6, empty green symbols). In RPMI with or without NaHCO 3 , PICs were stable for 32 h before precipitation (Figure 6, empty and closed red symbols). In DMEM and RPMI containing FBS (Figure 6, violet and pink symbols), the PICs were stable for respectively 27 h and 39 h before colloidal destabilization, which means that presence of proteins does not lead to spontaneous precipitation. Such behaviour in protein-containing media is promising regarding in vivo applications. Indeed, the time of circulation of nanoparticles in the bloodstream, from magnetic nanoparticles coated with citrate or dextran to PEGylated vesicles, does not exceed 100 minutes with half-lives of the order of 10 minutes. [57,[62][63][64] Thus, it is expected that γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs will be stable enough over the time-period required by biomedical applications. studies demonstrated the non-cytotoxicity of γ-Fe 2 O 3 -PAA, [33,34] and the low cytotoxicity of pSe-b-PEG. [36] PEG 10K , as well as the hydrolytic product serine, are nontoxic at low doses and would probably be excreted in vivo. [START_REF] Webster | PEGylated Protein Drugs Basic Sci. Clin. Appl[END_REF] Cell viability assays show convincing results regarding short term non-toxicity of γ-Fe objects into ≈ 500 nm dense clusters within 24 h, [33] nanoassembly involving hydrolysable poly(ester serine) leads to almost complete separation of the γ-Fe 2 O 3 -PAA nanoparticles from each other in the same duration. endosomes. [START_REF] Hillaireau | [END_REF][67][68] Remarkably, number and density of nanoparticles in the membrane-bound domain shown in Figure 8c seem lower than what could be expected from complete degradation of a whole γ-Fe 2 O 3 -PAA/pSe-b-PEG cluster.

A reason for that could be the fast erosion and dissolution of coated iron oxide nanoparticles in intracellular compartments. [69] An important consequence of rapid degradation of γ-Fe 2 O 3 -PAA/pSe-b-PEG clusters is the faster degradability of the γ-Fe 2 O 3 -PAA nanoparticles themselves as, generally, isolated nanoparticles erode faster than aggregated ones. [70]

Conflicts of interest

There are no conflicts to declare.

Transmission electron microscopy (TEM) analysis of γ-Fe 2 O 3 nanocrystals

TEM analysis was performed on a Zeiss Libra200FE microscope. Figure S1a shows a TEM image of the maghemite nanocrystals (γ-Fe 2 O 3 ). The particles are characterized by a dense structure and an almost spherical shape. For both bare and PAA-coated iron oxide particles, the (220), (311), (400), ( 511) and

𝑝𝑝(𝐷𝐷) = 1 �2𝜋𝜋(𝑠𝑠 𝑇𝑇𝑇𝑇𝑇𝑇 )𝐷𝐷 𝑒𝑒𝑒𝑒𝑝𝑝 �- 𝑙𝑙𝑙𝑙 2 �𝐷𝐷 𝐷𝐷 0 𝑇𝑇𝑇𝑇𝑇𝑇 ⁄ � 2×(𝑠𝑠 𝑇𝑇𝑇𝑇𝑇𝑇 ) 2 �. (
(440) diffraction peaks are indexed to cubic spinel structure (card number PDF#:39-1346) corresponding to maghemite. The average particle size is estimated from the most intense peak (311) by using the Debye-Scherrer formula. d = 0.9 λ/β cosθ, where d is the particle size, β is the full width at half-maxima, λ is the incident copper Kα wavelength of 1.546 Å, and θ is the maximum peak position. The average crystal size obtained from the Debye-Scherrer equation is found to be about 10 nm.

Determination of the molecular weight of the γ-Fe 2 O 3 nanoparticles by static light scattering (SLS)

SLS monitored on a Brookhaven 90plus was used to determine the molecular weight (M w ) of the γ-Fe 2 O 3 nanoparticles. Several dispersions at pH = 2 (HNO 3 ) were prepared at concentrations from 10 -3 wt% to 0.4 wt%. The non-aggregated state of these nanoparticles dispersions was asserted DLS. The Rayleigh ratio was obtained from the scattered intensity I(q,c) measured at the wave-vector q according to Equation S2:

𝑅𝑅(𝑞𝑞, 𝑐𝑐) = 𝑅𝑅 𝑠𝑠𝑠𝑠𝑠𝑠 𝐼𝐼(𝑞𝑞,𝑐𝑐)-𝐼𝐼 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤 𝐼𝐼 𝑇𝑇𝑇𝑇𝑇𝑇 � 𝑙𝑙 𝑙𝑙 𝑤𝑤𝑇𝑇𝑇𝑇 � 2 , ( S2
)
where R std and n Tol are the standard Rayleigh ratio and refractive index of toluene, I Water and I Tol the intensities measured for the solvent and for the toluene in the same scattering configuration and q = (4πn/λ) sin(θ/2) (n is the refractive index of the solution, θ is the scattering angle, and λ is wavelength 633 nm). 1 The scattering angle of the Brookhaven spectrometer was θ = 90°, corresponding to wave-vectors q = 1.87 × 10 -3 Å -1 .

The linear evolution of the Rayleigh ratio as a function of concentration, c, is given by Equation S3 and is experimentally verified.

𝑅𝑅(𝑐𝑐) = 𝐾𝐾𝑐𝑐𝑀𝑀 𝑊𝑊 𝑎𝑎𝑎𝑎𝑎𝑎 (S3) In this equation, app W M
is the apparent molecular weight of the diffusing species and

𝐾𝐾 = 4𝜋𝜋 2 𝑙𝑙 2 (𝑠𝑠𝑙𝑙 𝑠𝑠𝑐𝑐 ⁄ ) 2 𝑁𝑁 𝐴𝐴 𝜆𝜆 4
is the diffusion contrast with N A as Avogadro's number and (dn/dc) as the refraction index increment as measured using a differential refractometer Chromatix KMX-16.

The average molecular weight of the γ-Fe 2 O 3 nanoparticles is found to be 5.82 × 10 6 g mol -1 .

Acid-base titration of γ-Fe 2 O 3 -PAA nanoparticles

The ratio between carboxylic acid and carboxylate groups at the surface of the PAA coated particles depends on the pH of the dispersion. Acid-base titrations have been performed on γ-Fe 2 O 3 -PAA nanoparticles dispersed in pure water in order to determine their charge density as a function of pH. The pH-dependent protonation-deprotonation process has been characterized quantitatively following the method developed by Szekeres et al. 2 Measurements lead to the calculation of the net proton surface excess, which is equivalent to the amount of carboxylate groups, as a function of pH. Figure S3 displays the net surface proton excess expressed either in mmol per g of iron oxide or in number of proton per nanoparticle, as a function of the pH. Indeed, knowing the molecular weight of the γ-Fe 2 O 3 particles (M w = 5.8 × 10 6 g mol -1 ) and the mass of iron oxide in a sample, the number of nanoparticles can be calculated and net surface proton excess per g of iron oxide then converted in number of negative charges per nanoparticle. At pH = 7, the net surface proton excess is -0.577 mmol g -1 , corresponding to 3347 ± 50 negative charges per γ-Fe 2 O 3 -PAA 2K nanoparticle. Results are in good agreement with previous work. 4 The values of the magnetization at saturation, M S , are 46.2 emu g -1 , 44.1 emu g -1 and 42.5 emu g -1 for bare maghemite nanocrystals, PAA coated particles and γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs, respectively. Here, M S = ϕm S , where ϕ is the volume fraction of maghemite and m S is the specific magnetization of colloidal maghemite (m S = 3.5 × 10 5 A m -1 ), which is lower than that of bulk maghemite due to some disorder of the magnetic moments located near the surface. Absence of magnetization at H = 0 combined to significant values of M S are characteristic of superparamagnetic behavior.

Determination of the iron oxide concentration in dispersions by UV-vis spectroscopy

In this study, the iron oxide concentration of a colloidal dispersion of γ-Fe

Calculation of the charge ratio in reactive species

For a given pH, the amount of negative charges N -(in mol) brought by the carboxylate groups of a γ-Fe 2 O 3 -PAA nanoparticle dispersion is given by:

𝑁𝑁 -= 𝑐𝑐 𝑁𝑁𝑁𝑁 100 × 𝑉𝑉 𝑁𝑁𝑁𝑁 × 𝑐𝑐 - (S4),
where 𝑐𝑐 𝑁𝑁𝑁𝑁 and 𝑉𝑉 𝑁𝑁𝑁𝑁 are respectively the concentration in iron oxide in wt% and the volume in mL of the dispersion, and 𝑐𝑐 -is number of mol of negative charges per g of iron oxide of the dispersion. At pH = 7, 𝑐𝑐 -= 5.77 × 10 -4 mol g -1 (see "Acid-base titration of γ-Fe 2 O 3 -PAA nanoparticles" of this document).

The amount of positive charges N + (in mol) brought by the ammonium groups of a pSe solution is given by:

𝑁𝑁 + = � 𝑐𝑐 𝑝𝑝𝑇𝑇𝑇𝑇𝑝𝑝 100 � �×𝑉𝑉 𝑝𝑝𝑇𝑇𝑇𝑇𝑝𝑝 𝑀𝑀 𝑊𝑊 𝑝𝑝𝑇𝑇𝑇𝑇𝑝𝑝 × 𝐷𝐷𝐷𝐷 × 𝛼𝛼 (S5),
where 𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 and 𝑉𝑉 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 are respectively the concentration in polymer in wt% and the volume in mL of the solution. 𝑀𝑀 𝑊𝑊 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 is the molecular weight of the polymer in g mol -1 . DP is the average degree of polymerization of the polyelectrolyte block (average number of serine units

Determination of the numbers of cells during cell viability assessments

During the cell viability assessments, numbers of MC3T3 and HepG2 viable cells were determined using calibration curves. The "Optical density (OD) versus Number of cells" calibration curves were established as follow: cells were grown as described in "2.2.7 Cell culture", detached by 0.25 % trypsin/EDTA and pelleted by centrifugation. Then, supernatant was removed and cell pellets re-suspended in assay medium and counted using a cell-count board. 0, 10000, 40000, 60000, 80000 and 100000 MC3T3-E1 cells, and 0, 10000, 20000, 40000, 160000 and 200000 HepG2 cells in assay medium were deposited in well plates. ), the ratio between negative charges brought by the nanoparticles and the amount of ethylene oxide monomer units, equals 1.

