inﬂuence of the formulation process in electrostatic...

Influence of the Formulation Process in Electrostatic Assembly of Nanoparticles andMacromolecules in Aqueous Solution: The Interaction Pathway

Ling Qi,†,§ Jerome Fresnais,‡ Jean-Francois Berret,‡ Jean-Christophe Castaing,†Fanny Destremaut,§ Jean-Baptiste Salmon,§ Fabrice Cousin,| and Jean-Paul Chapel*,†,!

Complex Assemblies of Soft Matter Laboratory (COMPASS), CNRS UMI3254, Rhodia Center for Research andTechnology in Bristol, 350 Georges Patterson BouleVard, Bristol, PennsylVania 19007, Matiere et SystemesComplexes (MSC), UMR 7057 CNRS, UniVersite Denis Diderot Paris-VII, Batiment Condorcet, 10 rue AliceDomon et Leonie Duquet, 75205 Paris, France, Lab of the Future (LOF), UMR 5258 Rhodia, CNRS,UniVersite Bordeaux 1, 178 aVenue du Docteur Schweitzer, F-33608 Pessac cedex, France, Laboratoire LeonBrillouin (LLB), UMR CEA, CNRS 12, CEA Saclay, 91191 Gif-sur-YVette, France, and Centre de RecherchePaul Pascal (CRPP), UPR CNRS, UniVersite Bordeaux 1, 33600 Pessac, France

ReceiVed: July 16, 2010; ReVised Manuscript ReceiVed: August 24, 2010

The influence of the formulation process/pathway on the generation of electrostatic complexes made frompolyelectrolyte-neutral copolymers and oppositely charged nanocolloids is investigated in this work. Understrong driving forces like electrostatic interaction and/or hydrogen bonding, the key factor controlling thepolydispersity and the final size of the complexes is the competition between the reaction time of the componentsand the homogenization time of the mixed solution. The latter depends on the mixing pathway and wasinvestigated in a previous publication by tuning the mixing order and/or speed (Qi, L.; Fresnais, J.; Berret,J.-F.; Castaing, J.-C.; Grillo, I.; Chapel, J.-P. J. Phys. Chem. C 2010, 114 (30), 12870-12877). The formerdepends on the initial concentration of the individual stock solutions and the strength of the interaction andis investigated here on a system composed of anionic cerium oxide functional nanoparticles (CeO2-PAA) andcationic charged-neutral diblock copolymers (PTEA11K-b-PAM30K) or homopolyelectrolytes (PDADMAC100K).The electrostatic interaction was screened off completely by adding a large amount of salts. Desalting kineticswas then controlled by slowly decreasing the ionic strength from Ib ! 0.5 M, the minimum ionic strength tototally prevent the complexation of the two components, to lower values where the electrostatically screenedsystem undergoes an (abrupt) transition between an unassociated and a clustered state. Neutron scatteringdata evidenced differences in the nanostructure of complexes formed by either dilution or simple mixing.Furthermore, adsorption optical reflectometry experiments showed the impact of these different formulationprocesses on the wettability and antifouling properties of treated silica and polystyrene model surfaces. Bettercontrolled mixing processes were put forward at the end to improve the productivity and reproducibility ofthe complexes generation. In particular, a microfluidic chip coupled with dynamic light scattering was usedto better control the hydrodynamics of the complexation process.

Introduction

The electrostatic complexation of macromolecules andnanoparticles2,3 has attracted much attention in the past decade.Combining the advantageous properties of both the organic andinorganic worlds offers a great promise for engineering versatilefunctional structures with controlled physical and chemicalattributes at the nanometer scale. This synergy will certainlytrigger the emergence of a wide range of novel materials andprocessing techniques in various scientific and technologicalfields such as material science4-8 and biology.9-13

Compared to the abundant work on the mechanisms, structurecharacterizations, and functionalities, however, not much at-tention has been paid to the formulation process, which is akey issue for generating functional systems or devices on a largescale. Under a strong interaction as in the case of an electrostatic

complexation, it often takes a very long time to reach trueequilibrium leading to the formation of out-of-equilibrium“frozen” structures, which are not thermodynamically favored.It has been pointed out that the order of addition of inorganicions and polyelectrolytes affects the final structure of adsorbedpolyelectrolyte layers,14-16 i.e., the resulting structure dependsnot only on the bulk composition but also on whether thepolyelectrolyte or the salt was added first.

