LbL matrices

Colloids and Surfaces B: Biointerfaces 95 (2012) 178–185

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Influence of the type of phospholipid head and of the conformation of the polyelectrolyte on the growth of calcium carbonate thin films on LB/LbL matrices Ana P. Ramos ∗ , Daniela M. Espimpolo, Maria Elisabete D. Zaniquelli Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo 14040-901, Brazil

a r t i c l e

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Article history: Received 23 February 2012 Accepted 24 February 2012 Available online 6 March 2012 Keywords: Calcium carbonate Biomineralization Langmuir–Blodgett films Layer-by-Layer films

a b s t r a c t Calcium carbonate is one of the most important biominerals, and it is the main constituent of pearls, seashells, and teeth. The in vitro crystallization of calcium carbonate using different organic matrices as templates has been reported. In this work, the growth of calcium carbonate thin films on special organic matrices consisting of layer-by-layer (LbL) polyelectrolyte films deposited on a pre-formed phospholipid Langmuir–Blodgett (LB) film has been studied. Two types of randomly coiled polyelectrolytes have been used: lambda-carrageenan and poly(acrylic acid). A precoating comprised of LB films has been prepared by employing a negatively charged phospholipid, the sodium salt of dimyristoilphosphatidyl acid (DMPA), or a zwitterionic phospholipid, namely dimyristoilphosphatidylethanolamine (DMPE). This approach resulted in the formation of particulate calcium carbonate continuous films with different morphologies, particle sizes, and roughness, as revealed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The crystalline structure of the calcium carbonate particles was analyzed by Raman spectroscopy. The randomly coiled conformation of the polyelectrolytes seems to be the main reason for the formation of continuous films rather than CaCO3 isolated crystals. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Polymeric membranes and lipid thin films have been used as templates for the growth of inorganic crystals using a biomimetic approach [1–4]. The main goal of these studies has been the better understanding of the preferential nucleation of a desired mineral phase and the control of biomineral growth. As a consequence, a variety of new materials with peculiar features, like the mechanical resistance observed in seashells, have been obtained. The coating of metallic surfaces with these materials has been investigated, aiming at biomedical applications such as the achievement of materials for bone reconstitution or implants [5,6]. Among the templates, polymers [7,8] and polymeric membranes [2], proteins in solution [9–12], Langmuir monolayers [13–15], Langmuir–Blodgett (LB) films [16–18], layer-by-layer (LbL) films [19–21], and combinations of LB and LbL [22] have been reported. As for minerals, calcium phosphate (apatites) and calcium carbonate (calcite, aragonite, and vaterite) have been extensively investigated, due to their abundance in living organisms. It is generally accepted that the organic matrix directs the nucleation, depending on the molecular organization and the nature of the charged groups employed during the construction of the template. These properties affect

∗ Corresponding author. Tel.: +55 1636023828. E-mail address: [email protected] (A.P. Ramos). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.040

the binding ability of the calcium ions, thereby influencing the local supersaturation from which the precipitation starts. Different crystal shapes can be obtained from these systems, once the distance between charges within the matrix directs the growth of a certain axis of the crystal. This culminates in different morphologies and even in the generation of distinct polymorphs. For instance, Kotachi et al. [4] have shown that the distance between charges in chitosan–poly(acrylic acid) membranes, the temperature conditions, and the molecular weight of the poly(acrylic acid) (PAA) drive the formation of the CaCO3 polymorph. The unit cell of vaterite (c) ˚ is closer to the array of hydroxyl groups in anhydrous chi8.56 A, ˚ rather than to the array of hydroxyl tosan (a) 8.50 and (b) 8.62 A, ˚ Therefore, treatment of the groups in regular chitosan (a) 8.95 A. membranes at high temperatures leads to the production of the metastable polymorph vaterite, whereas the stable calcite phase is grown over chitosan membranes at room temperature. In living organisms, the process of biomineralization usually occurs in the presence of biomacromolecules such as proteins and carbohydrates. The formation of polyelectrolyte multilayers using the LbL technique is one way of introducing these macromolecules in model systems for biomineralization [23,24]. On the other hand, Langmuir–Blodgett (LB) films offer easy control of the surface charge densities, also allowing for changes in the superficial charge through the use of positively or negatively charged lipids or a combination of both [22]. Moreover, the surface packing of the molecules can be modified by altering the surface pressure at which the Langmuir monolayers are transferred to the solid

