Synthesis and modeling of calcium alginate nanoparticles in quaternary water-in-oil microemulsions

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Synthesis and modeling of calcium alginate nanoparticles in quaternary water-in-oil microemulsions Daniel G. Angelescu ∗ , Mihai Anastasescu, Dan F. Anghel Romanian Academy, “Ilie Murgulescu” Institute of Physical Chemistry, Splaiul Independentei 202, 060021 Bucharest, Romania

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Calcium alginate nanobeads were

AFM tapping-mode image of the calcium alginate beads.

obtained by mixing two w/o microemulsions containing sodium alginate and calcium chloride. • The two microemulsions had similar water droplet size. • An average size of 54 nm was found for the calcium alginate nanobeads. • Experimental results and Monte Carlo simulations support the templating role of the microemulsion droplets.

a r t i c l e

i n f o

Article history: Received 17 October 2013 Received in revised form 20 January 2014 Accepted 27 January 2014 Available online xxx Keywords: Monte Carlo simulations nanoparticle synthesis polimeric nanoparticle

a b s t r a c t Calcium alginate nanoparticles were prepared using the water-in-oil microemulsion route. Sodium alginate and CaCl2 were solubilised in the aqueous phase of two microemulsions, and their mixing induced the crosslinking of the alginate polymer by the calcium ions. The microemulsions were based on the nonionic ethoxylated surfactant Brij30 and the nonionic poly(ethylene oxide)-poly(propylene oxide)poly(ethylene oxide) (PEO-PPO-PEO) triblock-copolymer L64. Dynamic light scattering analysis revealed that the microemulsion droplet size was not affected by the species in the aqueous phase and that the alginate polymer was strongly confined in the microemulsion water pool. Atomic force microscopy of the collected alginate nanoparticles indicated a polydisperse population with an average size of 54 nm and hinted at an active template role of the microemulsion droplets. A detailed description of the conformation adopted by the alginate polymer in the water droplet in the presence of mono- and divalent counterions was provided by Monte Carlo simulations. The binding of both counterions was nonspecific, with the chain configuration allowing the carboxylic, hydroxylic, and ether groups to coordinate the small ions. Addition of divalent counterions did not modify the radial extension of the alginate chain, yet it mediated the transient bridging between strands. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Alginic acid is a linear polysaccharide of copolymers containing residues of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). The alginates have no regular repeating units, and their

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content in the two acids, as well as their distribution along the polymeric chain, vary and depend on the natural source they are extracted from [1,2]. They are rather polydisperse, with an average molecular weight above 100 KDa and a persistence length in the range of 9–15 nm [3,4]. The key feature for most of their technological applications, such as cosmetics, pharmaceuticals, food engineering, and drug delivery [3,5–7], is given by the fact that the alginates form waterinsoluble gels in the presence of Ca2+ ions [1,8,9]. These gels were