Similarly, the order of addition of two oppositely chargedpolyelectrolyte solutions17 determines the final net charge ofthe system, and that deviation from 1:1 stoichiometry in theformed aggregates increases with the ionic strength of thesystem. The mixing protocol seems to have a18,19 great impacton the size of the aggregates initially formed. These process-dependent features have of course important consequences intechnological applications. Why is such complexation process-dependent? The competition between the “reaction time”(depending on the initial concentration and the nature of theinteraction) and the “homogenization time” (ranging frommilliseconds20,21 to hours) of the mixed solution is certainly atthe origin of the “process hysteresis”.

* To whom correspondence should be addressed. E-mail: [email protected].

† Rhodia Center for Research and Technology in Bristol.§ UMR 5258 Rhodia, CNRS, Universite Bordeaux 1.‡ Universite Denis Diderot Paris-VII.| UMR CEA, CNRS 12, CEA Saclay.! UPR CNRS, Universite Bordeaux 1.

J. Phys. Chem. C 2010, 114, 16373–16381 16373

10.1021/jp106610t " 2010 American Chemical SocietyPublished on Web 09/13/2010

The “homogenization time” depends on the mixing pathwayand was investigated in a former publication1 by tuning themixing order and/or speed on a very similar system composedof cerium oxide nanoparticles and charged-neutral diblockcopolymers. The complexes final morphologies (size, shape,polydispersity) were found to depend strongly on the formulationprocess, while keeping at a smaller scale (clusters) the samenanostructure as shown via light and neutron scattering experi-ments. The impact of the structures of the complexes wereevaluated on some bulk (rheology) and surface (wetting/antifouling) properties. The results highlighted that a process-dependent formulation seen a priori as a drawback can be turnedinto an advantage: different properties can be developed fromdifferent morphologies while keeping the chemistry constant.

The “reaction time” depends on the initial concentration ofthe individual stock solutions and the nature of the interactionand is put under scrutiny here on a system composed of anioniccerium oxide functional nanoparticles (CeO2-PAA) and cationiccharged-neutral diblock copolymers or homopolyelectrolytes.The complexation is here purely electrostatically driven and bothbasic components have a good stability toward high ionicstrength (g1 M). These features enabled us to formulate“dormant solutions” in which the interaction between the twobuilding blocks is completely screened off allowing the strengthof the electrostatic interaction to be tuned by simple dilution ordialysis. The influence of the process on the nanostructures ofthe complexes together with their impact on the wettability andantifouling properties of treated model surfaces were studied.Finally, better controlled mixing processes were put forward atthe end to improve the productivity and reproducibility of thegeneration of the complexes. In particular, a microfluidic chipcoupled with dynamic light scattering was used to better controlthe hydrodynamics of the complexation process.

Materials and Methods

Chemicals. Nanoparticles. The nanoparticles used in thiswork were cationic cerium oxide nanocrystals, or nanoceria(CeO2). The CeO2 nanoparticles were synthesized by Rhodiachemicals. CeO2 dispersion is naturally stable only at pH <1.5and stabilization is provided by a combination of long-rangeelectrostatic forces and short-range hydration interactions. Atsuch a low pH, the ionic strength arises from the residual nitratecounterions present in the solution and acidic protons. This ionicstrength around 0.045 M gives a Debye screening length !D

-1

! 1.5 nm. An increase of the pH or ionic strength (>0.3 M)results in an irreversible aggregation of the particles, anddestabilization of the sols leading eventually to a macroscopicphase separation. For this system, the destabilization of the solsoccurs well below the point of zero charge of the ceria particles(pzc ) 7.9). The nanoceria particles have a !-potential ! )+30 mV and an estimated structural charge of QCeO2 ) +300e.22,23 The hydrodynamic radius RH of the CeO2 particles wasfound by dynamic light scattering to be 4.9 ( 0.6 nm.Furthermore, in order to complex with cationic polyelectrolytes,ceria nanoparticles were coated with poly(acrylic acid) with amolecular weight of 2000 g/mol, noted hereafter as CeO2-PAA2K

through a precipitation-redispersion process published previ-ously.24 The structural charge is estimated in this case asQCeO2-PAA2K ) -700 e (at neutral pH). The hydrodynamicdiameter RH of anionic CeO2-PAA2K particles (NP) was foundby dynamic light scattering to be 6 ( 0.8 nm.

Polymers. Both charged-neutral block copolymers andhomopolyelectrolytes were used in this work. Poly(trimethyl-ammonium ethylacrylate methylsulfate-b-poly(acrylamide)) was

used as a cationic-b-neutral block copolymer abbreviated asPTEA11K-b-PAM30K in the paper. The values in subscript arethe weight average molecular weight Mw obtained by thesynthesis (controlled radical polymerization process-RhodiaMADIX technology25) with a polydispersity index Ip ) Mw/Mn

) 1.6 ( 0.1. Poly(diallyldimethylammonium chloride) abbrevi-ated as PDADMAC100K with an Mw of about 100 000 g/molwas used as a cationic homopolyelectrolyte. It was purchasedfrom Sigma-Aldrich and used without further purification.