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supports during the preparation of the LB films. In our previous study [20], the growth of calcium carbonate particulate thin films on LbL films consisting of chitosan and PAA, both of which are randomly coiled macromolecules, has been verified. However, the growth of calcium carbonate on LB–LbL films comprised of chitosan and iota-carrageenan, a sulfated polysaccharide that exhibits helical conformation in the presence of calcium ions, resulted in the formation of large isolated crystals [22] rather than a continuous film. Furthermore, it was possible to modify the structure of these crystals by changing the supersaturation conditions of the surfaces. This was achieved by altering the electrical charge of the LB film used as precoating, with consequent change in the intermolecular forces between the lipids and the polyelectrolyte layers. Nevertheless, it is not known why a continuous film rather than isolated crystals is formed. Whether this is only related to the presence of the LB film as precoating as well as the type of the phospholipid charged group or if it also depends on the conformation of the polyelectrolyte (coil or helix) remains to be elucidated. In this context, we have decided to conduct a comparative study of the influence of organic matrices consisting of different randomly coiled polyelectrolytes assembled by means of the LbL technique on precoatings prepared by the LB technique. To this end, two differently charged phospholipids with the same carbon chain length have been employed: the negatively charged sodium salt of dimyristoilphosphatidyl acid (DMPA) and dimyristoilphosphatidylethanolamine (DMPE), a zwitterionic phospholipid. The LbL matrices were prepared by using one of the randomly coiled polymers: PAA, which contains carboxylate groups, or lambdacarrageenan (␭-car), which displays sulfate groups. The polycation was the biomacromolecule chitosan. These films were used as templates for the growth of calcium carbonate using the previously described “dry method” [20–22]. 2. Materials and methods 2.1. Materials DMPA and DMPE were purchased from Sigma® and used without further purification. Chitosan (cht), with a nominal molecular weight (Mn) of 1.5 × 103 g mol−1 , degree of acetylation 0.15, as determined by NMR [25], was purchased from Polyscience® . Lambda-carrageenan (␭-car) was supplied by Gelimar-Chile, and poly(acrylic acid) (PAA) Mn = 2.5 × 105 g mol−1 was acquired from Sigma® . Calcium chloride and zinc chloride were obtained from Merk® , and ammonium carbonate was purchased from Vetec® (Brazil). All the aqueous solutions were prepared using dust free Milli-Q® water. 2.2. Preparation of the support Aluminum supports were polished with sandpaper (1500–2000 3M Brazil) and pretreated in KH2 PO4 /NaOH buffer solution (pH 7.5) containing the non-ionic surfactant Span 20 (4.0 × 10−5 mol L−1 ) for 5 min, at 65 ◦ C, under ultrasound agitation. After the treatment the supports were exhaustively washed with Milli-Q® water. 2.3. Langmuir-monolayers Surface pressure–surface area (–A) isotherms were performed at 25 ◦ C in a 216 cm2 Langmuir trough (Insight, Brazil) by spreading a 1.0 mmol L−1 lipid (DMPA or DMPE) solution dissolved in chloroform/methanol (3:1, v/v) HPLC grade (J.T. Baker). Monolayers were spread on subphases containing either 0.1 mmol L−1 ZnCl2 aqueous solution or 0.05% (w/w) cht aqueous solution. Zinc ions were

