Papa, Antonio (2014) ENGINEERING TUNABLE TEMPERATURE AND pH RESPONSIVE PNIPAM-VAA MICROGELS FOR BIOMEDICAL APPLICATIONS. [Tesi di dottorato]

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Abstract

Sensing and recognition of bio-molecules is of extreme interest nowadays because it allows for detection of specific targets which can be the hallmarks of diseases. Colloidal particles can be used as a powerful platform to recognize bio-molecules and to perform a fast screening of them. Indeed, in most cases the surface of colloidal particles can be easily functionalized with agents which allow to control specific interaction between the particles and specific recognition. In addition, colloids with a switchable recognition mechanism, which can be externally triggered, would have a tremendous impact in the field of drug delivery and bio-sensing. “Smart” microgels are potentially suitable for these applications. They are extensively studied due to their swelling response to changes in specific environmental stimuli (i.e. pH, temperature, solute concentration, solvent composition, ionic strength, light, or electric field). While a variety of polymer systems have been explored, most attention has focused on microgels based on poly(N-isopropylacrylamide) (PNIPAM). They exhibit an extreme response to changes in temperature. Linear PNIPAM has a lower critical solution temperature (LCST) of 32 °C in aqueous solution, at which point the polymer reversibly switches from a fully soluble, hydrophilic random coil at lower temperatures to an insoluble globule at higher temperatures. When cross-linked into a colloidal gel, PNIPAM-based microgels exhibit this temperature responsiveness by undergoing a reversible de-swelling volume phase transition between 32 and 35 °C (the volume phase transition temperature, VPTT). Smart environmental triggers can be incorporated into the PNIPAM microgels by co-polymerization, to provide multivariable control over the particle swelling like temperature and pH. The functionalization of PNIPAM microgels with carboxylic acid groups can provide these proprieties and can achieve several objectives. The VPTT behavior of the microgel can be controlled via carboxylic groups incorporation. Both the value of the VPTT and the breadth of the deswelling transition can be influenced through copolymerization of more hydrophilic monomers. Functionalization can also provide reactive sites for post-modification of the gel, such as the bioconjugation of ligands. Our work has consisted in synthesizing tunable thermo- and pH-responsive core-shell microgels based on N-Isopropylacrylamide (NIPAM) coupled with vynil acetic acid (VAA) groups. Their volume sensitivity to pH and temperature were monitored by small-angle neutron scattering (SANS) and light scattering measurements. Ultra-structural analysis revealed core-shell architecture of the microgels with the core consisting of PNIPAM while the shell composed by PNIPAM and VAA. Volume change of the microgel in response to environmental pH and temperature were driven by separate mechanisms. Temperature sensitivity is conveyed mainly by the PNIPAM component while the pH sensitivity was imparted by the VAA component. As consequence, pH volume changes affected mainly the outer shell whereas the temperature volume change is localized both in the core and in the shell. Results indicated that by changing relative composition of NIPAM and VAA it is possible to tune the microgel VPTT and by changing the relative extension of core and shell compartment it is possible to tune the sensitivity of the gel to the environmental variation. Afterwards, we set out to investigate whether DNA conjugated microgels were compatible with hybridization process, which is commonly used for manipulations of DNA in the design of DNA bioassays or biosensors. First, we performed a Quenching experiment, in order to investigate the nature of the interaction of DNA fragments with microgel (physisorption vs hybridization) and thus, the specificity of hybridization on microgels. Cy5-labeled DNA oligonucleotide was conjugated with PNIPAM-VAA particles and by using a full complementary DNA oligonucleotide, opportunely modified with Black Hole Quencher 2 (BHQ-2), we performed the quenching experiment. Once confirmed the capability of DNA conjugated microgels to catch and recognize specifically complementary DNA strands we analyzed the effect of temperature and pH on the hybridization event and its stability. The hybridization process was performed and tested in terms of specific catching of a complementary oligonucleotide and successively, it was studied in details, looking at the effect of microgel structural changes. The effect of the shrinkage controlled by temperature changes does not drive any de-hybridization process. No dependence of the hybridization process was highlighted during microgel conformation changing, neither when the shell has collapsed nor when it is fully extended outside the microgel. Even analyzing the process as function of the oligonucleotide exposure towards the complementary oligo sequence, there is no direct evidence of its effects on the interaction process between the two DNA strands. Eventually, DNA conjugated PNIPAM-VAA microgels charge proprieties were exploited for a reproducible and facile approach optimized for physisorption of gold nanoparticles. The advance of this approach consists in the simple mechanism by which gold nanoparticles are adsorbed on microgels templates, without dealing with sophisticated chemical treatment for their conjugation with the microgel. The resulted PNIPAAm-40nm gold nanoparticles modes demonstrate that this approach provides the capability to tune the interparticle distance and therefore to control and modulate the Surface Enhanced Raman Spectroscopy (SERS) affinity upon temperature changing.

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