Formulation Protocol. Before mixing, dilute stock solutionsof nanoparticles and polymers were prepared separately at thesame concentration (c ) 0.1 wt %). The relative amount ofeach component was monitored by the volume ratio X, yieldingfor the final concentrations cNP ) cX/(1 + X) and cP ) c/(1 +X). Two different formulation paths were used:

Direct Mixing. Three different mixing methods were used:adding drop by drop with a pipet or pouring or high speedinjection (1-10 mL/s with a syringe) of a nanoparticle solutioninto a polymer solution.1 In all cases, magnetic stirring wasstarted only at the end to redisperse any sediment and facilitatethe sampling for further analyses.

Micromixer. The micromixer is comprised of a chamber withtwo entries and one exit. The volume of the mixer is 0.3 mL(diameter 18 mm, height 1.2 mm) with an overall flow rate of600 mL/h giving an average retention time of 2 s. Two stocksolutions containing the individual nanoparticles and polymersare driven by two syringe pumps into the chamber at a givenflow rate. Under the agitation of a ministirrer (600 rpm), bothsolutions are homogenized. The complexation occurs in thechamber. The final coacervates are collected at the exit. Thissetup minimizes any fluctuation and enables the tuning of themixing ratio X accurately and continuously by adjusting the flowrate of each feeding solution. The mixed solution at different Xcan be easily collected and analyzed by light scattering (Table1).

Desalting-Dilution. Sodium chloride (NaCl) or ammoniumchloride (NH4Cl) was added into the initial nanoparticle andpolymer solutions until the ionic strength reached a value wellabove the critical ionic strength Ib, where the interaction iscompletely screened or turned off.26 The two salted solutionsat the same ionic strength were then put together into a dialysisbag made of a cellulose membrane (MWCO ) 10 000 Da). Themixed solution was dialyzed against DI water for about 1 h.The water bath was 50 times larger than the dialyzed solution,ensuring a final ionic strength close to that of the bath.

Probing Techniques. Light Scattering. Static (SLS) anddynamic (DLS) light scattering measurements are performedon a BI-9000AT Brookhaven spectrometer (with a verticallypolarized laser operating at 488 nm). Rayleigh ratios R andhydrodynamic diameters are measured as a function of theconcentration c. R is obtained from the scattered intensity I(c):

TABLE 1: Main Characteristics of the Different MixingSystems Used To Bring the Stock Solutions into IntimateContact

directmixing desaltinga

simplemicromixer

microfluidicschip

mixing time, s 1 3600 2 0.2interacting vol, mL 1-10 10-100 0.3 0.1flow rate, mL ·h-1 3600 NA 600 2

a In the case of desalting, the mixing time is not relevant; theextent on which the electrostatic interaction is fully turned off ismentioned.

16374 J. Phys. Chem. C, Vol. 114, No. 39, 2010 Qi et al.

where Rstd and nTol are the standard Rayleigh ratio (31.6 " 10-6

cm-1 at 488 nm) and refractive index of toluene, and Is and ITol

the intensities measured for the solvent and for the toluene inthe same scattering configuration. To accurately determine thesize of the colloidal species, dynamic light scattering (DLS)was performed with concentration ranging from c ) 0.01 to 1wt %. In this range, the diffusion coefficient varies accordingto D(c) ) D0(1 + D2c), where D0 is the self-diffusion coefficientand D2 is a virial coefficient of the series expansion. The signof the virial coefficient, the type of interactions between theaggregates, either repulsive or attractive can be deduced. Fromthe value of D(c) extrapolated at c ) 0 (noted D0), thehydrodynamic radius of the colloids is calculated according tothe Stokes-Einstein relation, DH ) (kBT/3"#D0), where kB isthe Boltzmann constant, T is the temperature (T ) 298 K), and#S (#S ) 0.89 " 10-3 Pa · s) is the solvent viscosity. Theautocorrelation functions of the scattered light are interpretedby using the CONTIN fitting procedure.

