Mechanical and magnetic properties of biocompatible ferrogels

Résumé

1. INTRODUCTION Magnetic hydrogels are heterogeneous systems consisted of magnetic nano- or microparticles dispersed inside a network of hydrophilic polymer chains. The macroscopic physicochemical properties of these materials depend on a number of variables related with the properties of the solid particles, the polymer composition and conformation, and the water content of the whole system. If, in addition, we take in mind that the internal structure of the hydrogel can be modified by chemical or physical internal/external stimuli, we are facing nanomaterials with extraordinary versatile benefits in very different fields of science and technology. We can mention four main areas of potential applications: i) Drug delivery, ii) tissue engineering, iii) biological research, and iv) hydrogel machines. All these application lays on the possibility of regulating, even dramatically, the mechanical softness and water content of the hydrogels. Thus, the mechanical moduli (Young’s modulus, rigidity modulus) of the hydrogels can range from values less than those of brains, nervous or spinal cords to those of cartilage tissues. In the case of magnetic hydrogels, the possible rearrangement of their microstructure by remote action of a magnetic field confers to ferrogels of a smart nature, which improves even more the capabilities of these soft-wet materials. In this work we aimed to contribute to the materials science of magnetic hydrogels by incorporating magnetic particles with at least four main characteristics. a) Non-spherical shape. b) Low average density. c) Biocompatibility, facing their potential applications in tissue engineering. d) And all of these properties, without losing the control of their viscoelastic properties using external magnetic fields (the so-called magnetorheological effect, MRE) of not too high intensity (H in the order of 100 kA/m). 2. RESULTS AND MAIN CONCLUSIONS To fulfill those features, in this work, the magnetic hydrogels designed consisted of composite particles based on clay particles with two different shapes (fibers, platelets) covered by magnetic nanoparticles. These particles were imbedded into a polymer matrix of a natural polymer (alginate, a polysaccharide), which can be easily cross-linked by physical interactions among the alginate molecules and even these ones attached to the particles, if they are adequately functionalized. The functionalization of the particles can play a double role: the reinforcement of the internal network of the hydrogels and, in addition, conferring the required biocompatibity to the obtained ferrogels. The determination of the mechanical (rheological) properties of the ferrogels had been a central point of this work. Ferrogels behave as soft viscoelastic materials in which the corresponding viscoelastic moduli depends on a number of variables. These variables are: i) the length and crosslinking degree of the polymer chains; ii) the degree of attachment among the polymer chains and the solid particles; iii) the strength of the bond between the crosslinker agent and the polymer molecules; iv) the concentration and aggregation degree of the solid particles imbedded in the hydrogel; v) the magnetic properties of the particles; vi) the intensity of the applied magnetic field; vii) and finally, the shape of the solid particles. The main conclusions of this work can be summarized as follows. A) From the results in Chapter 2 (sepiolite-magnetite ferrogels) we can conclude: A.1) The synthesized composite particles are actually composed by a fiber or rod-like core of sepiolite and a shell of magnetite nanoparticles (SM). These composite particles were efficiently functionalized by adsorption of alginate molecules (SMA). A.2) The magnetic response of these composite particles (SM and SMA) was large enough in comparison of that of pure magnetite and the composite particles contained a volume fraction of magnetic material around 40 %. A.3) The MRE reached, even for non-concentrated suspensions was very high, with the additional advantage of their very low setting rate in comparison with those in suspensions of non-composite magnetic microparticles (magnetite, iron). A.4) The cross-linking among the polymer chains, and among these ones and the alginate functionalized SM particles, was achieved by controlled addition of calcium ions. The hydrogel network was formed by alginate chains bonded to the solid particles. The variation in the crosslinking degree and also in the particle concentration allowed to obtain ferrogels with different softness and magnetic response. A.5) We have prepared two different ferrogels, with different alginate concentrations in the pregel solutions. This variable, coupled with the particle concentration, determined the stiffness of the resulting ferrogel avoiding both an excessive softness and at the other end an excessively rigidity. We obtained two different ferrogels for which the elastic modulus ranged from G’  20 kPa up to G’  30 kPa. A.6) The MRE achieved in ferrogels containing SMA particles depended on the role played by both the magnetic forces among particles and the elastic forces developed inside the particle-polymer network. Thus, when the ferrogels suffered from a low shear strain the MRE-G’ –MRE quantified by the relative field-induced increase in G’– can reach a value as high as 64%. This relatively strong MRE value indicated that the mechanical response of the ferrogels was mainly dominated by the particle-particle magnetic interactions and it can be controlled by the external field. On the contrary, at larger shear strains, the MRE-G’ values were considerably reduced: MRE  22 %. Now, the enlarged gap between particles weakens the magnetic interactions, and the mechanical response of the ferrogel was mainly determined by the elastic resilience of the knotted particle-polymer network. A.6) The effect of the particles shape on the MRE has been studied by defining a