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included in the subphase solution, in order to promote adhesion between the DMPA phospholipidic layers in the LB films. 2.4. Langmuir–Blodgett films The monolayers were transferred to 10 mm × 5 mm metallic aluminum supports by the LB technique. Y-type LB films were prepared by initially immersing the solid support in the subphase solution before spreading the solution of the phospholipid. Three layers of pure DMPA monolayers were deposited by means of a sequence of one vertical withdrawal followed by an immersion, and the procedure ended with another withdrawal of the support at 0.038 mm s−1 , at a constant surface pressure of 30 mN m−1 . This resulted in a hydrophobic three-layer Y-type LB film. The DMPA mass deposited per layer was 180 ± 10 ng, as measured with the aid of a quartz crystal microbalance (QCM). A fourth layer was deposited by transferring one of the pure phospholipid monolayers, DMPA or DMPE from a subphase consisting of 0.05% (w/w) cht solution, at 40 mN m−1 . Because this last layer displayed a hydrophilic end, it was stabilized in the presence of the polyelectrolyte, thereby avoiding the “flip–flop” of the phospholipid molecules. 2.5. Layer-by-layer film To prepare the LbL films, either ␭-car or PAA was dissolved in a 0.01 mol L−1 CaCl2 solution, which had a final pH of 3.7. Dissolution of cht in acetic acid solution (pH 3.5) was accomplished for 24 h. After the complete solubilization of cht, the pH of the solution was adjusted to 5.4 by means of a NaOH solution. The final solution was filtered in a Millipore® membrane (0.45 ␮m pore size). Three and thirteen layers of LbL films alternating the negatively charged ␭-car or PAA and the positively charged cht were deposited on the LB–cht film, denoted hereafter as (LbL)3 and (LbL)13 , respectively. To this end, firstly the support containing the LB–cht film was immersed in a ␭-car or PAA solution for 20 min. Subsequently, the support was immersed in a 0.01 mol L−1 CaCl2 solution for 5 min, in order to remove non-adsorbed polymer chains and to keep the ionic strength constant. The support was then dried under N2(g) flow. Following this procedure, other immersion–withdrawal cycles in cht and ␭-car or PAA aqueous solutions were accomplished, until the desired number of layers was obtained. The top layer always consisted of the polyanion. 2.6. Formation of calcium carbonate Formation of CaCO3 on the mixed LB–LbL films was achieved in a closed container by means of the “dry method” [20–22]. This involved the reaction of the CO2 product from (NH4 )2 CO3 decomposition with the Ca2+ (aqueous) ions trapped in the polymeric LbL film and the Ca2+ ions present as counterion of the polyanion in the polymeric matrix. It is worth mentioning that this is not the conventional procedure for the precipitation of inorganic crystals over thin film templates. In the traditional approach, the supports containing the organic matrix are commonly immersed in a recipient containing a supersaturated solution of the precipitant, so that the precipitation from the bulk is not prevented. In the latter system, ions are surrounded by a huge amount of water, and the displacement of water molecules by the inorganic crystal is rather important for the properties of the precipitates, thereby rendering products that may exhibit rather different properties as compared to those obtained at the interface [26]. Conversely, the “dry method” contributes to the occurrence of surface processes.

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Fig. 1. Chemical structures of (A) ␭−carrageenan and (B) poly(acrylic acid).

2.7. SEM and AFM

3. Results and discussion

The morphology of the gold-coated films was investigated by scanning electron microscopy (SEM) using a Zeiss-EVO 50 microscope. The atomic force microscope (Shimadzu-SPM 9600) was used in the contact mode, to explore the roughness of the films.

3.1. Considerations about the macroanions forming the LbL matrix

2.8. Raman spectroscopy Vaterite, calcite, and aragonite CaCO3 polymorphs can be identified by means of their characteristic Raman spectra [27]. Dandeu et al. [27] have shown that three different regions of the spectra can be used for the identification of these different polymorphs. The most intense peak at ∼1100 cm−1 , corresponding to the symmetric stretching of the CO3 2− group, is observed in all the samples. The different polymorphs display bands with distinct frequency and shape in the near infrared region, which aids the identification of the samples. A single band at ∼280 cm−1 is typical of the polymorph calcite, while the polymorph vaterite exhibits a split band with maxima at 265 and 303 cm−1 . The Raman spectra of the supports containing the organic matrix and CaCO3 were collected using a micro-Raman spectrophotometer Renishaw-EPSRC JREI and a 514 nm laser as incident radiation.