Neutron Scattering. Small angle neutron scattering (SANS)experiments were performed on the PAXY spectrometer atLaboratoire Leon Brillouin (LLB, Saclay, France). Two con-figurations are used (D ) 1.35 and 6.70 m, both at $ ) 6 Å),covering a q-range from 5 " 10-3 to 0.2 Å-1. Exposure timesof 2 and 1 h for the small and large angle configuration,respectively, are necessary to obtain good statistics. Raw dataare radially averaged. Standard corrections for sample volume,neutron beam transmission, empty cell signal subtraction, anddetector efficiency have been applied to obtain the scatteredintensities with use of standard SANS procedures27 yielding theneutron scattering cross section (expressed in cm-1). Theincoherent background arising from the hydrogen atoms wascalculated by using test solutions containing a mixture of H2Oand D2O.

Cryo-TEM. Cryo-transmission electron microscopy (cryo-TEM) was performed on hybrid complexes prepared at con-centration c ) 0.1 wt %. For the experiments, a drop of thesolution was put on a TEM-grid covered by a 100 nm thickpolymer perforated membrane. The drop was blotted with filterpaper and the grid was quenched rapidly in liquid ethane inorder to avoid the crystallization of the aqueous phase. Themembrane was then transferred into the vacuum column of aTEM microscope (JEOL 1200 EX operating at 120 kV)maintained at the temperature of liquid nitrogen. The magnifica-tion for the cryo-TEM experiments was selected at 40 000".

Optical Reflectometry. The amount of adsorbed complexesissued from different formulation processes onto poly(styrene)(PS)28 surface was monitored by using stagnation point adsorp-tion reflectometry (SPAR). A complete description of this devicedeveloped by Wageningen University (Netherlands) can befound in ref 29. Fixed angle reflectometry measures thereflectance at the Brewster angle on the flat substrate. A linearlypolarized light beam is reflected by the surface and subsequentlysplit into a parallel and a perpendicular component with use ofa polarizing beam splitter. As material adsorbed at thesubstrate-solution interface, the intensity ratio S between theparallel and perpendicular components of the reflected light isvaried. The change in S is related to the adsorbed amountthrough

where S0 is the signal from the bare surface prior to adsorption.According to this model, the sensitivity factor (As), which isthe relative change in the output signal S per unit surface, wasfound to be proportional to dn/dc and very weakly dependentupon the amount of material adsorbed. In practice, it wasregarded as a constant. Furthermore, good accuracy and repeat-ability were obtained when As is larger than 0.005 m2/mg.Hydrophilic silica substrates were modeled by using smoothsilicon wafers covered with a layer of 100 nm SiO2 in order tomaximize the reflectometer signal. Hydrophobic poly(styrene)(PS) substrate was modeled by a PS thin layer of 100 nmdeposited on top of an HMDS (hexamethyldisilizane)-function-alized silicon wafer by spin-coating a toluene solution (2.5 wt%) at 5000 rpm. The final PS layer thickness was around 100nm to ensure a good sensitivity and presented water contactangles around 88° typical for PS-coated material. The sensitivityfactor As was found to range between 0.02 and 0.035 m2/mgfor silica surfaces, and between 0.017 and 0.025 m2/mg for PSsurfaces.

Results and Discussion

Manual Formulation. In a previous publication the influenceof the mixing pathway on the CeO2/PSS11K-b-PAM30K systeminteracting through a combination of electrostatic and H-bondinginteractions1 was investigated. In the current work, the influenceof the interaction itself on the formulation process was put underscrutiny on the CeO2-PAA2K/PTEA11K-b-PAM30K system inter-acting solely through electrostatic interactions.

As preliminary experiments, we first investigated any possibleinfluence of the mixing pathway on the final morphology ofthe complexes. Different combinations of mixing orders (CeO2-PAA2K into PTEA11K-b-PAM30K or PTEA11K-b-PAM30K intoCeO2-PAA2K) and homogenization speeds (high speed injectionor pouring) were performed and DLS measurements wereconducted on the final solutions to estimate the size of thegenerated structures (Figure 1). DLS results did not show largedifferences in the final size or size distribution of the complexesissued from the different mixing processes, however.

The insensitivity to the mixing pathway for the CeO2-PAA2K/PTEA11K-b-PAM30K complexes is due to the very low concen-tration of the stock solutions (e0.1 wt %). Indeed, onlyelectrostatic interactions are present here and the charged blockrepresents only one-third (by weight) of the polymer chains.When the density of “reactive sites” (positive charges) is ratherlow, the “reaction time scale” is longer and more comparableto ordinary mixing time scales (e.g., pouring one solution intoanother one); the morphology of the final complexes becomeshence less sensitive to the mixing pathway. At higher concentra-tion (1 wt % and up), however, the mixing stage will indeedinfluence the formulation features a lot.