normalized-MRE-G’ parameter that mainly accounted for the particle morphology. The comparison, among the normalized-MRE-G’ values in the fiber-like SMA ferrogels in this work with other ferrogels previously reported containing spherical particles, demonstrated that the imbedding of elongated particles significantly enhanced the MRE effect in ferrogels. For example, the maximum normalized-MRE-G’ value reached in the sepiolite-based ferrogels was 64% while in a similar ferrogel with spherical silica-iron particles was 15%. B) From the results in Chapter 3 (bentonite-magnetite ferrogels) we can conclude: B.1) Unlike the sepiolite employed in previous chapter, the bentonite particles employed in this chapter was a mineral ore. The so-called hydrocyclone separation was used to simultaneously remove the mineral impurities and also for substituting the Ca2+ ions by Na+ ones. The sodium bentonite particles so obtained had the typical platelet-like shape of the smectite clays. Nevertheless, the well-known strong van der Waals attraction between the clay faces favored the formation of aggregates with stacked platelets. This arrangement remained after the coverage by magnetite and even after their functionalization by alginate. Therefore, the MRE in the bentonite-based ferrogels had to be conditioned by the presence of these clusters. B.2) The bentonite particles were effectively recovered by magnetite nanoparticles using the same coprecipitation method employed in chapter 2. These heterogeneous particles (BMag) had a strong enough magnetic character and an internal volume fraction of magnetic material of approximately 25%. B.3) The BMag particles were functionalized by alginate adsorption. In this case, two functionalized magnetic composites were obtained by adsorption of alginate molecules from solutions of the polysaccharide with a concentration ratio 1:4. The first advantage of the functionalization was a very significant decrease in the settling rates of the aqueous suspension of the functionalized particles. This fact represents a key point to obtain homogeneous ferrogels. B.4) The MRE values were determined in ferrogels containing the largest coverage by alginate molecules. In this case, four different ferrogels were prepared varying the concentration of alginate in the pre-gel solution. The normalized MRE-G’(%) values obtained by comparing ferrogels with the same alginate concentration in the pre-gel solution (1% w/v) and different morphologies were: 21% (bentonite-magnetite platelets), 43% (silica-iron spheres), and 180% (sepiolite-magnetite fibers). Likely, the presence of bentonite stacked clusters provoked the lowest value in this ferrogel. C) The Chapter 4 was devoted to check the biocompatibility of the ferrogels based in sepiolite-magnetite composites. The cytotoxicity of these magnetic colloids was studied ex vivo following different techniques usually employed in histological essays and, more particularly, in tissue engineering. The first one was based in visualizing the morphology of the cells cultured in the presence of the magnetic dispersions. The second and third techniques –Live/Dead test, based in the intracellular estearase activity; WST-1 test, based in the reaction of mitochondrial dehydrogenase enzyme with a dye– were useful to count the number of live cells after 48 h of cell culture. The fourth technique quantified the cytotoxicity by measuring the DNA released to the culture medium when the cell membrane integrity is compromised. C.1) These fourth techniques were employed to evaluate the cytotoxicity on fibroblasts in different culture media containing: i) a non-toxic standard solution (positive control); ii) the same non-toxic solution including a proportion of the magnetic dispersions studied; and iii) as negative control group, the cells were incubated in a solution of a powerful surfactant. C.2) The ferrogels evaluated contained sepiolite-magnetite (SM) particles in an alginate hydrogel. The particles were functionalized by adsorption of alginate molecules. Alternatively, and for comparison, the particles were functionalized by adsorption of a polymer (polyethylene glycol, PEG) that in bibliography had been reported as appropriate for conferring biocompatibility to magnetic nanoparticles. Finally, a suspension containing bare SM particles was tested. In all these magnetic dispersions the particle volume fraction was 1%, which is large enough to provoke or not toxic effect on the cultured fibroblasts. C.3) The morphological analysis demonstrated that the cells in contact with bare SM particles or alginate-functionalized SM particles did not lose their characteristic elongated shape compatible with living fibroblast, while the cells in contact with SM-PEG particles had a rounded shape as a consequence of the possible toxic effect of the particles. C.4) The Live/Dead test demonstrated the cell viability (around 90 % of living cells) in the colloids containing bare SM or SM-alginate particles. On the contrary, in the cell culture in the presence of SM-PEG a percentage as high as 50% of the fibroblast present were dead cells. The results obtained by means of the WST-1 test gave similar results to those with the Dead/Live test, demonstrating the consistence between the two tests. C.5) Finally, the test that checked the integrity of the cell membrane showed that for all the magnetic dispersions tested the results obtained were similar to that in the positive control media (and opposite to the negative one). Thus, it seems that the SM particles (bare or recovered by alginate or PEG) did not provoke a significant damage of the cellular membrane. C.6) All these ex vivo essays demonstrated that the alginate ferrogels, with alginate-covered SM particles imbedded, could be an good (because of their biocompatibility) candidate for generating artificial tissues in which these soft wet biomaterial work as scaffold for tissue engineering purposes. 4. BIBLIOGRAPHY *Abrougui, M. 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