γ-Fe 2 O 3 -PAA mixtures with respectively serine and PEG 10K were prepared according to the formulations given in Table S3 and stored at T = 25 °C.

Solutions to mix Mixtures

γ-Fe ) of the mixtures.

Figure S8 presents D H measurements according to the time of the γ-Fe 2 O 3 -PAA/serine and γ-Fe 2 O 3 -PAA/PEG 10K mixtures described above (see Table S3).

  stability of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs in bio-fluids, amount of materials internalized by MC3T3-E1 cells and cell viability up to 72 h after incubation are investigated. Finally, rapid degradation of γ-Fe 2 O 3 -PAA/pSe-b-PEG

FeCl 2 •

 2 4 H 2 O; FeCl 3 in solution at 27 wt% in H 2 O, poly(sodium acrylate) (M w = 2000 g mol -1 , molar mass dispersity = 1.7), DL-serine (≥ 98 %), poly(ethylene glycol) (M W = 10000 g mol -1 ) (PEG 10K ), Dulbecco's Phosphate Buffer Saline CaCl 2 and MgCl 2 free (PBS1×, powder), Dulbecco's Modified Eagle Medium (DMEM) without glucose, Lglutamine, phenol red, sodium pyruvate and sodium bicarbonate (powder) and RPMI 1640 cell culture medium with L-glutamine, without glucose and sodium bicarbonate (powder) were purchased from Aldrich. Fetal Bovine Serum (FBS no.10099-141) used for Dynamic Light Scattering (DLS) experiments was purchased from Gibco, USA.

2. 1 . 2

 12 Synthesis and characterization of poly(acrylic acid) coated iron oxide nanoparticles (γ-Fe 2 O 3 -PAA)

  Microscopy (TEM) images of the obtained maghemite nanocrystals show almost spherical particles (Figure S1, Supporting Information). The size distribution of the particles is described by a log-normal function with a median diameter of 9.3 nm and a standard width of logarithm σ = 0.18. Average size of the particles and maghemite structure are confirmed by XRD (Figure S2, Supporting Information). DLS analysis of nanocrystal dispersion at pH = 1.8 reveals a single population of particles with a hydrodynamic diameter D H (γ-Fe 2 O 3 , pH = 1.8) = 29 ± 1 nm and a polydispersity index PDI = 0.25 ± 0.05. The positively charged surface of the particles at low pH with ξ p (γ-Fe 2 O 3 , pH = 1.8) = +30 mV ensures their colloidal

. 1 . 3

 13 Synthesis of poly(serine ester) (pSe) and poly(serine ester)-b-poly(ethylene glycol) (pSe-b-PEG)

2 O 3 -

 23 PAA/pSe and γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes (PICs) γ-Fe 2 O 3 -PAA/pSe (respectively γ-Fe 2 O 3 -PAA/pSe-b-PEG) PICs were prepared at room temperature by direct mixing of γ-Fe 2 O 3 -PAA nanoparticle dispersions and pSe (respectively pSe-b-PEG) solutions in the same solvent (water or Phosphate Buffer Saline (PBS1×)) and at the same weight concentrations ([γ-Fe 2 O 3 ] = [polymer]). The charge ratio in reactive species Z of a nanoparticle/polymer mixture is calculated as the amount of negative charges brought by the carboxylate groups of the γ-Fe 2 O 3 -PAA dispersion divided by the amount of positive charges brought by the ammonium groups of the polyelectrolyte solution. Desired values of Z are obtained by mixing nanoparticles and polymer solutions in appropriated volumes. Z are calculated considering 0.577 mmol of negative charges per g of iron oxide (determined by acid-base titration of γ-Fe 2 O 3 -PAA) and 80 positive charges (DP of pSe blocks) per polymer chain. Indeed, pKa of the amino group of serine being 9.15, approximation is made that all the amino groups of pSe blocks are charged (-NH 3 + ) at pH ≈ 7. Preparation and composition of the samples are detailed in the Supporting Information (Table

  Light Scattering (DLS) DLS experiments were made on a Brookhaven 90plus instrument to measure the collective diffusion coefficient D. The wavelength of the light source was λ = 632.8 nm, the scattering angle θ = 90°, and the temperature T = 25 °C. The autocorrelation functions of the scattered light were interpreted using both the method of 2 nd order cumulants and the CONTIN fitting procedure provided by the instrument software. Hydrodynamic diameters (D H ) of colloids were calculated according to the Stokes-Einstein relation, D H = k B T/3πηD, where k B is the Boltzmann constant, T the temperature and η the solvent viscosity (0.89 mPa s).

Fe 2 O 3 -

 23 PAA/pSe-b-PEG material) were digested in 35 vol% HCl and their contents in iron determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, ThermoFisher iCAP6500ICP-MS). The corresponding numbers of cells were determined during cell viability assessments (see Cell viability assessment in this part).

3. Results and discussion 3 . 1 . 1 .Fe 2 O 3 - 3 . 2

 3112332 Figure 2a presents hydrodynamic diameters (D H ) of mixtures of γ-Fe 2 O 3 -PAA and

2 O 3 -Fe 2 O 3 - 20 3. 3

 2323203 PAA/pSe PICs are formed, they slowly auto-degrade. At c = 6 × 10 -4 wt%, D H decreases from 220 nm at 235 h to 34 nm at t = 940 h, which corresponds to an average rate of -0.26 nm h -1 (Figure2a, dark red symbols). The decrease of the average size of the colloidal objects is the consequence of the fragmentation of the clusters into smaller and smaller units until single nanoparticles are recovered, as shown in Figure1. As ester groups of pSe undergo hydrolysis in aqueous solvent, pSe chains become shorter and unable to interconnect nanoparticles. Consequently, γ-Fe 2 O 3 -PAA/pSe edifices progressively collapse. Without acid-base catalysis, hydrolysis of ester bonds is slow, explaining the ≈ 30 days required to achieve complete degradation of the clusters into single nanoparticles. It is assumed that pSe is progressively degraded into biocompatible serine amino-acids. Time of degradation of clusters prepared at c = 6 × 10 -5 wt% and c = 6 × 10 -6 wt% are about 740 h and 1450 h with rates of -1.75 nm h -1 and -1.19 nm h -1 , respectively (Figure2a, orange and yellow symbols). For these two samples, the final stage of degradation process is characterized by the presence of stable colloids of D H = 85 nm and D H = 270 nm, respectively. This result could be a consequence of the tendency for the released serine molecules to coat the surface of γ-Fe 2 O 3 -PAA, which would decrease their ξ p and trigger aggregate formation. Such phenomenon would be 𝐼𝐼 𝑠𝑠 dependent and can explain why such colloidal assembly of nanoparticles exist at c ≤ 6 × 10 -5 wt% but not at c = 6 × 10 -4 wt. In an additional set of experiments, γ-Fe 2 O 3 -PAA were mixed with serine molecules from solutions at [γ-Fe 2 O 3 ] = [serine] = 6 × 10 -4 wt%, 6 × 10 -5 wt% and 6 × 10 -6 wt%, respectively, and as Z = 1 (Supporting Information). FigureS8presents the D H of the mixtures according to the time. At c = 6 × 10 -4 wt%, aggregation of γ-PAA and serine does not occur whereas at c = 6 × 10 -5 wt% and c = 6 × 10 -6 wt%, aggregates of respectively 190 ± 15 nm and 290 ± 20 nm are formed 45 h after mixing and remain stable over time. These results are in good agreement with what is observed for γ-Fe 2 O 3 -PAA/pSe mixtures after degradation (Figure2a) and comfort previously made hypothesis. Moreover, absence of aggregation between γ-Fe 2 O 3 -PAA and serine at 6 × 10 -4 wt% and the relatively small sizes of the colloidal objects obtained at c ≤ 6 × 10 -5 wt% as well as their stability suggest that polymeric architecture of the cationic moiety is mandatory for self-assembly of large auto-degradable PICs. Furthermore, study of the degradation of pSe-b-PEG in water at 50 °C (FigureS9, Supporting Information) shows that pSe blocks are only slightly degraded within a period of 240 h and suggests that pSe chains were indeed involved in PICs formation.To summarize, self-assembly of γ-Fe 2 O 3 -PAA nanoparticles and pSe into colloidal PICs is a concentration-and ionic strength-dependant electrostatically driven process. Optimum control on the complexation and on the degradation of the synthesized clusters is obtained at higher concentration, as long as physicochemical conditions do not forbid electrostatic interactions. Author manuscript version of paper accepted for publication in Mater. Sci. Eng. C, 2019, 109920, after peer review and technical editing by the publisher. DOI: 10.1016/j.msec.2019.109920 Synthesis and degradation of γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes γ-Fe 2 O 3 -PAA nanoparticles were complexed with pSe-b-PEG copolymer to form colloidal clusters. The purpose of adding a PEG block to pSe chains is manifold.