Instead of tuning the complexation processes via the mixingstage while keeping the interaction constant between thecomponents (see former publication1), we follow here analternate approach based on the concept of a dormant-reactiVesolution in which both building blocks are not initially interact-ing with each other in the aqueous solution. This process,inspired by molecular biology, was originally developed for invitro reconstitutions of chromatin;the DNA/histones macro-molecular substance that forms the chromosomes of our cells30,31

and consists of two steps. In the first step the components are

R(q, c) ) Rstd

I(c) - Is

ITOL( nnTOL

)2(1) !(t) ) 1

As

S(t) - S0

S0(2)

Assembly of Nanoparticles and Macromolecules J. Phys. Chem. C, Vol. 114, No. 39, 2010 16375

mixed together in a high ionic strength aqueous solution. Thesalt screens the electrostatic interactions between the individualcomponents preventing them from forming aggregates. In thesecond step, the salt is removed progressively by dialysis or bydilution effectively triggering the coassembly process.

Desalting kinetics results were very recently published onthe system of salted (NH4Cl) solutions of CeO2-PAA and PTEA-b-PAM.26 Both the particles and the polymers were shown tobe stable in the presence of 1 M NH4Cl. When the two saltedsolutions were mixed together, no association occurred due tothe charge screening effect. DI water was then added stepwiseat the average flow rate of 0.3 µL/s until the final saltconcentration reached 10 mM. The aggregation of the polymer/nanoparticle system was monitored by static and dynamic lightscattering. Figure 2 (left) shows the hydrodynamic diameter asa function of the ionic strength of the solution. An abrupttransition was observed at a critical ionic strength estimatedhere at Ib ) 0.43 M. As evidenced by cryo-TEM (insets), theelectrostatically screened polymer and nanoparticle systemunderwent an abrupt transition between an unassociated and acluster state. By fine-tuning the desalting kinetics, the size of

the clusters was varied from 100 nm to over 1 µm. Cryo-TEManalysis showed that with the “desalting” route, the structuresformed were much larger (RH ! 200 nm vs 30 nm from directmixing in salt free condition at c ) 0.1 wt %) and ratherspherical (vs “frozen” irregular shape).

The CeO2-PAA2k/PDADMAC100k system where the charged-neutral block copolymer PTEA11K-b-PAM30K was replaced witha cationic homopolyelectrolyte was then investigated. An abrupttransition was equally observed via light scattering measure-ments (Figure 3) with a critical salt concentration around Ib !0.5 M. An ionic strength similar to that of the previous systemsuggesting likely that the transition triggered by the salt iscontrolled by the particle size and charge as predicted theoreti-cally by Muthukumar et al.32 and via MC simulations by Stollet al.33,34

Neutron scattering experiments (Figure 4) were then per-formed on these differently generated coacervates. In the direct

Figure 2. Ionic strength dependence of the hydrodynamic diameter for a dispersion containing CeO2-PAA2K particles and oppositely chargedPTEA11K-b-PAM30K block copolymers (closed symbols). The dispersions were obtained by dilution. With decreasing ionic strength, an abrupttransition was observed at the critical value of Ib ) 0.43 M. The open symbols represent the hydrodynamic diameters for CeO2-PAA2K (circles) andPTEA11K-b-PAM30K (squares), respectively. Inset: Cryo-TEM images of particles and clusters apart from the transition. Upper right: Cryo-TEManalysis of CeO2-PAA2K/PTEA11K-b-PAM30K coacervates prepared via direct mixing (top).

Figure 1. Hydrodynamic radius (RH) distribution of CeO2-PAA2K/PTEA11K-b-PAM30K complexes prepared via different formulationroutes.

Figure 3. Scattering intensity and hydrodynamic diameter RH as afunction of the “bulk” ionic strength I (and the corresponding overallnanoparticle + polymer concentration c) for the CeO2-PAA2k/PDADMAC100K (50/50 ) wt/wt) system at c ) 0.1 wt %. Ib ! 0.5 Mis the critical ionic strength above which the electrostatic interactionbetween the particles and the polyelectrolytes in the bulk is turned off.