In a previous study [22] we found that LB–LbL films ending with iota-carrageenan (␫-car) promote the formation of isolated CaCO3 crystals rather than a continuous film, whereas LbL films consisting of PAA and cht produce thin films of nanoparticulate calcite [20]. The polyanions PAA and ␫-car differ in terms of the charged group and the conformation in solution: ␫-car is a sulfated polysaccharide that adopts helical conformation in the presence of calcium ions in solution [28]. PAA is a poly(carboxylic acid) present as a random coil, regardless of the ion present in solution. Schoeler et al. [29] have shown that the helical conformation adopted by ␫-car in solution is kept during the multilayer buildup. These differences can elicit changes in the local supersaturation and thereby lead to different modes of CaCO3 growth. To differentiate between the influence of the charged group and of the conformation of the template-forming macro-ion, herein we have prepared LbL films using another type of carrageenan, namely ␭-car, which is also a sulfated polysaccharide that does not bear the anhydro-galactose residue, pointed out as being the responsible for the helical conformation of carrageenans in aqueous solution [28–30]. The chemical

Fig. 2. SEM images of CaCO3 films grown on (LbL)13 matrices consisting of (A) cht and ␭-car; (B) cht and PAA. In the bottom are the enlarged sections of the top images.

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Fig. 3. Raman spectra of the CaCO3 films grown on (LbL)13 matrices consisting of (A) cht and ␭-car; (B) cht and PAA.

structures of the polyanions used in this work are represented in Fig. 1. The generation of continuous films or non-connected crystals can be attributed to differences in the nucleation and growth mechanisms that taking place for different LbL films. For example, one can suppose that the helical structures of ␫-car transferred to solid supports confines the CaCl2 solution, thereby accumulating calcium ions in isolated sites and enabling the growth of isolated CaCO3 crystals. However, the coiled PAA or ␭-car may provide a continuous distribution of Ca2+ ions along the matrix, thus allowing for production of homogeneous and continuous CaCO3 films. On the other hand, the electric charge separation at the LbL film interface and the different abilities of the negatively charged groups in terms of Ca2+ binding should be the reason for the preferential formation of continuous CaCO3 film or isolated CaCO3 crystals. The linear charge density of PAA depends on its ionization degree [31].

For the working pH 3.7, the charge fraction of the chains (f) is only about 0.01; i.e., one of 100 monomers is negatively charged. However, the sulfated ␫-car and ␭-car macro-ions correspond to strong electrolytes with approximately the same f [32]: 2 sulfate groups per disaccharide repetitive unit. Nevertheless, the linear charge densities depend on the separation between the charged groups (r), which in turn will depend on the conformation of the macromolecule. The distance between the carboxyl groups in PAA chains ˚ For ␭-car, in the coiled conformation, r is equal to 4.8 A, ˚ is 5.02 A. ˚ and for ␫-car, which exhibits the helical conformation, r is 4.3 A. The linear charge densities () of the ␭- and ␫-car, where  = lB /r and lB = Bjerrum length, are 1.49 and 1.52, respectively. In the case of PAA,  is 0·014· On the basis of the r values of these different polyelectrolytes, one can conclude that the linear charge densities of ␭- and ␫-car cannot be the reason for the differences observed

Fig. 4. SEM images of CaCO3 films grown on mixed DMPA/LB–(LbL)3 matrices consisting of (A and B) ␭-car and (C and D) PAA.