  2 O 3 -PAA/pSe-b-PEG PICs were studied by DLS and TEM. In particular, influence of the charge ratio in reactive species Z (see Experimental section) on size, nanostructure and morphology of the clusters was investigated. Figure 3 presents D H measurements according to the time of mixtures of γ-Fe 2 O 3 -PAA and pSe-b-PEG in PBS1× at [γ-Fe 2 O 3 ] = [pSe-b-PEG] = 6 × 10 -4 wt% and at charge ratio Z going from 0.1 to 100. The results show that size of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs strongly depends on Z. At Z = 0.1 (excess of pSe-b-PEG, Figure 3, blue symbols), large colloidal objects with D H = 759 nm are formed at t = 283 h. Then, D H of these PICs remains almost constant for ≈ 170 h before degradation occurs. TEM image at an early stage of degradation (t = 598 h, D H = 598 nm) shows a non-spherical dense cluster with smooth irregular edges (FigureS10a, Supporting Information). This aspect suggests that the cluster is mainly made of polymer. Decrease of D H from t ≈ 500 h to t ≈ 2000 h is the signature of the degradation process. At t > 2000 h, the dispersion contains colloidal objects of D H ≈ 180 nm in a quasi-equilibrium state. At Z = 0.5 (Figure3, red symbols), clusters of D H = 535 nm are formed at t = 260 h. This size is maintained for ≈ 260 h before degradation occurs at ≈ -1 nm h -1 for 200 h. Then, fragments are slowly degraded into single nanoparticles. TEM image at t = 598 h of a cluster made at Z = 0.5 shows compact assembly with irregular edges (FigureS10b).At Z = 1, 10 and 100 (Figure3, black, green and pink symbols, respectively), γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs with respective D H = 420 nm, 285 nm and 185 nm are obtained at t = 295 ± 10 h. TEM images of these clusters show very well defined spherical objects with densely packed maghemite nanocrystals (Figure4a

  degradation of the dense spherical PICs can be finely tuned by playing on the charge ratio Z of γ-Fe 2 O 3 -PAA/pSe-b-PEG mixtures. Regarding PICs made with pSe homopolyelectrolyte at c = 6 × 10 -4 wt%, complexation of the nanoparticles with pSe-b-PEG is not spontaneous. Delays before the start of self-assembly were found to vary from 22h for Z = 0.1 to 115 h for Z = 100. Mechanisms of degradation of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs in respectively PBS1× and deionized water were investigated by TEM. Time evolution of D H of γ-Fe 2 O 3 -PAA and pSe-b-PEG mixtures at Z = 1 in PBS1× and in water were found to be similar (Figure S11, Supporting Information) but TEM observation of the PICs at various times in these two media reveals different mechanisms of degradation (Figure4). In PBS1×, elongated fragments are first detached from outer part of spherical clusters while cores remain dense (Figure4b). Over time objects becomes increasingly fragmented into small aggregates and single nanoparticles (Figure4c). In water, blocks of nanoparticles are progressively detached from the spherical PICs. At an intermediate state of the degradation process, clusters are perforated and porous (Figure4e). As the process continues, clusters are dislocated in smaller dense fragments which are progressively degraded into single nanoparticles (Figure4f).

3. 4

 4 Magnetic performance of γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes and hyperthermia experiment Characterization of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs by Vibrating SampleMagnetometry confirms their superparamagnetic behaviour with a value of magnetization at saturation of 42.5 emu g -1 (FigureS4). This value is slightly lower than the 44.1 emu g -1 recorded for γ-Fe 2 O 3 -PAA, which was already decreased compared to the original 46.2 emu g -1 value of specific magnetization of bare γ-Fe 2 O 3 nanoparticles, presumably because stronger complexation of the iron oxide surface increase the disorder of the magnetic moments at the surface of the nanocrystals (spin canting effect). γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs ability to generate heat when exposed to high frequency alternating magnetic field (AMF) was investigated. In our magnetic hyperthermia experiment, temperature of a dispersion of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs at [γ-Fe 2 O 3 ] = 8.8 × 10 -3 wt% was measured by a fibre optic probe as a function of time (Figure 5b). The two successive applications of the AMF (separated by a return to thermal equilibrium) induced increases of temperature of ΔT = 0.4 °C and ΔT = 0.7 °C, respectively. Ability of the material to macroscopically increase the temperature of the whole sample under AMF gives a good indication about its potential ability to induce cell death. However, recent

  2 O 3 -PAA/pSe-b-PEG PICs were observed by TEM before and after hyperthermia (Figure 5a and 5c, respectively). The images reveal that the dense clusters were just slightly degraded during the cycles of hyperthermia as only ≈ 100 ± 50 nm fragments were detached while cohesion of the clusters was maintained. To support this result, thermal stability of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs (made at c = 6 × 10 -4 wt% and Z = 1 in water, t = 418 h) was studied by DLS (Figure S12, Supporting Information). The almost constant values of D H of the clusters during incubation at 60 °C and 95 °C for 12 h demonstrate that a substantial increase of the thermal energy available in the medium does not trigger the macroscopic degradation of the PICs. As shown on Figure S13, longitudinal (r 1 ) and transverse (r 2 ) proton relaxation rates of the γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs were measured before and after hyperthermia. These values of r 1 = 10.7 and r 2 = 54.1 s -1 mM (Fe) -1 before hyperthermia and r 1 = 11.2 and r 2 = 56.1 s -1 mM (Fe) -1 after hyperthermia, were almost unaffected by the application of the AMF, in accordance with the limited degradation of the clusters observed during the hyperthermia experiment. The longitudinal relaxivities are also almost the same as the original r 1 = 10.8 s -1 mM (Fe) -1 measured on individual γ-Fe 2 O 3 -PAA nanoparticles, meaning that the surface of iron oxides are highly accessible to water molecules within the clusters. On the contrary, there was a significant (almost two-fold) increase of transverse relaxivity in the clusters compared to r 2 = 30.3 s -1 mM (Fe) -1

3. 5

 5 Stability of γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes in bio-fluids Colloidal stability of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs in bio-fluids at 37 °C was investigated by DLS. Clusters with D H = 417 ± 15 nm (c = 6 × 10 -4 wt%, Z = 1, t = 342 h) were first synthesized in water and then transferred in Simulated Body Fluid (SBF), DMEM and RPMI with or without sodium bicarbonate, and DMEM and RPMI containing 10 vol% of fetal bovine serum (FBS).

Figure 6

 6 Figure 6 presents the evolution of D H according to the time for γ-Fe 2 O 3 -PAA/pSe-

3. 6

 6 Cellular uptake and toxicity assays γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs (synthesized at c = 6 × 10 -4 wt% as Z = 1, t = 304 h, D H = 414 ± 10 nm) were incubated with respectively MC3T3-E1 and HepG2 cells. Amounts of iron internalized or adsorbed by MC3T3-E1 cells at 24 h, and MC3T3-E1 and HepG2 cell viability at 24 h, 48 h and 72 h were determined as a function of concentration.

Figure 7a compares masses

  Figure 7a compares masses of iron taken up by MC3T3-E1 cells (empty black symbols) to masses of iron introduced (closed black symbols) in pg per cell at [Fe] = 7.51 × 10 -3 , 7.51 × 10 -2 , 0.751 and 7.51 mM, respectively. Corresponding mass fractions of internalized iron are given in wt% (Figure 7a, blue symbols). At [Fe] = 7.51 × 10 -3 mM,

2 O 3 -

 23 PAA/pSe-b-PEG PICs while low toxicity of the degradation byproducts has been recognized in literature reports. γ-Fe 2 O 3 -PAA/pSe-b-PEG based systems can therefore be considered as suitable tools for biomedical applications such as easily eliminable MRI contrast agents or nanomedicine. 3.7 TEM analysis of γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes in cells MC3T3-E1 cells were incubated with γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs (Z =1, t = 304 h, D H = 414 nm) at [γ-Fe 2 O 3 ] = 6 × 10 -3 wt% and observed by TEM 24 h, 48 h and 72 h after exposure. Representative images of the cells at these exposure times are shown in Figure 8. After 24 h of incubation, nanoparticles were found inside cells either as individual units and oligomeric aggregates gathered in round-shaped domains (Figure 8a, red arrows) or as partially degraded clusters. These internalized clusters were found at different stages of degradation: almost undegraded dense and spherical objects are present (Figure 8a', green arrow), some dense clusters have already undergone destructuration of their outer layer (Figure 8a', orange arrows) and others are at a more advanced stage of degradation as indicated by low nanoparticle densities and extended interparticle distances (Figure 8a', yellow arrow). Two arguments support the idea that single particles and fragments observed inside cells are degradation byproducts of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs and not the result of the internalization of already degraded material. First, no γ-Fe 2 O 3 -PAA/pSe-b-PEG clusters, fragments or single particles were found adsorbed on cell membranes which indicates a rapid internalization of the material by cells. Second, almost no single particles were found in the cytosol and only either nanoparticles-free (Figure 8a, violet arrows) or highly populated intracellular domains (Figure 8a, red arrows) are present in cells, which suggest a former internalization of large, undegraded, objects. 48 h and 72 h after exposure, no dense clusters were found in cells but only single nanoparticles or oligomeric aggregates gathered in domains as pictured in Figure 8b and 8c. Compared to the biodegradation of chemically stable γ-Fe 2 O 3 -PAA/PTEA-PAM PICs which mainly consists of fragmentation of micro-sized