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found to be very attractive as drug delivery vehicles despite the fact that, due to their intrinsic hydrophilic nature, many hydrophobic drugs cannot be solubilised in the calcium alginate gels. The gel formation involves ionic exchange, the starting point being the water-soluble alginate and the calcium counterions. Calcium alginate gel beads in the micrometric range, with good control of the gel mechanical strength, can be easily produced by either dripping or spraying a sodium alginate solution into a calcium salt solution [10–13]. Preparation of the nanometer-sized Ca alginate beads turned out to be more difficult. Some of the methods involved large input of mechanical energy, either by shearing or ultrasonication, which in turn may damage the sensitive biopolymer [14,15]. As an alternative mild approach, the micro- or nanoemulsion route has been reported. This method, extensively employed in the synthesis of inorganic nanoparticles and preparation of polymeric latexes [16–19], is based on the nano-sized reactors provided by the dispersed domains of the (micro)emulsions. For inorganic materials, salt precursors were generally used and they were conveniently solubilised in two water-in-oil (w/o) microemulsions [16,18]. The two w/o microemulsions were mixed afterwards and the fusion-fission process of the colliding droplets led to the formation of seed nuclei and to their further growth to the final nanoparticles. The synthesis of Ca alginate nanobeads via micro- or nanoemulsions have been scarcely reported so far [20,21] despite the appealing nature of the method. That is because the alginate containing micro- and nanoemulsions are expected to be much less stable than the ones containing the precursors of the inorganic nanoparticles due to the alginate polymer bulkiness. To date, ionic and nonionic surfactant ternary systems have been used to obtain calcium alginate via the emulsion route. Spherical beads of calcium alginate with sizes from 55 to 100 nm have been obtained in w/o microemulsions stabilized by the surfactant dioctylsulfosuccinate (AOT) [21]. Nanoemulsions produced from mixtures of the surfactant tetraethylene glycol monododecyl ether (C12 E4 ), decane, and water have been used for synthesizing Ca alginate nanoparticles having size less than 200 nm [20]. Notably, the preparation methods started from a w/o ternary system containing sodium alginate, to which CaCl2 was added as an aqueous solution. To the best of our knowledge, the capability of synthesizing nanometer-sized calcium alginate beads by mixing two microemulsions containing sodium alginate and CaCl2 , respectively, has not been investigated so far. The aim of this work is to demonstrate that the Ca alginate nanobeads can be prepared by mixing two nonionic w/o microemulsions (referred to as ␮e1 and ␮e2) containing as precursors the sodium alginate and CaCl2 , respectively. The results for the first time indicate the feasibility of this approach when a mixture of two amphiphiles, namely the nonionic ethoxylated surfactant Brij30 and the PEO-PPO-PEO triblock copolymer Pluronic L64 were employed. The ethoxylated surfactant Brij30 was chosen as the main microemulsion stabilizer because of the little effect of the charged species on the microemulsion structure and of the versatility of the surfactant Brij30 microemulsion for the preparation of either metallic or semiconductor nanoparticles [11,22,23]. In this work, it is shown that the water droplets of ␮e1 and ␮e2 have similar size, ensuring thus that the soft organic nanobeads are synthesized in a controlled manner, as in the case of the inorganic nanomaterials preparation. The templating role of the water domains is supported by the dynamic light scattering investigations of the precursor microemulsions and by the atomic force microscopy measurements of the synthesized organic nanoparticles. In addition, Monte Carlo simulations are carried out to obtain knowledge of the conformation adopted by the alginate chain in the w/o microemulsion droplet.

2. Experimental 2.1. Materials The oil (n-octane, 99.5%), was supplied by Fluka, and the surfactant Brij30 and calcium chloride (CaCl2 , 99.5%) were purchased from Sigma Aldrich. The surfactant contained an average of four ethylene oxide groups and a linear alkyl chain C12 H25 . The nonionic PEO-PPO-PEO)triblock copolymer Pluronic L64 was purchased from BASF. The copolymer has an average molecular weight of 2900 g mol−1 and consists of 26 ethylene oxide groups and 30 propylene oxide groups. Alginic acid was procured from Fluka. Sodium alginate was prepared by neutralizing the alginic acid with NaOH in aqueous solution. It was then purified by dialysis using an ultrafiltration membrane of molecular weight cutoff 10 KDa and finally freeze-dried. The average molecular weight determined was 1.4 × 105 g/mol [24]. The structures of the compounds are displayed in Fig. S1a of Supplementary Material. The other chemicals were all used without further purification and the water was treated with a Millipore-Q water purification system. 2.2. Preparation of the microemulsions and of the calcium alginate nanoparticles We used Brij30 as a less expensive and polydisperse alternative to monodisperse alkyl polyethylene oxide surfactant C12 E4 because such replacement can be done without significant effect on the microemulsion phase behavior [25,26]. The samples were prepared by weighing appropriate amounts of the aqueous phase A (water or solute-containing aqueous solution), nonpolar solvent B (n-octane), surfactant C (Brij30), and cosurfactant D (Pluronic L64). The composition of the quaternary sample was given by the following parameters: - the mass fraction of the oil phase (B) in the mixture of oil and aqueous phase ˛=

mB mA + mB

(1)

- the mass fraction of surfactant (C) and cosurfactant (D) in the oil phase b =

mC + mD mB + mC + mD

(2)

- the mass fraction of the co-amphiphile Pluronic L64 (D) relative to the mixture of surfactant (C) and cosurfactant ıc =

mD mC + mD

(3)

The probes were made into tubes sealed with stoppers, and they were homogenized under magnetic stirring in a thermostated water bath at 25 ◦ C. The phase boundaries of the reference microemulsion and of the two microemulsions of interest, ␮e1 containing 0.75 wt. % sodium alginate, and ␮e2 containing 0.50 wt. % CaCl2 , were determined by visual inspection. Starting from turbid samples, the concentration of surfactant C was increased stepwise until clear samples were obtained. As a consequence, we alternatively considered the mass fraction of Pluronic L64, in the water phase as ı=

mD , mA + mD

where ␦ is related to the eqs. 1–3 through ı =

(4) ˛b ıc (1−˛)(1−b )+˛b ıc

. The

Ca alginate particles were prepared by transferring rapidly ␮e1 to ␮e2 under vigorous stirring. The mixed microemulsion ␮e1 + ␮e2, found at the stoichiometric monovalent-to-divalent molar ratio,