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Fig. 5. SEM images of CaCO3 films grown on mixed DMPE/LB–(LbL)3 matrices consisting of (A and B) ␭-car and (C and D) PAA.

in terms of crystal growth. But one should realize that the linear charge density for PAA (f = 0.01 in the working conditions) is about two orders of magnitude lower than that verified for the sulfated polyanions. Therefore, the surface electric charge densities should be the relevant difference between the two matrices. 3.2. Effect of the surface charge densities of the matrices containing polyanions on the growth of CaCO3 The coiled polyelectrolytes PAA and ␭-car without the precoating consisting of LB film were employed in the preparation of the matrix to be used for CaCO3 precipitation. Fig. 2 depicts SEM images of the CaCO3 films formed on a thirteen-layered LbL matrix consisting of cht/␭-car (Fig. 2A) and cht/PAA (Fig. 2B). Both ␭-car and PAA yield thin particulate CaCO3 films on the entire polymeric matrix. However, the grain sizes are different: they are larger in the case of ␭-car (∼200 nm) as compared to PAA (∼50 nm). Concerning ␭car, each grain consists of an ensemble of smaller particles (see Fig. 2A enlargement in the bottom), whereas in the case of PAA the sintering of the particles is more pronounced, so it is not possible to distinguish the smaller particles forming the grain (see Fig. 2B enlargement in the bottom). The Raman spectra obtained for different regions of the films are illustrated in Fig. 3A and B. When ␭-car is the matrix, the spectrum reveals the exclusive formation of calcite, whereas for PAA a mixture of calcite and vaterite is detected, thereby confirming the results previously obtained by X-ray diffraction analysis [20]. As previously mentioned, the electric charge separation for ␭-car is about 4.8 A˚ and is practically coincident with that of carbonate ions ˚ [4], which accounts on the {1 0 4} and {1 1 0} faces of calcite (4.99 A) for the preferential formation of this polymorph. In the case of PAA ˚ (with f = 0.01), the average electric charge separation is about 500 A, which is far beyond the values of calcite and vaterite unit cells (for ˚ The result is a non-selective precipitation, with vaterite, c = 8.56 A). the production of a mixture of the two polymorphs on the films

formed by this polyanion. This result reflects the direct influence of the different charge densities of these two polyanions. Moreover, the smaller electrical charge separation in the ␭-car polymeric chain drives the formation of smaller CaCO3 particles. 3.3. Influence of the orientation of PAA and -car as driven by a phospholipidic LB film The supersaturation depends on the ability of the organic film to trap water (or CaCl2 aqueous solution). If this feature is changed systematically, one should expect differences in crystal growth [22]. Moreover, the arrangement of the polyanion in the polymeric matrix is essential for the control of the crystal structure during film growth [4]. On the other hand, it is possible to force the exposition of certain chemical groups of the polyanion, in detriment of others, by using a well-ordered thin film as the precoating. Thus, the formation of CaCO3 on PAA and ␭-car has also been studied by depositing the LbL film on a precoating prepared by the LB technique with selected phospholipids. In a previous work [22], we reported the influence of a mixed LB–LbL matrix on the formation of CaCO3 crystals using ␫-car in the helical conformation as polyanion. The presence of different DMPA/DMPE molar ratios in the LB film grown under the ␫-car LbL films affected the crystallization of CaCO3 , culminating in distinct shapes, roughness, and sizes for the resulting crystals. Using these coiled polyelectrolytes, we again obtained continuous particulate films upon CaCO3 precipitation on LbL films containing only 3 layers of cht and ␭-car deposited on an LB film containing 4 layers of DMPA as precoating (Fig. 4A and B). Elongated particles mostly oriented with the larger axis parallel to the surface were detected. It is important to emphasize that the growth of continuous CaCO3 films on single (LbL)3 films was not successful, but surprisingly this was possible when the LB precoating was present, evidencing that the LB film influences the packing of the polyelectrolytes present in the LbL film. The homogeneous and oriented precoating produced by the negatively