Figure

  Figure8cshows a degraded PIC in a membrane-enclosed compartment whereas

Fe 2 O 3 -

 23 γ-Fe 2 O 3 -PAA/pSe-b-PEG systems allow accelerating this degradation process with enhanced biocompatibility due to the chemical nature of pSe-b-PEG.These experiments demonstrate the rapid internalisation and degradation of γ-PAA/pSe-b-PEG PICs in cells. Therefore, diagnosis and treatments involving this type of degradable nanoassembly should be performed within the few hours after injection to take advantage of the aggregated state of the nanoparticles.4. ConclusionIn this study, auto-degradable and biocompatible SPION/polyelectrolyte colloidal polyion complexes (PICs) were developed to overcome potential long-term toxicity of this type of nanostructured hybrid materials while ensuring the high density of magnetic material required by biomedical applications. Poly(acrylic acid) coated maghemite nanoparticles (γ-Fe 2 O 3 -PAA) were complexed with either poly(serine ester) (pSe) or poly(serine ester)-b-PEG (pSe-b-PEG) as biocompatible and biodegradable polyelectrolytes. Once clusters were formed, hydrolysis of poly(ester) blocks triggered the disassembly of the whole edifices. The degradation process generated individual γ-Fe 2 O 3 -PAA nanoparticles, serine molecules and PEG blocks as biocompatible byproducts. Interaction between γ-Fe 2 O 3 -PAA nanoparticles and pSe homopolyelectrolyte was studied. Polyion complexation of these building blocks was found to be a concentration and ionic strength dependent process mainly driven by screened electrostatic interaction. Self-assembly of γ-Fe 2 O 3 -PAA nanoparticles in presence of pSe chains lead to relatively dense 200 -800 nm colloidal clusters yet with Author manuscript version of paper accepted for publication in Mater. Sci. Eng. C, 2019, 109920, after peer review and technical editing by the publisher. DOI: 10.1016/j.msec.2019.109920 31 irregular shape and inhomogeneous structure. Interestingly, kinetics of growth are particularly slow (> 250 h) and, at high concentration, the aggregation of nanoparticles started only 100 h after direct mixing of the reactive species. Degradation of γ-Fe 2 O 3 -PAA/pSe clusters in aqueous medium was confirmed by the decrease of the D H over several weeks. Formation of γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs as a function of the charge stoichiometry in reactive species Z was then studied. In spite of the slow rate of aggregation, perfectly defined dense and spherical clusters were obtained at Z ≥ 1 (excess of SPIONs), due to PEG blocks which regulate nanoparticle organisation at the surface of the growing clusters. Once formed, γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs started to auto-degrade into individual nanoparticles over several weeks in aqueous media. The results show that size and time of degradation of the clusters can be finely tuned by variation of the charge ratio Z during synthesis. Different mechanisms of degradation were identified depending on the solvent. In the first stage, clusters are "peeled" in PBS1× whereas in water, dense blocks are detached from the structure, leading to perforated clusters. Superparamagnetic γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs generated heat under magnetic stimulation with a SAR value of 31.0 ± 1.5 W g -1 of iron oxide. TEM, DLS and relaxometry analysis indicate that structure and properties of the clusters were not significantly affected by such magnetic hyperthermia cycles. Cell viability assessments conducted on MC3T3-E1 cells incubated with γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs over a period of 72 h demonstrate the non-cytotoxicity of the material, even at [Fe] = 7.51 mM, corresponding to 25 pg of iron internalized by cell. TEM observation of γ-Fe 2 O 3 -PAA/pSe-b-PEG in cells 24 h, 48 h and 72 h after exposure reveals that clusters were rapidly internalized and degraded. Most of the PICs were degraded into individual nanoparticles within 24 h and only a part of the clusters were found in a less advanced stage of degradation. 72 h after incubation, no clusters were found in cells but only single SPIONs and oligomeric aggregates. The present study showed a simple synthesis route to obtain dense spherical biocompatible and biodegradable SPION/polymer polyion complexes with great control of their size and time of degradation. The γ-Fe 2 O 3 -PAA/pSe-b-PEG clusters exhibit properties required for biomedical applications such as short-term colloidal stability, magnetic responsivity and nontoxicity. Finally, rapid degradation of the material in cells was demonstrated. For all these reasons, SPION/poly(serine ester) complexes are interesting tools for nano-architects designing dynamic multi-functional nanostructured systems for which both biocompatibility and high density of magnetic material are required.

Figure 1 .

 1 Figure 1. Schematic representation of the synthesis and auto-degradation of γ-Fe 2 O 3 -

Figure 2 .Figure 3 .

 23 Figure 2. DLS and SEM analysis of γ-Fe 2 O 3 -PAA/poly(serine ester) polyion complexes

Figure 4 .

 4 Figure 4. TEM study of the auto-degradation γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion

Figure 5 .

 5 Figure 5. Hyperthermia experiment performed on γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes in water at [γ-Fe 2 O 3 ] = 8.8 × 10 -3 wt%. a) TEM image of the polyion

Figure 6 .

 6 Figure 6. DLS study of the stability of γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes in

Figure 7 .

 7 Figure 7. Cellular uptake (a) and toxicity assays (b, c) of γ-Fe 2 O 3 -PAA/pSe-b-PEG

Figure 8 .

 8 Figure 8. TEM images of MC3T3-E1 cells 24 h (a, a'), 48 h (b) and 72 h (c) after incubation with γ-Fe 2 O 3 -PAA/pSe-b-PEG spherical clusters at [γ-Fe 2 O 3 ] = 6 × 10 -3 wt%

Figure S1 .

 S1 Figure S1. a) Maghemite superparamagnetic nanoparticles (γ-Fe 2 O 3 ) as observed by TEM. b)

  diffraction (XRD) analysis of the γ-Fe 2 O 3 and γ-Fe 2 O 3 -PAA nanoparticles Concentrated γ-Fe 2 O 3 and γ-Fe 2 O 3 -PAA nanoparticles dispersions were freeze-dried and the powders analyzed by XRD using a PANalytical X'Pert PRO X-ray diffractometer.The 2θ values were taken from 20° to 70° using Cu Kα radiation (λ value is 1.5416 Å). FigureS2presents the XRD patterns of the materials.

Figure S2 .

 S2 Figure S2. XRD patterns of γ-Fe 2 O 3 and γ-Fe 2 O 3 -PAA nanoparticles.

  Titration experiments were performed at 25 °C, under nitrogen flow and with constant stirring speed of 200 rpm. Dispersions of γ-Fe 2 O 3 -PAA particles (20 mL, [γ-Fe 2 O 3 ] = 0.05 wt%) were freshly prepared from degassed deionized water (30 minutes at boiling point and subsequent Argon bubbling during 30 minutes), and degassed again by Argon bubbling prior to titration. HCl and NaOH titrant solutions were introduced using micropipette, and pH and E (mV) measured after each injection once equilibrium was reach. Experiments were performed using an Accumet XL200 pH-meter. pH probe was washed with concentrated HCl and linearity of E (mV) vs pH (Nernst slope: R 2 > 99.98) checked before each titration. γ-Fe 2 O 3 nanoparticles were coated with PAA 2K using precipitation redispersion process. 3 PAA 2K chains were adsorbed to iron oxide cores in acidic conditions (pH = 2) and then redispersed at pH = 10 using NH 4 OH before their purification by dialysis. γ-Fe 2 O 3 -PAA used in the study are therefore surrounded by NH 4 + counter-ions. As ammonium counter-ions could be involved in proton exchange processes or could chemically interact with γ-Fe 2 O 3 -PAA surface, it was required to replace them by Na + counter-ions prior to titrations. Coated nanoparticles with ammonium counter-ions were precipitated in HCl and washed sequentially with large amount of HCl solution (pH = 2) in order to remove NH 4 + ions, and deionized water. γ-Fe 2 O 3 -PAA were then redispersed with NaOH to pH = 9, ensuring presence of Na + counter-ions, and finally purified by dialysis versus deionized water. pH of purified γ-Fe 2 O 3 -PAA dispersion with Na + counter-ions is 6.5.

Figure S3 .

 S3 Figure S3. Net surface proton excess (expressed in mmol of H + per g of iron oxide (left ordinate axis) and in H + per nanoparticle (right ordinate axis)) as a function of pH for γ-

Fe 2 O 3 -

 23 Figure S5 (black curve).

Figure S5 .

 S5 Figure S5. UV-vis spectra of γ-Fe 2 O 3 (black curve, used as master curve for determination of

  per pSe or pSe-b-PEG per chain) and α is the proportion of the amino groups positively charged at a given pH. M W (pSe with Acetate counter-ions) = 11840 g mol -1 and M W (pSe-b-PEG with Acetate counter-ions) = 21840 g mol -1 . The pKa(-NH 2 /-NH 3 + ) of serine being 9.15, α was approximated to 1 in the study as pSe and pSe-b-PEG solutions were used at pH = 7 or pH = 7.4 when in PBS1× buffer. The charge ratio of a mixture of γ-Fe 2 O 3 -PAA and either pSe or pSe-b-PEG is defined by 𝑍𝑍 = 𝑁𝑁 -𝑁𝑁 + ⁄ . Preparation of γ-Fe 2 O 3 -PAA/pSe polyion complexes Before mixing, purified γ-Fe 2 O 3 -PAA were dispersed in deionized water and pH was adjusted to 7 by addition of a tiny amount of NaOH. pSe polyelectrolyte was dissolved in deionized water, giving solutions at pH = 7. γ-Fe 2 O 3 -PAA/pSe PICs were obtained by mixing stock solutions prepared at the same weight concentrations: 𝑐𝑐 𝑁𝑁𝑁𝑁 = 𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 .

  dispersions and pSe solutions mixed to obtain γ-Fe 2 O 3 -PAA/pSe mixtures, and, concentrations in iron oxide and in pSe, and charge ratio Z of the mixtures.Preparation of γ-Fe2 O 3 -PAA/pSe-b-PEG polyion complexes γ-Fe 2 O 3 -PAA dispersions in water or PBS1× at various concentrations were prepared by dilution of a γ-Fe 2 O 3 -PAA dispersion at [γ-Fe 2 O 3 ] = 0.984 wt% in water. pH of diluted dispersions in water was adjusted to 7 by addition of a tiny amount of NaOH. pSe-b-PEG solutions were prepared as follow: pSe-b-PEG at 𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 = 1.3 × 10 -3 wt% in deionized water was first prepared and vigorously stirred for 12 h at 50°C until complete dissolution. The as-prepared stock solution was then diluted in either water or PBS1× to achieve the desired concentrations. The pH value of pSe-b-PEG solutions in water is 7. γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs were obtained by mixing stock solutions prepared at the same weight concentrations: 𝑐𝑐 𝑁𝑁𝑁𝑁 = 𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 = 6 × 10 -4 𝑤𝑤𝑤𝑤% .

Figure S6 .Fe 2 O 3 -

 S623 Figure S6. Calibration curves used for the determination of the number of MC3T3 (a) and

  The D H of γ-Fe 2 O 3 -PAA nanoparticles mixed with serine at c = 6 × 10 -4 wt% (Figure S8, red symbols) and of γ-Fe 2 O 3 -PAA mixed with PEG 10K (Figure S8, grey symbols) remain at constant values corresponding to the D H of single nanoparticles over periods of 425 h, demonstrating that these mixtures do not lead to the formation of aggregates. Mixtures of γ-Fe 2 O 3 -PAA and serine at c = 6 × 10 -5 wt% (Figure S8, blue symbols) and c = 6 × 10 -6 wt% (Figure S8, green symbols) lead to spontaneous formation of aggregates with D H = 112 nm and 190 nm, respectively. 45 h after mixing, stable colloidal objects with D H = 190 ± 15 nm and 290 ± 20 nm for mixtures at 6 × 10 -5 wt% and 6 × 10 -6 wt%, respectively, are obtained.

Figure S8 .

 S8 Figure S8. DLS analysis of the interactions between γ-Fe 2 O 3 -PAA nanoparticles and,

FigureFigure S9 .Figure S10 .

 S9S10 FigureS9presents the GPC chromatograms of pSe-b-PEG copolymer at t = 0 h, 120 h,

γ-Fe 2 O 3 -

 23 PAA/pSe-b-PEG in PBS1× and water: comparative Study by DLS Mixtures of γ-Fe 2 O 3 -PAA and pSe-b-PEG at Z = 1 in either PBS1× or deionized water were prepared from solutions at [γ-Fe 2 O 3 ] = [pSe] = 6 × 10 -4 wt% following the protocol described in the Experimental Section of the article and in "Preparation of γ-Fe 2 O 3 -PAA/pSe and γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes" and Table S1 of this document.