mixing case, frozen core-shell coacervates (RH ! 50 nm) wereformed under a strong electrostatic interaction as expected,2,28,35

with cores composed of clusters of nanoparticles wrapped byPTEA11K blocks. The structure of densely packed particles inthe core was evidenced by the appearance of a scattering peakat q ) 0.075 Å-1, corresponding to an interparticle distance of8.4 nm, the dimension of CeO2-PAA2K particles.2 It should benoted that direct mixing is here equivalent to a quench in thedesalting method as pointed out recently by Fresnais et al.26 Inthe case of a slow dialysis, much larger (RH ! 300 nm) sphericalaggregates were built up under a weaker interaction. Becausethe extended contour length of the copolymer chains (#25 nm)is shorter than the size of such aggregates, a core-shell structurecannot be obtained. The coacervate is then a mixture of neutralPAM and charged PTEA chains and CeO2 nanoparticles. Nodiscernible nanostructure signature was observed in the case ofthe dialysis. The disappearance of the scattering correlation peakmight be due either to the presence of the neutral part of thediblock throughout the spherical complexes leading to a dilutionof the nanoparticles packing or to a lower contrast as suggestedby a lower scattered intensity. To assess this hypothesis the blockcopolymer was replaced by a cationic homopolyelectrolyte. Verylarge aggregates were generated (which eventually sedimented)giving rise to a very similar yet more pronounced correlation

peak in the scattering intensity, a signature of densely packednanoparticles wrapped by the cationic homopolyelectrolytes.

Surface Modifications. Reflectometry experiments wereperformed with fresh solutions at 0.1 wt % prepared throughdifferent processes (direct mixing and dialysis-desalting) ontosilica and polystyrene surfaces. DI water was used to obtainthe baseline and a lysozyme protein solution (0.01 g/L) wasused to evaluate any subsequent antifouling effect. Figures5 and 6 show the adsorption of the complexes, DI water rinsing,and protein adsorption stages. It can be seen that on both silicaand PS surfaces, the saturated amount of adsorption is higherin the case of dialyzed structures (2.48 mg/m2 vs 1.05 mg/m2

for silica; 1 mg/m2 vs 0.7 mg/m2 for PS). The antifoulingefficiency is pretty good in both cases, however. Lysozymeadsorption was negligible on the treated silica surface. On thetreated PS surface, the lysozyme adsorption is largely reducedcompared to that of bare PS (0.38 mg/m2 vs 1.02 mg/m2).Furthermore it can be totally removed by a gentle water rinsing.

Adsorption kinetics similarities and differences were observedon different surfaces. In the case of silica surfaces, bothstructures adsorb very quickly at the beginning up to ! ! 1.1mg/m2, with a sharp increase in adsorption (Figure 5). The“direct mixing” adsorption then reaches a plateau while the“dialysis” goes through a step before reaching a new plateau

Figure 4. SANS cross sections of CeO2-PAA2K/PTEA11K-b-PAM30K hybrid complexes formulated in different ways: direct mixing in DI water(open circles) or dialyzed from salted solution (black diamonds). For comparison, the complexes made with CeO2-PAA2K nanoparticles and PDADMAChomopolyelectrolytes prepared by dialysis are also shown (black circles).

Figure 5. Adsorption onto silica of the CeO2-PAA2K/PTEA11K-b-PAM30K system monitored by optical reflectometry and subsequent evaluation oflysozyme adsorption onto the treated surface. Inset: Lysozyme adsorption onto a bare silica surface.


twice higher (! ) 2.48 mg/m2). The “step” is likely due to thepolydispersity of the complexes (bimodal distribution as seenby DLS) leading to two different adsorption kinetics. Thesmaller structures (RH ) 65 nm) made via dialysis and directmixing have a faster adsorption kinetics than the larger ones(RH ) 205 nm) as expected. This feature was not observed,however, when the adsorption occurred on PS surfaces (Figure6). It seems that both small and large structures adsorbed muchmore slowly than in the case of silica surfaces with no visibleadsorption steps.

Toward Better-Controlled Mixing Processes. The previousresults highlighted the possibility of developing differentmorphologies and surface properties from a limited number ofchemicals by tuning some formulation parameters: a crucialpoint for economical and environmental reasons and towardnowadays strict regulatory requirements during new productsdevelopment. Dialysis-desalting processes are reproducible. Buta longer associated processing time scale does not necessarilymeet industrial constraints (couple of hours for dialysis). Direct

mixing processes do not have such drawbacks, but they are verysensitive to experimental conditions such as the mixing path-way.1 Both approaches call then for more efficient and bettercontrolled processes.

Simple Micromixer. In the micromixer configuration, themixing volume ratio X should equal the ratio between the flowrates. Compared with the previous “manual” formulation, thiswell-controlled process makes possible the building of acontinuous phase diagram for a given system in a very shorttime. We made a test experiment with a 1 wt % CeO2-PAA2K

solution to verify this hypothesis. The mixing process is here asimple dilution of the CeO2-PAA2K stock solution. We chosethree different flow rates VCeO2-PAA2K/VH2O ) 120/480, 300/300,and 420/180 (with the overall flow rate kept constant at 600mL/h). We then measured the Rayleigh ratio R% of the mixtureprepared at each different rate by light scattering. Mixtures withthe same X values were also prepared manually and thecorresponding R%(c) were measured. The micromixer resultsmatched perfectly the manual reference curve as seen in the

Figure 6. Adsorption onto PS of the CeO2-PAA2K/PTEA11K-b-PAM30K system monitored by optical reflectometry and subsequent evaluation oflysozyme adsorption onto the treated surface. Inset: Lysozyme adsorption onto a bare PS surface.