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was transparent initially, and, after stirring ceased, a white, visible, precipitate developed overnight. The precipitate was separated by mild centrifugation and purified repeatedly by subjecting the microemulsion to washing cycles with ethanol/water mixtures. 2.3. Microemulsions characterization by dynamic light scattering measurements The water droplet size of the w/o microemulsions was determined by DLS measurements. They were performed on a Malvern Zetasizer NanoZS apparatus equipped with laser emitting at 0 = 633 nm and operating at a scattering angle of 173◦ . Data analysis was performed by determining the distribution of the relaxation rate  from the relaxations times g(1) (t). The apparent translational diffusion coefficient, Ddiff , was calculated as Ddiff =

Table 1 Specification of the confined species considered. Number of uronate units in alginate chain, Nb Bond force constant, kbond (N m−1 ) Angular force constant, kangle (J deg−2 ) Number of small ions, NNa+ , NCa+ , NCl− Cavity radius Rsph (nm)

εij =



(5)

400 3.33 32.7 × 10−24 400, 400, 800 10

εi εj and εi , i given in Table S1 of Supplementary Mate-

rial. The interacting potential parameters of the uronate chain are based on the atomistic description given in ref. [27]. The second and third term of eq. 7, the bond energy Ubond , and the angular energy, Uangle , are the harmonic potentials modeling the alginate chain connectivity and stiffness, respectively. They are given by

k

Nb −1

 , q2

3

Ubond =

bond



2

ri,i+1 − r0

2

,

(9)

i=1

where q is the magnitude of the scattering vector and the hydrodynamic diameter DH of the assumed hard sphere-like water droplets was estimated from the Stokes-Einstein equation DH =

kT , 3Ddiff

(6)

where k is Boltzmann’s constant, T the absolute temperature, and  the viscosity of the solvent. 2.4. Calcium alginate beads characterization by atomic force microscopy AFM measurements were carried out to determine the size of the purified calcium alginate nanobeads. They were performed in the noncontact mode with a XE-100 apparatus from Park Systems equipped with flexure-guided, cross-talked eliminated scanners, using ultra-sharp tips (<7 nm tip radius; PPP-NCHR type from NanosensorsTM ) of 125 ␮m length, 30 ␮m width, and 42 N/m spring constant/∼330 kHz resonance frequency. In order to prepare the specimens for AFM investigations, a small quantity of the calcium alginate suspension was ultrasonically dispersed and then, a drop from this suspension was deposited on silica surface and dried at room temperature. 2.5. Monte Carlo simulations The alginate chain was modeled as a single-chain polymer containing uronate residues linked with beta-1,4 bonds. The uronate monomer architecture and its atoms description are given in Figure S1b. All interactions in the system were taken as pair-wise additive. The total potential energy U of the system is given by

Unonbond =

uij rij =

i
+

 i


4εij

ij rij

12

 −

ij

6 

rij

 Zi Zj e2 i
2

(˛i − ˛0 )2

(10)

i=2

where ˛i is the angle between the vectors ri+1 –ri and ri-1 –ri made by center-of-masses of three consecutive residues, ˛0 the equilibrium angle (˛0 = 180◦ ), and kangle the angular force constant. The chosen value for the kangle angular force constant led to the experimentally reported persistence length of 15 nm for the modeled charged chain and neutralizing Na+ counterions under an unrestricted geometry, i.e., large ellipsoid. The torsional potential, taken into account in previously reported simulation studies of free alginate chain [28–32], was not considered for this reduced model system. Last term in eq. 7 Ucell represents the water droplets where the alginate chain is confined, and is given by Ucell =

 i



ucell (ri ) =

0, ri < Rsph ∞, ri ≥Rsph

(11)

An overview of the parameters that model the alginate chain and small ions in the microemulsion water droplet is shown in Table 1. Simulations employing two different initial chain configurations (referred to as S1 and S2 ) were carried out. Apart from the single particle move, the trial displacements included two collective moves: (i) pivot rotation of a randomly selected part of the chain and (ii) translation of the entire chain. All simulations were performed using the integrated Monte Carlo/molecular dynamics/Brownian dynamics simulation package MOLSIM [33].