Figure S11 .

 S11 Figure S11. DLS analysis of γ-Fe 2 O 3 -PAA/poly(serine ester)-b-PEG polyion complexes

Figure S12 .

 S12 Figure S12. DLS analysis of the thermal stability of γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion

  Author manuscript version of paper accepted for publication in Mater. Sci. Eng. C, 2019, 109920, after peer review and technical editing by the publisher. DOI: 10.1016/j.msec.2019.109920 11

  Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy(SEM) analysis of γ-Fe 2 O 3 -PAA/pSe and γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs were performed on a Zeiss Libra200FE microscope (TEM) and a Zeiss EVO 18 (SEM). Cell culture and Incubation of cells with γ-Fe 2 O 3 -PAA/pSe-

	For TEM study of cells, the following protocol was applied. γ-Fe 2 O 3 -PAA/pSe-b-
	PEG PICs (synthesized at c = 6 × 10 -4 wt% and Z =1 in PBS1×, t = 304 h, D H =
	414 nm) were incubated with MC3T3-E1 cells at [γ-Fe 2 O 3 ] = 6 × 10 -3 wt% for 24
	h, 48 h and 72 h (see:

b-PEG for details). After incubation, excess of medium was removed, and cells were washed in 0.2 M PBS (pH = 7.4) and fixed in 2% glutaraldehyde-phosphate buffer 0.1 M for 1 h at room temperature. Fixed cells were washed in 0.2 M PBS.

.5 Transfer of γ-Fe 2 O 3 -PAA/pSe-b-PEG polyion complexes in bio-fluids for DLS analysis

	Author manuscript version of paper accepted for publication in Mater. Sci. Eng. C, 2019, 109920,
	after peer review and technical editing by the publisher. DOI: 10.1016/j.msec.2019.109920
	wt% and Z = 1 in water) were concentrated by solvent evaporation to [γ-Fe 2 O 3 ] = CPMG sequence, while choosing an echo time (TE) around T 2 /50 and a number of
	8.8 × 10 -3 wt% 374 h after direct mixing. Sample (2 mL in a closed polystyrene data points from 150 to 250, the decay curve of transverse magnetization being
	cuvette with an immerged optic fibre temperature probe, Opens ™ , Quebec city, fitted by a mono-exponential law. Relaxivities were obtained from the slope of the
	Quebec CA) was placed inside the coil. The macroscopic temperature of the linear variation with [Fe] in mM of the longitudinal (respectively transverse) decay
	sample was measured every second. Once thermal equilibrium was reached to an rate of water 1 H after subtraction of respective value measured on pure water.
	initial temperature about 38 °C set by a water circulation jacket, the alternating
	magnetic field was applied for 33 min and then switched off. After recovery of thermal equilibrium, magnetic field was applied a second time for 51 min. 2.2γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs at Z = 1 were prepared in water at T = 25 °C from
	2.2.4 Relaxometry solutions at [γ-Fe 2 O 3 ] = [pSe] = 6 × 10 -4 wt%. 342 h after mixing, cluster dispersion with
	Longitudinal (r1) and transverse (r2) relaxation rates of hydrogen nuclear spins D H = 417 ± 15 nm was concentrated to [γ-Fe 2 O 3 ] = 7.2 × 10 -3 wt% by solvent evaporation
	were measured at 1.41 T (60 MHz 1 H resonance, i. e. close to the 1.5 Tesla clinical and transferred into Simulated Body Fluid (SBF), DMEM, DMEM supplemented with
	MRI scanners) and 37 °C on a Bruker Minispec mq60 relaxometer using 3.7 g L -1 of NaHCO 3 , DMEM containing 10 vol% of fetal bovine serum (FBS), RPMI,
	respectively an inversion-recovery (IR) and Carr-Purcell-Meiboom-Gill (CPMG) RPMI supplemented with 2 g L -1 of NaHCO 3 and RPMI containing 10 vol% of FBS as
	spin-echo sequences. Repetition time (TR), echo time (TE), and number of data [γ-Fe 2 O 3 ] = 6 × 10 -4 wt%. Samples were then incubated at T = 37 °C and shaken at 79
	points were adjusted carefully to minimize the experimental uncertainty on the rpm. D H measurements were performed at T = 37 °C.
	measured T 1 and T 2 values for a series of iron concentrations [Fe] of 5, 2, and 0.4
	mM for individual γ-Fe 2 O 3 -PAA nanoparticles, and of 0.0752, 0.0251 and 0.00835
	mM for γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs. The receiver gain was adjusted between
	45 and 53 dB to optimize signal-to-noise ratio without saturation. For the IR
	sequence, initial delay time was adjusted around T 1 /10, while the final delay was at
	least 3 T 1 , and the recycling delay (RD) at least 5 T 1 , according to
	2 O 3 -PAA/pSe-b-PEG PICs (synthesized at c = 6 × 10 -4 recommendations for relaxometry measurements. [51] RD was kept constant for the
	13

2.2.6 Cell culture, incubation with γ-Fe 2 O 3 -PAA/pSe-b-PEG, cell viability assessment and amount of iron oxide internalized

	serum (FBS, PAN Biotech, Germany) and DMEM with 10 vol% FBS and 1% enabling the determination of the number of viable cells by UV-vis spectrometry.
	Penicillin-Streptomycin solution (Gibco, USA) were respectively applied for After incubation of cells with γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs, the PBS washes
	MC3T3-E1 and HepG2 cell cultures. Media were replenished daily. After 4 were slowly sucked out and CCK-8 working solution (120 μL) was directly added
	passages for MC3T3-E1 and 7 passages for HepG2, cells were detached by 0.25 % into each well. Subsequently, the plates were incubated at 37 °C for 1 h. Then,
	trypsin/EDTA (Gibco, USA) as soon as they were confluent in the bottom of the supernatant (100 μL) was extracted from each well and transferred into a new 96-
	culture flasks and used for the seeding and viability assessments. Cells were well plate. Optical Densities (OD), which correspond to the absorbance of the
	directly seeded onto 96-well tissue culture plates with an initial density of 2 × 10 4 isolated supernatants at 450 nm, were measured using a microplate reader
	cells per well. (Varioskan Flash Multimode Reader, Thermo Scientific, USA). The numbers of
	Incubation of cells with γ-Fe 2 O 3 -PAA/pSe-b-PEG: γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs living cells corresponding to the respective OD values were determined using
	(synthesized at c = 6 × 10 -4 wt% and Z = 1 in PBS1×, t = 304 h after direct mixing, calibration curves (Figure S6, Supporting Information).
	D H = 414 ± 10 nm) were concentrated by solvent evaporation to [γ-Fe 2 O 3 ] = 7.2 ×
	10 -2 wt%, sterilized by UV irradiation and transferred into culture media. After the
	24 h required for cellular adhesion, culture media were replaced by freshly
	prepared media containing the γ-Fe 2 O 3 -PAA/pSe-b-PEG PICs (200 μL per well)
	so that concentrations in iron oxide in wells were respectively 0, 6 × 10 -5 , 6 × 10 -4 ,
	6 × 10 -3 and 6 × 10 -2 wt% (corresponding to [Fe] = 0, 7.51 × 10 -3 , 7.51 × 10 -2 ,
	0.751 and 7.51 mM, respectively). After being co-cultured with γ-Fe 2 O 3 -PAA
	/pSe-b-PEG PICs for respectively 24 h, 48 h and 72 h, the supernatants were
	collected and cells gently washed eight times with PBS (200 μL per well).
	Cell culture: MC3T3-E1 (CRL-2593, ATCC, USA) and HepG2 cells (HB-8065, Cell viability assessment: quantitative cell viability assessments were conducted
	ATCC, USA) were maintained in cell culture flasks and sub-cultivated in a cell using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan). This assay is based on the
	incubator at 37 °C (5 % CO 2 atmosphere, 95 % relative humidity). Minimum conversion of WST-8 (monosodium salt) into a highly water-soluble formazan dye
	Essential Medium Alpha Basic (α-MEM, Gibco, USA) with 10 vol% fetal bovine upon bioreduction in the presence of an electron carrier (1-Methoxy PMS),

Table S1 .

 S1 Table S1 resumes the parameters characterizing the γ-Fe 2 O 3 -PAA/pSe mixtures of the study. Concentrations, volumes and amounts of charge of γ-Fe 2 O 3 -PAA

			Solutions to mix				Mixtures
	γ-Fe 2 O 3 -PAA dispersions		pSe solutions	γ-Fe 2 O 3 -PAA/pSe mixtures
	𝑐𝑐 𝑁𝑁𝑁𝑁 (wt%)	𝑉𝑉 𝑁𝑁𝑁𝑁 (mL)	𝑁𝑁 -(mol)	𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 (wt%)	𝑉𝑉 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 (mL)	𝑁𝑁 + (mol)	𝑐𝑐 𝑁𝑁𝑁𝑁 (wt%)	(wt%) 𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝	𝑍𝑍 =	𝑁𝑁 -𝑁𝑁 +
	6.10 -3	10	3.4.10 -7	6.10 -3	0.85	3.4.10 -7 5.5.10 -3 4.7.10 -4	1
	6.10 -4	10	3.4.10 -8	6.10 -4	0.85	3.4.10 -8 5.5.10 -4 4.7.10 -5	1
	6.10 -5	10	3.4.10 -9	6.10 -5	0.85	3.4.10 -9 5.5.10 -5 4.7.10 -6	1
	6.10 -6	10	3.4.10 -10	6.10 -6	0.85	3.4.10 -10 5.5.10 -6 4.7.10 -7	1

Table S2 .