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Fig. 6. Raman spectra of CaCO3 films grown on mixed LB–(LbL)3 matrices consisting of (A) DMPA–(cht/␭-car)3 , (B) DMPA–(cht/PAA)3 , (C) DMPE–(cht/␭-car)3 , and (D) DMPE–(cht/PAA)3 .

charged phospholipid probably directs the polyelectrolyte binding in a homogeneous way even in the first layers of the LbL film, thereby allowing for the growth of continuous structures. Fig. 4C and D shows the formation of CaCO3 on three-layered cht/PAA LbL films also grown on a DMPA–LB film. This matrix drives the formation of larger particles, as compared to the particles obtained when ␭-car is employed as polyanion (Fig. 4A and B). Particulate films can also be produced when the precoating LB film contains the zwitterionic phospholipid DMPE in the outer layer. From the micrographs presented in Fig. 5, it is possible to distinguish prolate ellipsoids oriented in different angles, with the larger axis mostly perpendicular to the surface, resulting in a flower-like structure when ␭-car is used in the LbL film (Fig. 5A and B). For PAA (Fig. 5C and D), the orientation is mostly parallel to the surface, and the crystals are larger. PAA is only partially ionized in the pH used herein, so it should be a random coil with low  and low persistence length. On the other hand, ␭-car is completely ionized and, due to its higher , the chains should be fully stretched with a higher persistence length. This should be the main reason for the different geometries observed for the particles formed using these two polyanions. Moreover, the CaCO3 polymorph grown on the DMPA–LbL film is calcite, independently of the polyanion employed in the matrix, and vaterite in the case of the DMPE–LbL film, as seen from the results obtained from Raman spectra recorded for these films (Fig. 6). Table 1 summarizes the types of polymorph grown on each organic matrix. These results show that, despite the importance of the chemical nature of the uppermost layer of the matrix where the CaCO3 is

grown, the way in which the polyelectrolyte is bound is important and can furnish quite different morphologies and crystal type for the nanoparticulate films. From our results, the LB film used as precoating and prepared with DMPA or DMPE can direct the binding of cht, so it influences the LbL film and the subsequent growth of CaCO3 . DMPA is negatively charged, and the polycation cht, used in the first layer and transferred together with DMPA, may bind to the phospholipid mainly through electrostatic, thus leaving the NH2 and OH groups of cht available for interaction with the polyanion deposited onto the next LbL layer via hydrogen bonds. In turn, the polyanion has its charged groups ( OSO3 − in the case of ␭-car, or COO− in the case of PAA) exposed to binding with Ca2+ ions. When the zwitterionic DMPE is used in the preparation of the outer layer of the LB film, cht binds to the phospholipid mainly through its NH2 or OH groups, hence leaving the NH3 + available for binding to the polyanion. In this case, the polyanions will preferentially expose their uncharged groups ( OH, in the case of ␭-car, or COOH in the case of PAA) in the outer layer. It is important to mention Table 1 CaCO3 polymorph grown on each specific organic matrix. Matrix

CaCO3 polymorph

(cht/␭-car)13 (cht/PAA)13 DMPA–(cht/␭-car)3 DMPA–(cht/PAA)3 DMPE–(cht/␭-car)3 DMPE–(cht/PAA)3

Calcite Calcite/Vaterite Calcite Calcite Vaterite Vaterite

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Fig. 7. AFM images of the (A) DMPA/LB–(LbL)13 film without CaCO3 , (B) the DMPA/LB–(LbL)13 film with CaCO3 , (C) the top view of the (LbL)13 film with CaCO3 , and (D) the (LbL)13 film with CaCO3 .

that the precipitation of CaCO3 does not occur only from the Ca2+ ions directly bound to the charged groups, but it also occurs within the aqueous solution that is solvating the hydrophilic groups of the polyelectrolyte. Moreover, different chemical groups have different affinities for water or aqueous solutions. Thus, driving the binding of the polyanion in a specific way means generating different supersaturation conditions. Consequently, assembly of the particles will take place in distinct ways. Kotachi et al. [4] have shown that vaterite is preferentially formed on cht membranes that display exposed OH groups, because distances between these groups are compatible with the distances in the vaterite unit cell. The exposure of OH also improves the ability of the polymeric layer to trap the CaCl2 solution, thereby increasing the local supersaturation and leading to the formation of vaterite, the most kinetically stable CaCO3 polymorph, as evidenced by the Raman spectra presented in Fig. 6.