(7)

The first term can be expressed as a sum of Lennard-Jones and electrostatic potential interactions according to

 

 kangle

Nb −1

Uangle =

3. Results and Discussion

U = Unobond + Ubond + Uangle + Ucell



where ri,i+1 is the distance between C1  and O4 atoms belonging to adjacent uronate residues, r0 the corresponding equilibrium distance and kbond the bond force constant.

(8)

4ε0 εr rij

where rij is the distance between the centers of atoms i and j, Zi the charge of the atom i, e the elementary charge, ε0 the permittivity of the vacuum, εr the relative permittivity of the solvent. εij , ij , define





the interacting potential of ij atom pair, with ij = 0.5 i + j ,

The preparation of Ca alginate nanobeads via the microemulsion route required the presence of two microemulsions, ␮e1 and ␮e2, containing 0.75 wt. % sodium alginate and 0.50 wt. % CaCl2 , respectively. While the solubilization of the inorganic salt in the latter microemulsion occurred easily for aqueous phase-n-octane-Brij30 system, only turbid samples were found when the aqueous phase of the former microemulsion contained the sodium alginate. With this perspective, the formulation with an additional amphiplile as cosurfactant, namely the triblock copolymer Pluronic L64, enabled us to solubilise the bulky alginate polymer in ␮e1. The structure of the w/o H2 O–n-octane–Brij30/L64 reference microemulsion investigated by dynamic light scattering is first presented and thereafter the light scattering experiments will be extended to the ␮e1 and ␮e2. The size of synthesized calcium alginate nanoparticles will

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Fig. 1. Distribution of the relaxation time P( ) of H2 O–n-octane–Brij30/L64 microemulsions prepared at ˛ = 0.95 and ␥b , ␦C = (䊉) 0.137, 0.045, () 0.159, 0.078, () 0.177, 0.156, and () 0.226, 0.306; (inset) the mean hydrodynamic diameter Dh vs. ␦C .

Fig. 2. Distribution of the hydrodynamic diameter of solute–n-octane–Brij30/L64 microemulsions containing (䊉) water, () 0.75 wt % alginate and () 0.55 wt % CaCl2 at ␣ = 0.95, ␦ = 0.22 and ␥b = 0.150.

3.2. Characterization of ␮e1 and ␮e2 microstructure be then evidenced by AFM measurements and correlated with the water droplet size. The experimental results will be eventually complemented by the computational investigation of the alginate conformation in the water droplet in the presence of monovalent and divalent counterions. 3.1. Light scattering characterization of the w/o H2 O–n-octane–Brij30/L64 microemulsions The measurements were performed at the oil content ␣ = 0.95 and at the fraction of the Pluronic L64 in the water-L64 mixture ␦ ranging from 0.12 to 0.63. As mentioned in the Experimental Section, we considered the samples containing the lowest amount of Brij30 needed for the water uptake. It turned out that the fraction of Pluronic L64 in the amphiphile mixture ␦c (or the fraction of amphiphiles in the oil phase ␥b alternatively) at the onset of the one-phase region increased from ␦c = 0.045 (or ␥b = 0.137) at ␦=0.12 to 0.306 (or ␥b = 0.226) at ␦=0.63. The relaxation time distributions along the phase boundary found at the oil content ␣ = 0.95 are presented in Fig. 1. The distributions are all monomodal and they are attributed to the diffusion of the water droplets stabilized by a mixed Brij30/L64 interfacial layer. Notably, the narrowness of the relaxation time distributions implies that the presence of two water droplet populations, stabilized preponderantly by Brij30 and L64, respectively, is ruled out. The dispersed water phase at ␣ — — 0.95 corresponds to 3%–5% of the total microemulsion volume, and the obstructive effect on the translational diffusion coefficient of the water droplets can be neglected. It should be also mentioned that the presence of triblock copolymer may lead to the droplets bridging when the PEO terminal groups rest on different droplets [34,35]. However, such clustered droplets are not expected for dilute microemulsions at ␣ = 0.95 and we consider that the dispersed droplets with two PEO moieties residing in the same water pool are favored. As a consequence, one could estimate the apparent hydrodynamic diameters, Dh , of spherical water droplets from eq. 6. Notably, Dh varied linearly with the fraction of L64 in the amphiphile mixture, from 10 nm at ␦C = 0.045 to 68 nm at ␦C = 0.306 as seen in the inset of Fig. 1. This finding was noted at increasing the fraction of amphiphiles in the oil phase ␥b . Consequently, the increase of the water droplet size was due to the progressive radial extension of the PEO corona of the interfacial L64 triblock copolymer.

