Biodegradable Polymer/Clay Nanotubes Composites - Nanocompositi a Base di Polimeri Biodegradabili e Nanotubi Alluminosilicatici

This work of thesis reports on the preparation and physico-chemical characterization of nanostructured polymer-based systems. Clay nanotubes, namely imogolite and Halloysite, have been extensively investigated and proposed as inorganic fillers of biodegradable polymer matrices with potential biomedical applications, especially in the bone medication field. Instead of paying attention to a specific system, different materials and processing techniques have been investigated, focusing each time on specific physico chemical aspects, such as stability and degradation in physiological conditions, mechanical properties, rheology, nano- and micro-structure, release/uploading properties. In particular, four case-studies on the use of nanocomposite systems in different biomedical areas are described in the thesis. The first case study reports on the preparation and characterization of a bioactive hybrid film made of Sr-loaded halloysite embedded within a biopolymer matrix. The adopted polymer (3-polyhydroxybutyrate-co-3-hydroxyvalerate, PHBV) is a biocompatible and biodegradable material already used in several environmental and biomedical applications, while the inorganic component is a clay nanotube with peculiar mechanical and cation-exchange properties. In particular, strontium(II) ions were adsorbed on the surface of the clay nanotube in order to enhance its bioactivity. In fact, strontium-based drugs have been widely prescribed over the years to treat osteoporosis, but when Sr(II) is orally administered at high doses it could increase the risk of heart attack and other side effects. Rather than through its systemic administration, it is therefore crucial to deliver strontium(II) at the treatment site, so that its bioactivity could be locally exploited, avoiding the associated collateral effects. A possible carrier for Sr(II) is thus represented by the synthetic graft matrix constituted by the clay nanotube and PHBV. Halloysite nanotubes introduce mechanical resistance (i.e., resistance to compression evaluated by means of AFM nanoindentation experiments) in the composite, as well as providing Sr(II) uploading properties. The biodegradability of the polymer matrix is a key parameter, as the composite is able to progressively expose the Sr(II)-loaded inorganic scaffold. The in vitro biocompatibility of the composite was demonstrated through cytotoxicity tests on fibroblast cells, highlighting the crucial role of the biopolymer film in acting as a binder and as a diffusional barrier to the Sr(II) release. Furthermore, the fabrication method described to obtain the nanocomposite is highly versatile, and it could address different biomedical and physico-chemical needs. In particular, the amount of uploaded Sr(II) could be easily adjusted by varying the surface functionalization of HNT, and the time before the bioactive inorganic scaffold is exposed to the physiological environment could be tuned by the thickness of the biopolymer coating. The second case study reports on the study of the upload/release capacity of engineered Halloysite and imogolite nanoclays. In a first example, halloysite nanotubes (HNT) were integrated with chitosan and hyaluronic acid to obtain hybrid nanocomposites with opposing charges, and their potential in the controlled release of drug model probes was investigated. The high surface area and the hollow nanometric sized lumen of HNT allowed for the efficient loading of rhodamine 110 and carboxyfluorescein, used as models for oppositely charged drugs. In the case of chitosan, the preparation of the nanocomposite was carried out exploiting the electrostatic interaction between the polymer and HNT in water, while with hyaluronic acid a covalent functionalization strategy was employed to couple the polymer and the clay. For the release experiments, a fixed amount of nanocomposite material loaded with the fluorescent dyes was placed in water and kept under agitation. At specific intervals of time (up to a period of 48 hours) an aliquot of the dispersion was centrifuged, and the concentration of the released probes was evaluated from the fluorescence intensity of the supernatant solution. Results showed that the polymeric coatings were successful in modulating the charge of the halloysite surface and altering the release kinetics of the probes. In particular, the model adopted to fit the release kinetics described very well the experimental data, indicating that both charge and coating composition play a key role in the desorption process from halloysite-based composites. In a second example, the adsorption of different amino acids onto imogolite nanotubes was investigated by means of turbidimetry, ζ-potential measurements, and FT-IR spectroscopy. A high affinity of glutamic acid (Glu) for imogolite surface was observed, and this finding was exploited to prepare a composite material made of lauroyl glutamate (C12Glu) adsorbed onto imogolite nanotubes. The obtained hybrid was then used in a proof of concept experiment for the upload of a model drug. The amount of uploaded drug drastically increases when C12Glu is present, highlighting the crucial role of the surfactant’s alkyl chains as hydrophobic pockets. The obtained results strongly point out towards the possibility of using glutamate-based surfactants and imogolite nanotubes for the design of hybrid systems for biomedical applications, and that the approach used can be generalized towards any aluminum oxide surface. Furthermore, the interaction of glutamate with imogolite surface could be exploited for modulating the chemical affinity between polymers bearing glutamate-based functionalities and the nanotube wall, paving the way to a more extended use of imogolite in the fabrication of nanocomposites. In another case study, a nanocomposite material made of Halloysite nanotubes (HNT) and carboxymethyl cellulose was prepared and characterized from a structural and rheological perspective. Chemical modification of the clay surface followed by an hydrazide-aldehyde coupling protocol with partially oxidized cellulose successfully produced a nanocomposite in which the inorganic nanotubes are homogeneously distributed, able to form a hydrogel when dispersed in water. Rheological properties of the composite are driven by chemical and structural design, with nanotubular fillers playing a crucial role as rheo-modifiers. The introduction of HNT in the polymer matrix, due to their chemical functionalities favoring inter-chain interactions, substantially modifies the rheological behavior of the system, making it significantly more viscous (at least twice as much) in the entire range of explored shear rates. A key aspect of the work was the careful evaluation of the material’s injectability, which represents a critical factor for the use of new materials in the biomedical field (e.g., minimal invasive surgery). In spite of the presence of inorganic nanostructures that could in principle aggregate and obstruct the orifice, the injectability was mostly dictated by the cellulose matrix, with 1D clays tending to align with the direction of the flow. Injection, i.e. high stress, produced a strong change in the structure of the polymer matrix at the microscale and this was reflected in the rheological properties of the material. The last example reports on the preparation and mineralization properties of macroporous composite hydrogel made of chemically cross-linked gelatin and imogolite nanotubes. Imogolite nanotubes have been synthesized and successfully integrated in a cross-linked gelatin matrix, then an extensive characterization has been conducted highlighting the role of the cross-linking agent and the inorganic filler in determining the stability in physiological conditions and the mineralization properties. The composite hydrogel was properly designed so to address multiple requisites in the bone tissue engineering field: the polymer matrix, as well as the adopted chemical cross-linking agent (glycerol diglycidyl ether), is fully biocompatible and biodegradable; the inorganic matrix has high surface area, low toxicity, a remarkable mechanical strength and stability; the composite material has a multi-scale macro-sized porosity that should favor osteoconductivity and cell permeation in vivo. The key finding of the work is that the presence of imogolite nanotubes in the hydrogel enhances the formation of hydroxyapatite crystals, as observed by means of thermogravimetry and X-ray diffraction after simulation of a mineralization process with a standard protocol, presumably acting as crystallization seeds. The obtained results clearly highlight the potentials of imgolite when introduced in the design of hydrogel-based scaffolds, especially thanks to the combination of their nanotubular shape and their affinity towards apatite mineral phases.