 S2 Table S2 resumes the parameters characterizing the γ-Fe 2 O 3 -PAA/pSe-b-PEG mixtures of the study. Concentrations, volumes and amounts of charge of γ-Fe 2 O

			Solutions to mix				Mixtures
	γ-Fe 2 O 3 -PAA dispersions	pSe-b-PEG solutions	γ-Fe 2 O 3 -PAA/pSe-b-PEG
	𝑐𝑐 𝑁𝑁𝑁𝑁 (wt%)	𝑉𝑉 𝑁𝑁𝑁𝑁 (mL)	𝑁𝑁 -(mol)	𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 (wt%)	𝑉𝑉 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝 (mL)	𝑁𝑁 + (mol)	𝑐𝑐 𝑁𝑁𝑁𝑁 (wt%)	(wt%) 𝑐𝑐 𝑎𝑎𝑝𝑝𝑙𝑙𝑝𝑝	𝑍𝑍 =	𝑁𝑁 -𝑁𝑁 +
	6.10 -4	4	1.38.10 -8 6.10 -4	6.28	1.38.10 -7	2.3.10 -4 3.8.10 -4	0.1
	6.10 -4	10	3.45.10 -8 6.10 -4	3.14	6.90.10 -8	4.6.10 -4 1.4.10 -4	0.5
	6.10 -4	10	3.45.10 -8 6.10 -4	1.57	3.45.10 -8 5.18.10 -4 8.1.10 -5	1
	6.10 -4	10	3.45.10 -8 6.10 -4 0.157	3.45.10 -9 5.91.10 -4 9.3.10 -6	10
	6.10 -4 10.191 3.52.10 -8 6.10 -4 0.016 3.51.10 -10 5.99.10 -4 9.4.10 -7	100

3 -PAA dispersions and pSe-b-PEG solutions mixed to obtain γ-Fe 2 O 3 -PAA/pSe-b-PEG mixtures, and, concentrations in iron oxide and in pSe-b-PEG, and charge ratio Z of the mixtures.

Table S3 .

 S3 Concentrations, volumes and amounts of charge or monomer units (for PEG 10K ) of γ-Fe 2 O 3 -PAA dispersions and either serine or PEG 10K solutions mixed to obtain γ-Fe 2 O 3 -PAA/serine and γ-Fe 2 O 3 -PAA/PEG 10K mixtures. Concentrations in iron oxide and concentrations in either serine or PEG 10K , and charge ratio Z or ratio between negative charges brought by the nanoparticles and the amount of ethylene oxide monomeric units

	2 O 3 -PAA dispersions	Serine solutions	γ-Fe 2 O 3 -PAA/serine mixtures
	𝑐𝑐 𝑁𝑁𝑁𝑁 (wt%)	𝑉𝑉 𝑁𝑁𝑁𝑁 (mL)	𝑁𝑁 -(mol)	𝑐𝑐 𝑠𝑠𝑠𝑠𝑀𝑀𝑠𝑠𝑙𝑙𝑠𝑠 (wt%)	𝑉𝑉 𝑠𝑠𝑠𝑠𝑀𝑀𝑠𝑠𝑙𝑙𝑠𝑠 (mL)	𝑁𝑁 + (mol)	𝑐𝑐 𝑁𝑁𝑁𝑁 (wt%)	𝑐𝑐 𝑠𝑠𝑠𝑠𝑀𝑀𝑠𝑠𝑙𝑙𝑠𝑠 (wt%)	𝑍𝑍 =	𝑁𝑁 -𝑁𝑁 +
	6.10 -4	2	6.8.10 -9	6.10 -4	0.119	6.8.10 -9	5.7.10 -4 3.4.10 -5	1
	6.10 -5	2	6.8.10 -10 6.10 -5	0.119	6.8.10 -10	5.7.10 -5 3.4.10 -6	1
	6.10 -6	2	6.8.10 -11 6.10 -6	0.119	6.8.10 -11	5.7.10 -6 3.4.10 -7	1
	γ-Fe 2 O 3 -PAA dispersion	PEG 10K solution	γ-Fe 2 O 3 -PAA/PEG 10K mixture
	𝑐𝑐 𝑁𝑁𝑁𝑁	𝑉𝑉 𝑁𝑁𝑁𝑁	𝑁𝑁 -	𝑐𝑐 𝑁𝑁𝑃𝑃𝑃𝑃	𝑉𝑉 𝑁𝑁𝑃𝑃𝑃𝑃	𝑁𝑁 𝑠𝑠𝑝𝑝𝑙𝑙𝑝𝑝𝑠𝑠𝑠𝑠𝑀𝑀	𝑐𝑐 𝑁𝑁𝑁𝑁	𝑐𝑐 𝑁𝑁𝑃𝑃𝑃𝑃	𝑁𝑁 -
	(wt%)	(mL)	(mol)	(wt%)	(mL)	(mol)	(wt%)	(wt%)	𝑁𝑁 𝑠𝑠𝑝𝑝𝑙𝑙𝑝𝑝𝑠𝑠𝑠𝑠𝑀𝑀
	6.10 -4	2	6.8.10 -9	6.10 -4	0.050	6.8.10 -9	5.9.10 -4 1.5.10 -5	1
				𝑁𝑁 -𝑁𝑁 𝑚𝑚𝑇𝑇𝑚𝑚𝑇𝑇𝑚𝑚𝑤𝑤𝑤𝑤 (

Acknowledgements

This research was supported by the ''1000 talents'' program (Sichuan, China) under the Southwest University of Science and Technology (SWUST) program 16QR003, by the National Natural Science Foundation of China (NSFC, project numbers: 41650110481 and 41630646) and by the SWUST Postgraduate

Author manuscript version of paper accepted for publication in Mater. Sci Vibrating sample magnetometry measures the magnetization, M, as a function of excitation, H, of a liquid dispersion containing a volume fraction of magnetic material from the signal induced in detection coils when the sample is moved periodically in an applied magnetic field (via synchronous detection and with an appropriate calibration). Figure S4 shows the evolution of the macroscopic magnetization M(H) of the γ-Fe 2 O 3 nanoparticles, γ-


Guanzheng Zhuang

Maguy Jaber
email: maguy.jaber@upmc.fr

Francisco Rodrigues

Baptiste Rigaud

Philippe Walter

Zepeng Zhang

A new durable pigment with hydrophobic surface based on natural nanotubes and indigo: interactions and stability

Keywords: hybrid pigments, clay minerals, organic dyes, chemical stability, photostability

Covering with polyorganosilane (POS) was proved as an effective way to enhance the chemical and thermal stability of clay/dye hybrid pigments. But the photostability and interactions with clay minerals, dyes and POS layer has never been reported. In order to investigate above issues, new organic-inorganic hybrid pigments based on halloysite (Hal) and indigo (In) were prepared by grinding method. X-ray diffraction, transmission electron microscopy, thermogravimetry, Fourier transform infrared spectroscopy were applied to characterize the structure of In-Hal (without POS layer) and In-Hal-POS (with POS layer) pigments. Solid state nuclear magnetic resonance (NMR) was employed to reveal the interactions between Hal, In and POS. Reflection spectra and CIE 1976 color space system were used to evaluate the color parameters and color changes of pigments. Thermal stability, chemical resistance to ethanol, 1 mol•L -1 HCl and 1 molL -1 NaOH, and light fastness to visible light were tested.

Indigo molecules dispersed on the surface of Hal nanotubes. POS layer homogeneously covered on the surface of hybrid pigments, without changing the crystal structure and morphology of Hal. Covering with POS layer seldomly affect the color of hybrid pigments. However, In-Hal-POS exhibited better stability than In-Hal, due to hydrophobic surface which can prevent indigo molecules from chemical reactions and degradation. A new route was proposed to prepare organic-inorganic hybrid pigments, ignoring the interaction between dye molecules and substrates.

Introduction

Maya blue (MB) is a fantastic pigment which was extensively used in ancient times in Mesoamerica. The MB was nearly extended to all Mesoamerican cultures. Many Maya cultures were kept by MB in the forms of murals, figurines and decoration of buildings. MB keeps its color well after centuries of vicissitudes. The remarkable resistance to weather, light, acids, bases, heat, as well as biodegradation had attracted much attention from scientists and archeologists. MB was confirmed as a hybrid pigments of palygorskite and natural indigo dye [START_REF] Doménech | Chemometric study of Maya Blue from the voltammetry of microparticles approach[END_REF][2][START_REF] Río | Chapter 18 -The Maya Blue Pigment[END_REF]. In the past decades, many efforts were taken to reveal the structure and stabilization mechanism of MB [START_REF] Chiari | Crystal structure refinements of palygorskite and Maya Blue from molecular modelling and powder synchrotron diffraction[END_REF][START_REF] Hubbard | Structural study of Maya Blue: textural, thermal and solidstate multinuclear magnetic resonance characterization of the palygorskite-indigo and sepiolite-indigo adducts[END_REF][START_REF] José-Yacamán | Maya blue paint: an ancient nanostructured material[END_REF][START_REF] Polette | Maya blue: application of XAS and HRTEM to materials science in art and archaeology[END_REF][START_REF] Reinen | The nature of the colour centres in 'Maya blue'-the incorporation of organic pigment molecules into the palygorskite lattice[END_REF][START_REF] Tilocca | The Color and Stability of Maya Blue: TDDFT Calculations[END_REF][START_REF] Van Olphen | Maya blue: a clay-organic pigment?[END_REF]. The interaction between palygorskite and indigo is believed to result in the amazing stability of MB, but the real nature of the interaction is still controversial.

The most acknowledged interactions encompass hydrogen bonding, van der Waals forces, steric shielding and interaction between indigo and octahedral cations of palygorskite [START_REF] Río | Chapter 18 -The Maya Blue Pigment[END_REF].

Despite the controversial mechanism, MB pigments provides a strategy of inorganic-organic hybrids to enhance the stability of organic dyes in applications.