The two microemulsions ␮e1 and ␮e2 containing 0.75 wt% sodium alginate and 0.50 wt% CaCl2 , respectively, were prepared in a similar manner as the reference microemulsion. Thus, Brij30 was added stepwise at ␣ = 0.95 and constant ␦· In contrast to the reference microemulsion, ␮e1 was turbid irrespective of the amount of Brij30 added when ␦ < 0.22. At ␦ = 0.22, the one-phase region could be attained for the narrow range ␥b = 0.150–0.159. Notably, the presence of the polysaccharide polymer shrunk considerably the one-phase area, yet the onset of the one-phase domain was not altered. As for ␮e2, the presence of inorganic salt in the aqueous phase did not modify the microemulsion stability. A comprehensive phase behavior study of the two microemulsions systems is postponed to a forthcoming investigation. It can though be stated that a given amount of L64 is needed for the alginate solubilization in the w/o microemulsion. The role of the triblock copolymer for accommodating the bulky alginate is either to increase the water droplet size, as suggested by Fig. 1, or to provide specific interactions between the alginate and the droplet interface. The distribution profiles of the hydrodynamic diameter of the reference microemulsion, ␮e1 and ␮e2 prepared at ␣ — — 0.95, ␦ = 0.22, and ␥b = 0.150 are displayed in Fig. 2. The data showed that the distributions were all monomodal. The presence of a significant population of large droplets containing the alginate polymer is ruled out, as the corresponding scattering intensity, which depends strongly on the droplet size, was not detected in the DLS experiment. Thus, both ␮e1 and ␮e2 contained a single population of droplets. Notably also, the mean hydrodynamic diameter Dh was not affected by the presence of either sodium alginate or CaCl2 in the aqueous phase. For the case of ␮e1, this implies that the size of the water domain is still controlled by the amphiphiles properties and not by the alginate bulkiness. Thus, one could prepare water droplets of similar size containing the calcium alginate precursors, an important feature for controlling the nanoparticle synthesis via the w/o microemulsion route. If one takes into account the molecular weight of the alginate and the hydrodynamic radius in Fig. 2, we estimated that about one alginate chain, exhibiting a hydrodynamic diameter in neat water of ∼400 nm, was solubilised in a water droplet with a diameter of ∼20 nm. We then can state that the alginate chain is strongly confined in ␮e1. This feature can be understood by considering that the penalty resulting from the chain confinement is overcome by the specific interactions between the alginate and the interfacial amphiphiles. Thread-like complexes of

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alginate and PEO chains were recently reported [36,37] and it was claimed that the H-bonds between the hydroxyl groups of the alginate and the ether oxygen atoms of PEO chains were responsible for complexation. Intriguingly, the H-bonds formation was elusive in the w/o C12 E4 -based nanoemulsions that contained sodium alginate [20] as the polysaccharide did not dramatically change the phase behavior. However, one can infer for the herein quaternary w/o microemulsion that the H-bonds may be established between the alginate and PEO moieties of the triblock copolymer. The finding that the presence of the triblock copolymer induces the alginate solubilization can be also ascribed to a shift of the cloud point. The cloud point of Brij30 and L64 in water is 7 ◦ C [38] and 60 C◦ [39], respectively, and though these values are expected to be affected by the alginate, the cloud point of the mixed amphiphiles remained above 25 ◦ C, which may account for the one-phase region. 3.3. Characterization of calcium alginate particles Having determined the structure of the precursor microemulsions, they were further mixed. The calcium alginate beads were formed as a result of the material exchange occurring during the fusion-fission process in the mixed ␮e1 + ␮e2. The sample appearance started turning slightly turbid after one hour and a well defined precipitate with a clear top phase was formed overnight. The precipitate was separated from the microemulsion and then purified according to the procedure given in Experimental Section. The final material was insoluble in water, and could be dispersed into visually homogeneous dispersion by sonication. When it was left for resting, the precipitate started appearing after one hour. A typical AFM image of the calcium alginate beads, deposited from the suspension onto silica surface, is shown in Fig. 3. It is seen that spherical or nearly-spherical shaped particles coexisted with clustered larger aggregates. The distribution of the particle size is rather broad, with a mean diameter value of 54 nm. It is important to note that a certain fraction of nanoparticles with diameter about 20 nm, similar to the size of the water droplets, were formed. This observation clearly indicates the water droplet acted as a template for the preparation of the calcium alginate beads. As to the particles and aggregates of larger size, their presence denotes a tendency of individual nanoparticles to attach to each other. The calcium alginate is water insoluble and after leaving the water droplet the individual nanobeads may coagulate in the continuous phase. In addition, they are expected to manifest strong adhesion during the separation and purification procedure. 3.4. Monte Carlo simulations of the alginate polymer confined in the microemulsion droplet The DLS experiments revealed that the alginate chains underwent strong confinement as a result of solubilization in the microemulsion droplets. To gain further insights on the chain conformation and the counterions binding, Monte Carlo simulations of an alginate chain bearing 400 units and confined in a spherical cavity of 20 nm diameter were performed. Typical snapshots obtained in the absence and in the presence of CaCl2 are illustrated in Fig. 4. In the both cases, the chain was distributed across the cavity. On short range, the chain was stretched locally because its intrinsic persistence length of 15.0 nm was larger than the mean separation between the center-of-mass of adjacent uronate units, 0.57 nm. On long range, U-turn conformations were adopted in order to accommodate the alginate to the available space. This long-range arrangement, with variable number and orientation of local loops, is typically for a long chain confined in a spherical cavity, whose radius is comparable with the chain persistence length [40]. As the equilibrium configuration is difficult to

