Development of biocompatible and “smart” porous structures using CO2-assisted processes

Dissertação apresentada para a obtenção do grau de Doutor em Engenharia Química, especialidade Engenharia da Reacção Química, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Over the past three decades the use of supercritical carbon dioxide (scCO2) has received much attention as a green alternative in the synthesis and processing of polymers. The scope of this thesis is the development of biocompatible and “smart” porous structures using CO2-assisted processes. This thesis is organized in four main chapters. The first one reviews and highlights some potentialities of supercritical fluid technology and the following ones compile the experimental work developed. The work is divided in three main parts: in the first part (2nd chapter) a CO2-assisted phase inversion method was developed in order to prepare porous structures, namely membranes. In the second part (3rd chapter) the focus was the synthesis of “smart” polymers,especially thermo and pH sensitive polymers. Finally, these two areas were combined (4th chapter) for the preparation of “smart” porous structures. The common guide line was the preparation or processing of biodegradable and/or biocompatible materials with special emphasis on the preparation of porous matrices, namely membranes and scaffolds, with controlled morphology. For membrane preparation a new high pressure apparatus and a new high pressure cell were developed. Polysulfone membranes (a biocompatible polymer with numerous applications in the medical field) were prepared and the effect of the solvent affinity and depressurization rate in the morphology and in the performance in terms of pure water flux of the membranes was investigated. The incorporation of a foaming agent was also analyzed and the high pressure CO2 capability to swell and melt polycaprolactone (PCL) was used to produce and control the porosity and the properties of the membranes. Finally, a natural and water soluble polymer (chitosan) was processed. The presence of water in the casting solution introduced extraordinary difficulties due to the low affinity between water and CO2. To induce the phase inversion a co-solvent (ethanol)was introduced in the CO2 stream. The obtained devices (membranes and beads) were fabricated using moderate temperatures and “green” solvents (ethanol, water and CO2). The morphology and the three dimensional (3D) structures were controlled by altering the co-solvent (ethanol) composition in the CO2 non-solvent stream during the demixing induced process. Microarchitectural analysis by scanning electron microscopy identified the formation of particulate agglomerates when 10% of ethanol in the scCO2 stream was used and detected the development of porous membranes with different morphologies and mechanical properties depending on the programmed gradient mode and the entrainer percentage (2.5-5%) added to the scCO2 stream. These chitosan matrices exhibited low solubility at neutral pH conditions, with no further modifications, demonstrating their applicability in bioreactors as static (membranes) or stirred (beads) culture devices. It was also demonstrated that the current method is able to prepare, in a single-step, an implantable antibiotic release system by co-dissolving gentamicin with chitosan and the solvent. In addition, the cytotoxicity as well as the ability of these structures to support the adhesion and proliferation of human mesenchymal stem cells (hMSC) in vitro were also addressed. After 2 weeks in culture, a 9-fold increase was obtained (versus 6 of the control). More importantly, cells maintained their clonogenic potential and immunophenotype (>95% CD 105+ Cells after 7 days of culture). In this chapter, a hypothetical schematic ternary diagram for the systems polymer–solvent–CO2 is used to discuss and explain the results. Another goal of this thesis was the synthesis of “smart” polymers. Chapter 3, addresses the precipitation polymerization of a thermoresponsive hydrogel, poly(N-isopropylacrylamide)(PNIPAAm), in scCO2. This hydrogel has a transition temperature, hereinafter called low critical solution temperature (LCST), around 32 ºC in an aqueous solution, close to body temperature. A strategy of solvent-free impregnation/coating of polymeric surfaces with PNIPAAm was suggested, in order to further extend the applications of membranes or porous bulky systems. The in situ synthesis of PNIPAAm within a chitosan scaffold was tested as a proof of concept, in order to produce smart partially-biodegradable scaffolds for tissue engineering applications. The LCST was tuned by copolymerization or graft polymerization of NIPAAm with other monomers. Copolymerization with hydroxyethyl methacrylate (HEMA) was used to decrease the LCST temperature from 32.2 ºC to approximately 27.7 ºC. Cloud point measurements of CO2 + HEMA system were used to optimize the polymerization temperature. Experimental data were obtained at 40 ºC, 50 ºC and 65 ºC and pressures up to 21.1 MPa. Soave-Redlich-Kwong equation of state with Mathias-Klotz-Prausnitz mixing rule was used to model experimental results and a good correlation was achieved. To increase the LCST, polyethylene oxide (an hydrophilic polymer) was grafted to PNIPPAAm. Dual stimulus (thermo and pH responsive) hydrogels were also prepared by copolymerizing methacrylic acid with PNIPAAm. As a proof of concept fluorouracil was incorporated in the hydrogels network and their release was controlled by temperature and pH stimulus. In chapter 4 the concepts of the previous chapters were put together envisaging the preparation of“smart” functional polymeric devices with targeted physical and chemical properties namely: (i) chitosan-based dual stimulus scaffolds (temperature and pH responsive); (ii) polysulfone-based thermoresponsive membranes and (iii) polymethylmethacrylate-based membranes. The chitosan scaffolds (pH sensitive) were coated/impregnated with a thermoresponsive polymer,poly(N-isopropylacrylamide) (PNIPAAm), using scCO2 as a carrier to homogeneously distribute the hydrogels monomer within the chitosan scaffolds and as a solvent to perform the polymerization reaction.

Fundação para a Ciência e Tecnologia através da bolsa de Doutoramento (SFRH/BD/16908/2004) e do projecto PTDC/CTM/70513/2006