Figure 7. Variation of Rayleigh ratio R% as a function of the volume ratio X for CeO2-PAA2k/PTEA11K-b-PAM30K coacervates obtained manuallyand with the micromixer. Inset left: Schematic view of a simple micromixer. Inset right: Dilution curve of a 1 wt % CeO2-PAA2k via both manualand micromixer mixing.


inset of Figure 7. X is hence equal to the flow rate ratio.Furthermore, we plotted the CeO2-PAA2k/PTEA11K-b-PAM30K

phase diagrams at 0.1 wt % obtained both manually and withthe micromixer. Clearly both approaches are in very goodagreement (Figure 7).

Generally the dilution curves can help us determine severalimportant bulk parameters for a given system, such as the phasediagram, the coacervate critical aggregation concentration(CAC), the diffusion coefficient of the particles at c f 0, etc.With a micromixer, the “mixing order” parameter is not pertinentanymore. The two initial solutions are supposed to come intocontact always at the same volume ratio X (small volume). Thisprocess should then minimize any gradient during the mixingand be similar to the “high speed injection” process.1 Experi-mentally, however, the flow rate and the stirring speed willinfluence the final result. For example, when the flow rate istoo high or the stirring speed is too low, both feeding solutionswill not mix inside the chamber postponing the complexation

further down in the outlet tube generating large polydisperseaggregates. One needs definitely to pay specific attention to thehydrodynamic conditions during the complexation.

Microfluidic Chip Coupled with DLS. Recently Destremautet al.36 have developed a dynamic light scattering setup arounda microfluidic chip enabling the measurement of the size ofBrownian scatterers flowing in a PDMS-based microchannel.This particular chip can mix two reactants in 200 ms, and allowssize measurements using DLS at about 300 ms after completemixing. It is typically possible to measure sizes up to #500nm at a flow rate of Q ! 2 mL/h in a microchannel of cross-section h " w ! 500 " 500 µm2 (Figure 8).

Besides the control of the transport phenomena, the develop-ment of such laboratories on chip offers new interestingpossibilities such as the high-throughput screening at the nLscale like the continuous monitoring of the viscosity of a two-fluid mixture for example.

This setup was used here to generate and characterize thecomplexation of the CeO2-PAA2k nanoparticles with both theblock copolymers (PTEA11k-PAM30K) and the homopolyelec-trolytes (PDADMAC100K) via the desalting-dilution approach.

CeO2-PAA2K/PTEA11K-b-PAM30K. If the two nonsalted stocksolutions (c ranging from 0.1 or 1 wt %) are injected into thechip, the interaction is so strong and rapid that the complexationhappens before the two species are fully mixed leading to theformation of large frozen fractal structures. To avoid localinhomogeneities and to weaken the interaction, individualsolutions of nanoparticles and polymers were mixed in thepresence of 0.6 M NH4Cl, where the interaction is totallyscreened. A homogeneous solution was easily obtained asdescribed previously. Both the dormant solution and DI waterwere then injected via the two entries into the chip. Theinteraction can in this manner be fine-tuned by adjusting theflow rate of both feeding solutions. Keeping the total flow rateconstant at 300 µL/h, we tuned the ratio between the dormantsolution and the DI water rate, and monitored the formedcomplexes with the online DLS. By plotting the scattering lightintensity and the hydrodynamic radius RH vs the ionic strengthI or the “mass fraction of colloids” (equals the ratio “rate ofthe premixed solution”/“rate of the premixed solution + DIwater”), we obtained the phase diagram of Figure 9. It is a

Figure 8. Schematic representation of the microfluidic chip coupledwith online DLS.36 It consists of a 1 cm Y-shaped microchannel ofheight 40 µm, which drives two reactants into a 1 cm long channel ofwidth w ) 200 µm. Chaotic mixing37 in such a laminar flow isperformed with grooves of height 20 µm engineered on the mainchannel. DLS measurements are performed in the large channel(observation window 500 " 500 µm2) at an apparent angle % ) 60°.