5

reach under this circumstance [41], we investigated the ergodicity by performing alternative simulations that started from the different initial random chain configuration S2 . The conformational features described above were also found for the additional simulations (see snapshots in Fig. S2), with the observation that the location and number of U-turn varied. Though the snapshots of simulations starting from S1 and S2 configurations had different appearance in terms of U-turns, the difference of the corresponding mean potential energy was small as can be seen in Table S2. This implies that the enthalpy associated with the chain bending energy is in the same range as the entropy from the chain configurational fluctuations. We can then argue that the performed simulations enable us to reach meaningful conclusions for the alginate confinement in w/o microemulsions. The number density of the chain units at the Ca2+ /Na+ charge ratio ␤ = 0.0 and 2.0 is displayed in Fig. 5 >as a function of the radial distance. The profiles were given for the two simulations carried out from the initial configurations S1 and S2 . The functional form of the alginate radial density is characteristic for the confinement of a long semi-flexible charged chain in a cavity whose radius is comparable with the effective persistence length of the unconfined chain [42]. In the absence of divalent counterions, the distribution was nonmonotonous; the radial profile was poorly recovered at intermediate radial distance, where alternating maximum and minimum local densities were found, whereas low overlapping densities were observed at the void center and near the boundary (Fig. 5a). The chain depletion at r > 9 nm is given by the reduced available space for the chain conformation and for the counterions located between the chain and the cell wall. The distribution of the alginate residues at r < 9 nm represents the configuration of the long chain that tries to minimize the bending energy. The alginate chain would first start accommodating the available space near the void boundary. However, this region is rapidly saturated by the chain bearing 400 uronate units, and the remaining strands occupy the available space toward the center cavity. These chain sections made loops at intermediate distance to avoid high bending energy at the void center. As a result of these U turns, the density may increase locally owing to the chain crossing. The position of these maxima found at intermediate distance depended on the initial chain configuration, yet the radius of gyration of the two chain conformations were rather similar, as can be seen in Table S2. The radial density distribution of the uronate units in the presence of CaCl2 followed also a non-monotonous behavior (Fig. 5b). The void center and the space near the cavity wall were depleted, whereas local maxima were found at the intermediate distance and with their positions depending on the initial chain configuration. If one compares the radial profiles at Ca2+ /Na+ charge ratio ␤ = 0.0 and 2.0, as well as the radius of gyration (Table S2), it can be stated that the presence of the divalent counterions did not alter significantly the alginate radial distribution. This result infers that the chain did not shrink upon Ca2+ complexation, which is in contrast to the formation of the macroscopic gel formation, where a significant decrease in the volume was reported [43]. The number densities of the counterions, Na+ and Ca2+ counterions as a function of the radial distance are displayed in Fig. S3 >at ␤ = 0.0 and 2.0. The Na+ counterion binding to the alginate chain is reflected by the fact that their radial distribution followed nearly the alginate one at ␤ = 0.0 (Fig. 5a). With the presence of divalent counterions, Ca2+ became the main bound species, yet Na+ was not fully released as its radial distribution was not homogeneously distributed throughout the cavity, a feature which was observed only for Cl− coions (not shown). Note that all small ions densities exhibited a weak decrease at the cavity wall owing to the lack of polarizability on the other side of the borderline [44]. The location of the small ions with respect to the uronate units was additionally investigated by determining the uronate–small

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Fig. 3. (a) AFM tapping-mode image and (b) particle size distribution of the calcium alginate beads obtained in ␮e1 + ␮e2 at ␣ = 0.95, ␦ = 0.22 and ␥b = 0.150.