Many scientists tried to re-create the MB and MB-like pigments by using clay minerals and dyes. Adsorption in solvents and grinding in solid state were taken. A serious MB-like pigments with different colors were prepared using natural or synthetic dyes (such as methyl red, alizarin, murexide, Sudan red, cationic red X-GRL, methylene blue, and methyl violet etc.) [START_REF] Fan | Facile preparation of stable palygorskite/cationic red X-GRL@ SiO "Maya Red" pigments[END_REF][START_REF] Liu | Attapulgite/bentonite interactions for methylene blue adsorption characteristics from aqueous solution[END_REF][START_REF] Giustetto | Sorption of red dyes on palygorskite: Synthesis and stability of red/purple Mayan nanocomposites[END_REF][START_REF] Giustetto | Spectroscopic characterization and photo/thermal resistance of a hybrid palygorskite/methyl red Mayan pigment[END_REF][START_REF] Zhang | A comparative study about adsorption of natural palygorskite for methylene blue[END_REF]. Palygorskite [START_REF] Giustetto | Sorption of red dyes on palygorskite: Synthesis and stability of red/purple Mayan nanocomposites[END_REF][START_REF] Giustetto | Spectroscopic characterization and photo/thermal resistance of a hybrid palygorskite/methyl red Mayan pigment[END_REF][START_REF] Wang | Palygorskite-based hybrid fluorescent pigment: preparation, spectroscopic characterization and environmental stability[END_REF], sepiolite [START_REF] Giustetto | Chemical stability and dehydration behavior of a sepiolite/indigo Maya Blue pigment[END_REF][START_REF] Ovarlez | Indigo chemisorption in sepiolite. Application to Maya blue formation[END_REF][START_REF] Ovarlez | The Incorporation of Indigo Molecules in Sepiolite Tunnels[END_REF][START_REF] Ovarlez | Indigo-sepiolite nanohybrids: Temperature-dependent synthesis of two complexes and comparison with indigo-palygorskite systems[END_REF] and zeolite [START_REF] Dejoie | Indigo@Silicalite: a New Organic-Inorganic 39 Hybrid Pigment[END_REF] were used as the matrix of hybrid pigments because of the nanosized porous structure which may accept more dye molecules and interact with each other. Besides, montmorillonite was reported as the matrix to prepare hybrid pigment by intercalating the dyes into the interlayer space [START_REF] Fournier | Physico-chemical characterization of lake pigments based on montmorillonite and carminic acid[END_REF][START_REF] Mahmoodi | A hybrid dye-clay nano-pigment: Synthesis, characterization and application in organic coatings[END_REF][START_REF] Raha | Dye/Clay intercalated nanopigments using commercially available non-ionic dye[END_REF]. These hybrid pigments, however, exhibited inferior stability to the ancient MB. In addition, the previous literature mostly concerned about the chemical stability of the hybrid pigments, but ignored the photostability, which is very important for the conservation of colorful products, especially for arts and constructions.

Herein, new substrates for hybrid pigments are considered. Halloysite (Hal) (Al 2 (OH) [START_REF] Chiari | Crystal structure refinements of palygorskite and Maya Blue from molecular modelling and powder synchrotron diffraction[END_REF] Si 2 O 5 2H 2 O) [START_REF] Joussein | Chapter 2 -Geology and Mineralogy of Nanosized Tubular Halloysite[END_REF], a natural nanotubular clay mineral (see Fig. 1), is a promising host for organic-inorganic hybrid pigments. Generally, the length of halloysite nanotubes varies from ca. 0.02 to 30 µm and the external diameter from ca. [START_REF] Dong | Colorful Superamphiphobic Coatings with Low Sliding Angles and High Durability Based on Natural Nanorods[END_REF] to 190 nm, with an internal diameter range of ca. 10-100 nm [START_REF] Bate | Morphology and structure of endellite and halloysite[END_REF][START_REF] Churchman | Characteristics of Fine Pores in Some Halloysites[END_REF]. The very large diameter of the halloysite lumen makes it potentially suitable for the accommodation of a range of guests. As a naturally occurring hydrated polymorph of kaolinite, it has a similar structure and composition, but the unit layers are separated by a monolayer of water molecules. Hydrated halloysite has a basal spacing (d 001 ) of 10 Å, which is ∼3 Å larger than that of kaolinite. The interlayer water is weakly held, so that halloysite-(10 Å) can readily transform to halloysite-(7 Å) by dehydration.

Tubular structure of halloysite results from the wrapping of the clay layers around onto themselves to form hollow cylinders under favorable geological conditions. This wrapping process is driven by a mismatch in the periodicity between the oxygen sharing tetrahedral SiO 4 sheets and adjacent octahedral [A1O 6 ] sheets in the 1:1 layer.

The previous work about MB and MB-like pigments always concern about the interaction between dye molecules and clay substrates. However, the secret of MB has never been revealed by convincing evidences. Furthermore, hybrid pigments with the same stability to MB has not been re-produced by modern people. Another strategy to improve the stability of hybrid pigments, i.e., covering the hybrid pigment surface with a protective layer, should be considered. In this way, the interaction between dye molecules and substrates is insignificant. A substrate supports the dye molecules, which gives the color, and a protective layer packs the hybrid of substrates and dyes. Some attempts about a layer of silica [START_REF] Fan | Facile preparation of stable palygorskite/cationic red X-GRL@ SiO "Maya Red" pigments[END_REF][START_REF] Zhang | Learning from ancient Maya: Preparation of stable palygorskite/methylene blue@SiO 2 Maya Blue-like pigment[END_REF][START_REF] Zhang | Facile preparation of stable palygorskite/methyl violet@SiO2 "Maya Violet" pigment[END_REF] or polyorganosilane (POS) [START_REF] Dong | Colorful Superamphiphobic Coatings with Low Sliding Angles and High Durability Based on Natural Nanorods[END_REF][START_REF] Dong | Photochromic and Super Anti-Wetting Coatings Based on Natural Nanoclays[END_REF][START_REF] Tian | Mechanically Robust and Thermally Stable Colorful Superamphiphobic Coatings[END_REF][START_REF] Zhang | From Maya blue to biomimetic pigments: durable biomimetic pigments with self-cleaning property[END_REF] covering the hybrid pigments surface were carried out. The authors confirmed that surface modification is an effective method to improve the stability of pigment.

Particularly, hybrid pigments with a POS layer showed excellent chemical and thermal stability. The colorimetric properties, chemical and heat resistance are very significant for arts, architectures, industrial and daily products. In the literature, the chemical and thermal stability of hybrid pigments were discussed, however their photostability and the interactions between clay minerals, dyes and POS layer have never been revealed.

In this work, Hal was employed as a new substrate due to its nanotubular structure and large specific surface area. Indigo was chosen as the color giving agent.

Polycondensation between two different organosiloxanes occurred on the surface of the hybrid of Indigo/Hal, and finally resulted in a protective layer. In comparison, the hybrid pigment without covering with POS was also prepared. The structure, thermal stability, chemical resistance and photostability of these pigments were characterized to verify this new strategy for preparing durable hybrid pigments. What is more important, the interaction among Hal, indigo and POS was investigated.

Fig. 1 The structure of halloysite.

Materials and methods

Materials

Hal, indigo (abbreviated as 'In' in the names of pigments), n-hexadecyltrimethoxysilane (HDTMS, purity of 85%) tetraethoxysilane (TEOS, purity of 99%) were purchased from Sigma-Aldrich, Inc, France. These materials were used without further purification. HCl (37%), NaOH (98.8%) and ethanol (95%) were purchased from VWR Co. Ltd. NaOH solution (1 molL -1 ) was prepared by dissolving NaOH solids into water. HCl solution (1 molL -1 ) was prepared by diluting concentrated HCl.

Preparation of hybrid pigments

In-Hal pigment was obtained by grinding halloysite and indigo (2% of the mass of halloysite) together in an agate mortar by hand for 20 min (Fig. 2 (A)). Then, In-Hal-POS pigment were prepared by polycondensation of HDTMS and TEOS in the presence of In-Hal pigment (Fig. 2 (B)). 0.50 g of the In-Hal pigment was fully dispersed in 9 mL of ethanol and 1 mL of ammonia saturated ethanol, magnetically stirring for 3 min in a vial. Then, proper amounts of TEOS and HDTMS (concentration of 0.15 molL -1 ) were charged into the mixture with stirring. The dispersion was ultrasonicated for 30 min at 50 °C, and then 1.44 mL of water was injected quickly into the dispersion under ultrasonication. The vial was sealed and ultrasonicated at 50 °C for 1 h. The products were centrifuged at 6000 rpm for 5 min to remove the supernatant followed by washing 3 times each with ethanol. Then, the hydrophobic In-Hal-POS pigment was obtained after drying at 50 °C for 24 h.

Characterization

X-ray diffraction (XRD) test was conducted at a Bruker D8 diffractometer from 5° to 70° at a speed of 4 s per step (step size of 0.02°). Fourier transform infrared (FTIR) spectra were collected from a Cary 630 FTIR spectrometer in the range of 4000-650 cm -1 . Thermogravimetric (TG) analysis was carried out at a TA-SDTQ600 type analyzer from room temperature to 900 °C in the air atmosphere, with the heating rate of 10 °C/min. Transmission electron microscopy (TEM) was performed on a Tecnai Spirit G2 transmission electron microscope with the voltage of 120 kV. The samples were dispersed in ethanol with ultrasound for 30 min, then the dispersions were dropped on copper supports with carbon grid. C, 1 H and 27 Al Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra were obtained on a Bruker Advance III spectrometer equipped with a 2.5 mm and 4 mm H-X MAS probe, operating at frequency of 500.07 MHz ( 1 H), 125.76 MHz ( 13 C), 99.36 MHz ( 29 Si) and 130.30 MHZ ( 27 Al). Chemical shifts were calibrated using the carboxyl signal of adamantane (38.52 ppm) for 13 C and (1.85 ppm) for 1 H, TMS (0 ppm) for 29 Si and Al(NO 3 ) 3 (0 ppm) for 27 Al as external standard. The 1 H experiment using 90° pulse was recorded with a spinning rate of 14 kHz, with a number of scans of 128 and 1 s of recycle delay. The 13 C Cross-Polarization spectra were acquired with a MAS rate of 14 kHz, a ramp-CP contact time of 2 ms and a 2 s of recycle delay and with a 1 H decoupling spinal. Over an acquisition time of 40 ms, the number of scans to obtain the spectra depends on the S/N obtained for each sample. Spectra were processed with a zero-filling factor of 2 and with an exponential decay corresponding to a 10 Hz line broadening in the transformed spectra. Only spectra with the same line broadening are directly compared. The 27 Al experiment using 30° pulse was recorded in the same condition of spinning rate (14 kHz), with a number of scans of 2048, 263 kHz spectral width and with 500 ms of recycle delay.

The 29 Si experiment using 90° pulse with a 1 H decoupling spinal was recorded in exactly the same condition of spinning rate. The number of scans was 8192 and the recycling time D1 was 10 s. The Spectra were processed with a zero-filling factor of 2 and without an exponential decay. . Similar calculation can be found in the literature [START_REF] Trigueiro | When anthraquinone dyes meet pillared montmorillonite: Stability or fading upon exposure to light?[END_REF][START_REF] Zhuang | Dressing protective clothing: stabilizing alizarin/halloysite hybrid pigment and beyond[END_REF]. To avoid the influence of humidity and temperature, the tableted pigments were placed in a special apparatus (see Fig. 3) with desiccants at 20 °C.