Figure 9. Light scattering intensity (Int) and hydrodynamic radius (RH) as a function of the ionic strength (I) and the corresponding colloid massfraction through online DLS measurements coupled with microfluidics chip for CeO2-PAA/PTEA11K-b-PAM30K system. The ionic strength at whichan abrupt transition between an unassociated and a clustered state is observed at I ! 0.42 M. Inset: Example of a typical DLS correlation functiontaken in the microchip.


typical phase diagram for electrostatic complexation with thepresence of a peak around I ) 0.40 M. Unassociated nanopar-ticles and polymers are present in the solution at I > 0.42 M,and coacervates of #100 nm are formed at I < 0.40 M. In bothcases, the intensity varies linearly with the colloid fraction (andionic strength) following a simple dilution law. A transition zonewas observed at I ) 0.40 to 0.42 M, where the interaction wasswitched back on but remained very weak. Some polydispersestructures were likely to be formed in such condition. A slowkinetics implies furthermore that not all the individual compo-nents can be integrated in the complexes within the observationtime scale (#500 ms) of the microfluidic chip, leading to anapparent lower scattered intensity. Interestingly, the critical ionicstrength Ib (0.42 M) found in this phase diagram was very similarto one found “manually” by Fresnais et al.26 on the same system(Figure 2), which highlights the potential of such microfluidictools for investigating interacting systems.

CeO2-PAA2K/PDADMAC100K. The stock solutions were pre-pared and mixed at X ) 1 in the presence of 0.8 M NH4Cl,where the electrostatic interaction is totally screened for thissystem (see Figure 3). The phase diagram shown in Figure 10is very similar to that of the CeO2-PAA2K/PTEA11K-b-PAM30K

system. A peak in the intensity and a critical ionic strength Ib

were found at 0.4 and 0.45 M, respectively, in agreement withmanual off-line DLS measurements shown in Figure 3.

Charged complexes were obtained with a size around 140nm, a result different from the manual formulation in whichmuch larger (#>1000 nm) and polydisperse aggregates weregenerated due to a relatively slow dilution stage (drop-by-dropaddition of DI water in the vial test). This drawback was avoidedwith the microfluidics formulation, where a minute amount ofmaterials and a specific mixing geometry enabled a better andfaster homogenization.

These experiments showed that microfluidics setup coupledwith DLS is a powerful tool for generating phase diagrams forelectrostatic coassembly of nanoparticles and different chargedmacromolecules in the presence of a variable ionic strength.This microfluidics approach enables fast, reproducible, andaccurate results to be obtained with a minute amount ofmaterials: a key advantage over manual formulation in vial tests.

Conclusions and Outlook

Different formulation pathways and mixing protocols wereinvestigated in three coassembled systems composed of ceriumoxide based nanoparticles and oppositely charged doublehydrophilic diblock copolymers or homopolyelectrolytes. Undera strong driving force like the electrostatic interaction (alongwith other interactions like hydrogen bonding etc.), the keyfactor that controls the final morphology of the complexes (size,structure, and polydispersity) is the competition between thereaction and the mixing time needed to homogenize theformulation. Two approaches were put under scrutiny to shedsome light on the crucial role of the formulation process:

(i) Tuning the mixing stage (or the way individual compo-nents come into intimate contact) with specific protocols or toolswas investigated in a former publication.1

(ii) Tuning the strength of the (electrostatic) interaction byvarying the ionic strength in the initial stock solutions containingeach individual building block was investigated in the currentpublication.

The resulting structures from various formulation processeswere characterized by cryo-TEM, AFM, light, and neutronscattering techniques. Some bulk and surface properties werealso evaluated via rheology, wetting, and optical reflectometrymeasurements. Better controlled mixing processes were putforward at the end to improve the productivity and reproduc-ibility of the complexes generation. In particular, a µ-fluidicchip coupled with dynamic light scattering was used to bettercontrol the hydrodynamics of the complexation process.

The impact of the formulation pathway clearly evidenced theresulting complexes morphologies at different length scaleleading to different bulk and surfaces properties. All these resultssuggest that a process-dependent formulation seen a priori as adrawback can be turned into an advantage: different propertiescan be developed from different morphologies while keepingthe chemistry constant, certainly a key advantage for any productdevelopment in today’s strictly regulated world.

References and Notes

(1) Qi, L.; Fresnais, J.; Berret, J.-F.; Castaing, J.-C.; Grillo, I.; Chapel,J.-P. J. Phys. Chem. C 2010, 114 (30), 12870–12877.

Figure 10. Light scattering intensity (Int) and hydrodynamic radius (RH) as a function of the ionic strength (I) and the corresponding particlefraction through online DLS measurements coupled with microfluidics chip for the CeO2/PDADMAC system. The transition occurs at I ! 0.45 M.


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