Fig. 4. Snapshot illustrating typical configurations of an alginate chain of Nb = 400 inside the microemulsion droplet at Ca2+ /Na+ charge ratio ␤ = (a) 0.0 and (b) 2.0; color key: black = carbon, red = oxygen, dark blue = hydrogen, green = natrium and light blue = calcium; chloride was omitted for the sake of clarity.

ions radial distribution function, g(r). The three distribution functions g(r) obtained at ␤ = 0.0 and 2.0 are displayed in Fig. S4. When both type of counterions were in the cavity, Ca2+ bound with higher affinity than Na+ , as considered from the peak height, that is consistent with the valency effect on the alginate counterions binding [29]. The correlation effects among the strongly bound Ca2+ ions also brought coions to the region with high alginate density, resulting in a shallow maximum found for gb-Cl (r) at short separation. A further insight into the intermolecular structure was obtained from the small ion–uronate atom radial distribution function, rdf, where an upper separation of 0.5 nm for counterions–uronate (center-of-masses) was considered. The rdf of Na–O and Ca–O pairs obtained at ␤ = 0.0 and ␤ = 2.0 are shown in Fig. S5 and Fig. 6,

respectively. Peaks located at a distance of ∼0.3 nm and exhibiting similar broadness were found for pairs of the counterion and one of the three types of oxygen atoms in the uronate chain. It is also noted that the rdfs profiles of Na–O pairs were not affected by the presence of divalent counterions. These results imply that the counterion binding is not a specific one, as all the three groups (carboxylic, hydroxylic, and ether) contributed to the counterions binding. The presence of other shallow peaks at longer distances indicates that the arrangement of the two adjacent uronates enabled the counterion interactions with the carboxylate and ether moieties of one uronate unit and the hydroxyl group of the other unit. This result is in agreement with previous investigations reporting that many equally favorable positions for binding counterions existed

Fig. 5. Number density of the uronate units u as a function of the radial distance r at Ca2+ /Na+ charge ratio ␤ = (a) 0.0 and (b) 2.0 for two simulations started from different initial random configurations.

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Fig. 6. Radial distribution function for (a) Ca-O pair gCa-O (r), and (b) Na-O pair gCa-O (r), O pair gCa-O (r) at ␤ = 2.0; (䊉) carboxyl, () hydroxyl, and () ether oxygen.

Fig. 7. Snapshots illustrating two uronate strands connected via Ca2+ ions (light blue); the chain strands are arranged (a) parallel and (b) perpendicular and the oxygen atoms belongs to (red) carboxyl, (green) hydroxyl, and (dark blue) ether groups.

[27,29,32]. From the height of the dominant rdf peaks, it can be stated that (i) the ether oxygen appears to interact more strongly with Ca2+ , and (ii) the uronate chain is reorganized on short range as a result of the replacement of the monovalent ions by the divalent ones. The former observation was based on the model system construction where both carboxylate oxygen atoms could bind equal amount of counterions, whereas the latter feature resulted from the different gains noted for the height of each dominant peak of the three rdfs. It is also important to bear in mind that the ability of the alginate solutions to form gels in the presence of Ca2+ ions resides in the formation of so-called junction zones created when two alginate chains are connected via Ca2+ ions [30,31]. For the herein used alginate model, this strands association governed by the electrostatic interactions was found only for Ca2+ ions, and two typical configurations are illustrated in Fig. 7. The two strands were arranged parallel to each other with several counterions in the available middle space or the Ca2+ mediated bridging at some crossing points where two strands were oriented approximately perpendicular to each other. The former structure resembles the egg-like box model [45,46] and it seems that the carboxyl oxygens belonging to two different polyuronate strands are mainly involved in the coordination of calcium ions. The later inter-strand association is presumably weaker as fewer uronate groups coordinate Ca2+ . Also important, the location of the bridging configurations along the polyuronate chain, and implicitly the Ca2+ binding in the junction zones depended on the initial chain configuration used in the simulation, a feature that explains the height variation of the main peak of gCa-O rdf seen in Fig. S4.