The absorbance, reflectance and CIE (Commission

Fig. 3 The apparatus for photostability test. The TG and corresponding derivative TG (DTG) curves of Hal, Indigo and hybrid pigments are displayed in Fig. 6. Hal presents three steps of mass loss. The mass loss before 120 °C, corresponding to the DTG peak at 56 °C, is attributed to the loss of adsorbed water on the Hal surface [START_REF] Yuan | Chapter 7 -Thermal-Treatment-Induced Deformations and Modifications of Halloysite[END_REF][START_REF] Joussein | Halloysite clay minerals-a review[END_REF]. The small mass loss at the 152 °C (DTG peak) is due to the mass loss of the small amount of interlayer water [START_REF] Joussein | Differences in the dehydration-rehydration behavior of halloysites: new evidence and interpretations[END_REF]. Although XRD pattern demonstrates a dehydrated Hal, the wide (001) reflection proves a little interlayer water remains. The adsorbed and interlayer water amount is about 3.5%.

The third step at the range of 350-600 °C, corresponding to the DTG peak at 485 °C, is assigned to the dehydroxylation of Al-OH and Si-OH groups [START_REF] Joussein | Halloysite clay minerals-a review[END_REF][START_REF] Joussein | Differences in the dehydration-rehydration behavior of halloysites: new evidence and interpretations[END_REF][START_REF] Yuan | Chapter 1 -General Introduction[END_REF][START_REF] Yuan | Functionalization of halloysite clay nanotubes by grafting with γ-aminopropyltriethoxysilane[END_REF] In-Hal shows the similar spectrum with Hal, except for a more intense and sharper signal at around 2. The phenomenon of changing to greenish color was also found in MB pigment.

Antonio et al. [START_REF] Doménech | Dehydroindigo: A New Piece into the Maya Blue Puzzle from the Voltammetry of Microparticles Approach[END_REF][START_REF] Doménech | Indigo/Dehydroindigo/Palygorskite Complex in Maya Blue: An Electrochemical Approach[END_REF] reported oxidation of indigo in MB pigment and found the presence of dehydroindigo (the oxidized form of indigo) contributed the greenish color of Maya blue. The FTIR spectra (Fig. 11) reveal the structural changes of the hybrid pigment. The FTIR spectrum of Hal demonstrates the presence of inner-surface -OH (3693 cm -1 ), inner -OH (3622 cm -1 ) and surface adsorbed water (3548 cm -1 ) [START_REF] Cheng | Infrared and infrared emission spectroscopic study of typical Chinese kaolinite and halloysite[END_REF][START_REF] Cheng | A spectroscopic comparison of selected Chinese kaolinite, coal bearing kaolinite and halloysite-A mid-infrared and near-infrared study[END_REF][START_REF] Theo Kloprogge | Raman microprobe spectroscopy of hydrated halloysite from a Neogene cryptokarst from Southern Belgium[END_REF]. The bands at 1652 cm -1 and 1632 cm -1 correspond to the In summary, a new strategy to enhance the thermal stability and photostability of dye/clay hybrid pigments by covering with POS layer was verified. It is a promising method to produce durable organic-inorganic hybrid pigments. The grafting reaction of organosilanes with clay surface and the hydrophobicity play important roles in improving the stability of hybrid pigments. In the future work, the compatibility between this hybrid pigment and organic solvents, and the application in art works (e.g., oil paintings) should be attractive.

Fig. 2

 2 Fig. 2 Experimental procedure to prepare (A) In-Hal and (B) In-Hal-POS pigments.

13

 13

  Internationale de L'Eclairage) parameters were obtained from an Ocean Optics spectrometer USB4000. The light source of Ocean Optics DH-2000-BAL was used for absorbance, and HL2000 light source was employed for reflectance and CIE results. CIE 1976 color space system was applied to evaluate the color of the pigments. Lightness (L*) represents the brightness (+) or darkness (-) of the color, i.e., more positive L* values refer to whiter while more negative L* values represent darker. The values of a* and b* indicate the color details: +a* is the red direction, -a* the green direction, +b* the yellow direction, and -b* the blue direction. Before the reflectance and CIE test, the samples were pressed into tablets with the pressure about 30 MPa. The total change in color was calculated as , where L 1 * refers to the L* value of a sample, L 0 * refers to the L* value of the reference, and similar to the other two parameters. Thermal stability of hybrid pigments was tested by calculating the total color change between raw pigment and that heated at high temperatures for 4 h. Photostability was evaluated under visible lights for 360 h using an Advanced Illumination SL164 type LED light. The working distance between the light and the samples is 10 cm, corresponding to a diameter of 3.6 cm of working area, with the illuminance of 50 kLux. Hence, the fading test applied for the prepared hybrid pigments gave a total light dose of 18000 kLux•h (50 kLux × 360 h). This light dose equals to about 30 years in a common museum gallery illuminated at 200 Lux (10 h of light exposure per day, 6 days per week, and 50 weeks per year)

3. Results and discussion 3 . 1

 31 Structure and morphologyThe XRD pattern of Hal (Fig.4) exhibits the typical reflections of halloysite, with a little amount of quartz and albite. Hal shows a basal spacing of 0.75 nm, demonstrating a dehydrated halloysite. In-Hal and In-Hal-POS pigments present the similar XRD patterns with the raw Hal, without any change of the basal spacing. This phenomenon indicates that indigo molecules did not intercalate into the interlayer space of halloysite. XRD pattern of indigo proves a crystalline structure of indigo solid. But no reflection belonging to indigo crystals was observed in the XRD patterns of hybrid pigments. This fact demonstrates that indigo molecules re-organized on the surface (including external and inner surface) of hybrid pigments. As Fig. 5 shows, halloysite exhibits the tubular morphology. The length of Hal nanotubes ranges from dozens to hundreds of nanometers. Hal shows an inner diameter of more than 10 nm. Although the indigo molecules cannot insert into the interlayer space, they can stay on both the external and inner surface. In-Hal hybrid pigment shows the similar morphology with Hal. Covered with POS, In-Hal-POS pigment still exhibits the tubular morphology and similar surface. This fact indicates that polycondensation between TEOS and HDTMS happened homogeneously on Hal surface, uniformly embedding indigo molecules and Hal.

Fig. 4

 4 Fig. 4 XRD patterns of (A) Hal and hybrid pigments and (B) indigo.

  . The oxidation of indigo starts at about 350 °C. Continuous oxidation goes until 580 °C. In-Hal exhibits the similar TG and DTG curves with raw Hal. It is difficult to distinguish the oxidation process of indigo dyes in In-Hal sample, due to the overlap of dehydroxylation process. But 2% more mass loss above 300 °C of In-Hal than Hal indicates the including of indigo in the hybrid pigment. In the TG and DTG curves of In-Hal-POS, a new mass loss step at the range of 300 to 400 °C emerged, with the mass loss of 9.9%. This is assigned to the thermal decomposition of POS which covers the surface of hybrid pigment. Continuous oxidation of POS at higher temperatures mixes with the dehydroxylation of Hal and oxidation of indigo, resulting in the mass loss of 15.2%.

Fig. 6

 6 Fig. 6 TG and DTG curves of (A) Hal, (B) indigo, (C) In-Hal and (D) In-Hal-POS.

Fig. 7 13 C

 13 Fig. 7 13 C MAS NMR spectra of indigo, In-Hal and In-Hal-POS.

Fig. 8 1 HFig. 9 (

 19 Fig. 8 1 H MAS NMR spectra of (A) indigo, (B) Hal, (C) In-Hal and (D) In-Hal-POS.

Fig. 11 FTIRFig. 13 A

 1113 Fig. 11 FTIR spectra of (A) Hal and Hal-190, (B) indigo and indigo-190, (C) In-Hal

3. 6 15 .

 615 Fig. 14 Photostability of the hybrid pigments under visible light.

Fig. 15

 15 Fig. 15 Stabilization mechanism of hybrid pigments with the covering of POS layer.

Fig. 17

 17 Fig. 17 Digital pictures of (A) In-Hal, (B) In-Hal-150, (C) In-Hal-170, (D) In-Hal-190,

  3 ppm. The 1 H MAS NMR spectra of In-Hal can be decomposed into four individual signals, i.e., 7.3 ppm, 4.6 ppm, 2.3 ppm and 2.2 ppm. The signal at 7.3 ppm corresponds to the C-H groups from indigo. The signals at 4.6 ppm and 2.3 ppm are assigned to water and -OH groups in Hal, respectively. The N-H signal disappeared while a new signal emerged at 2.2 ppm, very close to the signal of -OH.This phenomenon demonstrating the interaction happened between N-H groups and Hal. Indeed, it is possible that hydrogen bonds can be formed between the N-H groups and Si-O or Al-O groups. Consequently, the chemical shift decreased to 2.2 ppm. The 1 H MAS NMR spectrum of In-Hal-POS can be decomposed into 6 signals.

Signals at 1.0 ppm, 1.5 ppm and 3.6 ppm are attributed to C-H groups of organosilanes. Signal at 2.1 ppm is assigned to the -OH groups of Hal and hydrogen bonged N-H groups. The signal at 4.6 ppm is attributed to water. Weak signal of C-H groups from indigo at 7.2 ppm is also noticed.

Table 1

 1 The chroma parameters of hybrid pigments and the corresponding heated pigments.

	Sample	Heating	L*	a*	b*	△E*
		temperature (°C)
	In-Hal	Without heating	44.1	-1.1	-23.9	0.0
	In-Hal-150	150	46.1	-3.1	-24.9	3.0
	In-Hal-170	170	50.6	-6.4	-21.0	8.9
	In-Hal-190	190	54.2	-6.9	-11.4	17.1
	In-Hal-POS	Without heating	42.1	1.4	-21.8	0.0
	In-Hal-POS-150 150	44.8	-1.2	-21.4	3.7

Acknowledgement

The support provided by China Scholarship Council (CSC) during the visit of Guanzheng Zhuang (No. 201706400010) to Sorbonne Université is acknowledged.