3.4. AFM analysis In order to better understand the nature of CaCO3 nanoparticulate films coating on the organic matrix, we have performed AFM analysis of the matrices themselves, with a higher resolution than that obtained for SEM micrographs. The AFM images of the films formed with and without calcium carbonate are significantly different, as shown in Fig. 7. The film containing only the organic matrix (cht/␭-car)13 displays roughness peaks with up to 181 nm (Fig. 7A), whereas the film containing CaCO3 particles exhibits roughness peaks up to 43 nm (Fig. 7D), and the film containing DMPA and (LbL)13 with ␭-car (Fig. 7B) presents roughness peaks up to 61 nm. Additionally, the top view of the hybrid film (Fig. 7C) reveals complete coating of the organic matrix with the inorganic film as well as the clear formation of particles, thereby evidencing a continuous film. Therefore, it is very likely that the precipitation of calcium

Fig. 8. SEM images of CaCO3 films grown on mixed DMPA/(LbL)13 matrices consisting of ␭-car.

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carbonate occurs in the voids present in the organic matrix, making it smoother.

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formed in the presence of PAA. Therefore the shape and orientation of the particles as well as the crystalline structure are dependent on the phospholipid LB precoating/polyanion LbL matrix set.

3.5. Effect of the number of layers present in the LbL film Acknowledgments SEM images of the LB/LbL films containing increasing number of layers in the LbL films were also acquired. Comparing the images shown in Fig. 4A and B to the images given in Fig. 8, the formation of a more closed structure in the DMPA/(LbL)13 film can be seen (Fig. 8A and B). These results show that a more condensed packing of the polyelectrolyte layers is achieved when the number of LbL layers is increased. However, the features related to the shape and size of the CaCO3 particles grown on these matrices were similar to those of the particles grown on the DMPA/(LbL)3 film. 4. Conclusions In this work, we have attested that continuous particulate films of CaCO3 , and not isolated CaCO3 crystals, are formed on organic matrices produced by the LbL technique and in which the uppermost layer consists of a randomly coiled polyanion. The production of continuous films does not depend on the chemical nature of the negatively charged group, and the selection of polymorph seems to depend on the linear charge density () of the polyanion. This study proofs that the randomly coiled conformation of the polyelectrolytes is the main reason for the CaCO3 nanoparticulate continuous films formation, consisting in a new knowledge for in vitro biomineralization. Moreover, the morphology of the films and the crystal nature of the particles are influenced by the presence of a precoating consisting of an LB film onto which the LbL matrix has been deposited. The nature of the head group of the phospholipid present in the LB film drives the binding of the polycation to the first layer of the LbL matrix, thus culminating in a cascade effect as well as in changes to the available binding sites and to the local supersaturation that induce the crystallization. An LbL film comprised of only three layers does not render a CaCO3 continuous film, whereas the same three-layered LbL film deposited on a precoating consisting of an LB film does produce such a continuous film. The AFM analysis revealed that smother surfaces are obtained when the LB precoating is used under the LbL film. When the zwitterionic DMPE is employed in the preparation of the LB film, the CaCO3 particulate films consist of elongated vaterite particles oriented perpendicular or parallel to the surface, depending on the polyanion present in the LbL film. With the negatively charged DMPA, though, elongated particles are only generated in the presence of ␭-car in the outmost layer of the LbL film, whereas flat calcite particles, apparently without a preferential orientation, are

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