4. Conclusions The w/o microemulsions stabilized by a mixture of two amphiphiles, namely nonionic surfactant Brij30 and triblock copolymer Pluronic L64, have been utilized to synthesize calcium alginate nanoparticles. They were obtained by mixing two microemulsions, ␮e1 and ␮e2, that contained as precursors the sodium alginate and CaCl2 , respectively. At water content ␣ = 0.95, the water droplet size of the reference microemulsion could be tuned in the range 10–60 nm by varying the fraction of the copolymer in the amphiphile mixture. The microemulsion stability was considerably affected by the presence of the alginate in the aqueous phase. One-phase region of ␮e1 could not be found at the triblock copolymer fraction in the waterL64 mixture ␦ < 0·22. At ␦ — — 0.22 a one-phase region was identified for a narrow ␥b range. The mean hydrodynamic diameters of the water droplets in ␮e1 and ␮e2 were 20 nm, similar to the size of the droplets in the reference microemulsion. Thus, the semi-flexible alginate chain was strongly confined in the water droplet. The collected calcium alginate nanoparticles were polydisperse as they exhibited a strong tendency for agglomeration. The mean nanobead size was 54 nm, and a fraction of smaller nanoparticles with diameters in the same range as the size of the water droplet was found, supporting thus an active templating role of the droplet confinement. The simulation results provided a detailed description of a polyuronate chain modeling the alginate conformation in the water droplet cavity and of the alginate interaction with the monoand divalent counterions. The radial distribution of the chain was

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inhomogeneous. Lower densities were obtained near the center of the cavity and at the cavity wall, whereas local maxima denoting chain crossing were reported at intermediate distances. The radial extension was not strongly affected when both mono- and divalent ions were present, suggesting that the gel formation did not shrink further the biopolymer already confined. The counterions binding to uronate strands was ascribed to the nonspecific small ions condensation, with contributions of carboxylic, hydroxylic, and ether groups to the Na+ and Ca2+ coordination. The computational results also support the formation of calcium alginate nanobeads as bridging of two uronate strands was observed in the presence of divalent counterions. This Ca2+ mediated link occurred between strands oriented either parallel or perpendicular to each other. Because the water droplet size in the reference microemulsion could be adjusted by varying the triblock copolymer Pluronic L64 content in the amphiphile mixture, the herein synthesis can be extended to investigate the potential of obtaining tunable-size beads or of modulating the properties of the calcium alginate nanoparticles. Additionally, one could also investigate the possibility of enhancing the synthesis yield by increasing the water content in the microemulsion formulation. These emphasized advantages which may be offered by the synthesis in the w/o nonionic microemulsions stabilized by mixed amphiphiles will constitute the scope of forthcoming articles. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project number PN-II-RU-TE-2012-3-0036. POS-CCE 2.2.1 Project INFRANANOCHEM-19/01.03.2009 funded by EU (ERDF) and Romanian Government is gratefully acknowledged for the light scattering equipment. DA thanks Profs. Per Linse and Georgios Staikos for helpful discussions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa. 2014.01.083. References [1] O. Smidsrod, G. Skjakbraek, Alginate as immobilization matrix for cells, Trends Biotechnol. 8 (1990) 71–78. [2] F.A. Johnson, D.Q.M. Craig, A.D. Mercer, Characterization of the block structure and molecular weight of sodium alginates, J. Pharm. Pharmacol. 49 (1997) 639–643. [3] T. Andersen, B.L. Strand, K. Formo, E. Alsberg, B.E. Christensen, Alginates as biomaterials in tissue engineering, Carbohydr. Chem. 37 (2012) 227–258. [4] B.T. Stokke, D.A. Brant, The reliability of wormlike polysaccharide chain dimensions estimated from electron micrographs, Biopolymers 30 (1990) 1161–1181. [5] D.N. Bevan, C.D. Gilson, A. Thomas, An improved method for preparing microorganism laden alginate bead specimens for accurate Scanning Electron Microscope examination, Biotechnol. Tech. 9 (1995) 913–916. [6] C.K. Kuo, P.X. Ma, Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties, Biomaterials (2001) 511–521. [7] A. Jen, M.C. Wake, A.G. Mikos, Review: Hydrogels for cell immobilization, Biotechnol Bioeng 50 (1995) 357–364. [8] A. Martinsen, I. Storro, G. Skjakbraek, Alginate as immobilization material: III. Diffusional properties, Biotechnol. Bioeng. 39 (1992) 186–194. [9] A. Banerjee, D. Nayak, S. Lahiri, A new method of synthesis of iron doped calcium alginate beads and determination of iron content by radiometric method, Biochem. Eng. J 33 (2007) 260–262. [10] P. Degen, S. Leick, F. Siedenbiedel, H. Rehage, Magnetic switchable alginate beads, Coll. Polym. Sci. 290 (2012) 97–106.

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