IMMOBILIZATION OF BIOACTIVE PROTEINS ON SOLID SUPPORTS : APPLICATIONS TO FOOD PACKAGING AND FOOD NANOTECHNOLOGY

Much of the interest concerning bioactive protein immobilization onto solid supports can be attributed to the many potential applications protein-modified surfaces may have in the area of biotechnology. Immobilized bioactive proteins (e.g. antibodies, enzymes) have been used in countless immunoassays, as well as in clinical analysis and diagnostics, in biosensors, and in various industrial applications, including food processing. In this frame, this PhD thesis research project aimed at developing strategies to immobilize food-relevant bioactive proteins onto various solid supports, and at exploiting the interactions between the proteins and the solid matrices considered in this peculiar project to design either innovative packaging materials or novel nanotech-based analytical tools. Within the overall objective mentioned above, this project was subdivided into the following activities: 1. preparation of an antimicrobial biodegradable packaging material by binding lysozyme onto papers opportunely modified with polyelectrolytes; 2. preparation of biofunctionalized magnetic nanoparticles by binding specific food bioactive proteins to a conveniently activated dextran coating on the particle surface, for cellular targeting of conjugates; 3. preparation of magnetic nanotracers for addressing molecular recognition events, and use of the nanotracers to improve current analytical protocols. Novel food processing and new packaging strategies are being developed as a response to both consumer demand for and industrial trends towards mildly preserved, tasty, and convenient food products with prolonged shelf-life and controlled quality. Globalization of the food trade and recent food-borne microbial outbreaks are driving forces in the search for innovative ways to inhibit microbial growth in foods while maintaining quality, freshness, and safety. In this frame, one of the most innovative developments in the area of food packaging is the design of antimicrobial biopolymer-based active packaging materials incorporating biocides into or onto the surface of polymers themselves. In our studies, we addressed the interactions between charged polysaccharides and lysozyme, monitoring the protein structural changes and rearrangements consequent to noncovalent bonds with the polysaccharides. Lysozyme was incubated for different times and at different temperatures with soluble carboxy-methyl cellulose (CMC), and polygalatturonic acid (PGA), in the presence/absence of salts (NaCl), non-ionic chaotropes (urea), and anionic detergents (sodium dodecyl sulphate). The various systems were then analysed by a number of spectroscopic methodologies that demonstrated that the charged polyelectrolytes do not impair the structural and functional properties of lysozyme. Tryptophan fluorescence measurements showed that soluble CMC improves the thermal stability of the tertiary structure of lysozyme above 60°C, and has little if any effect on the stability of its secondary structure, as demonstrated by far-UV circular dichroism measurements. This data paved the way to the addition of CMC and PGA to paper-based packaging materials intended to incorporate lysozyme as antimicrobial not only because this improves the incorporation yield (mainly due to electrostatic interactions) and allows modulation of protein release, but also because it results in stabilization and preservation of the protein structure and activity during drying at 100°C, mimicking the heating steps in the paper making process itself, and would likely prolong the operative shelf-life of the resulting active packaging. The second part of this project dealt with the preparation of nanotechnological food-related applications. Nanotechnology is an emerging multidisciplinary field of applied science and technology which provides the methods and a sound framework for understanding and developing materials and products with at least one dimension smaller than 100 nm. Advancement in processes for producing nanostructured materials has led to the development of biocompatible magnetic supports with potential biochemical and biotechnological applications. This kind of support is generally synthesized by encapsulating magnetic materials within a polymeric layer. The interest for magnetic nanosupports is not only limited to the obvious ease of their separation under micro- and nanofluidic conditions, or to their entrapment in coatings and films. Most relevant is the fact that nanostructures can be conjugated to biologically active molecules, including hormones, antibodies, and various peptides, taken up by cells, and circulated among tissues expressing their cognate receptors. Given their intrinsic magnetism, and the perturbation induced on nuclear magnetism by other ferromagnetic species, magnetic nanoparticles may be used as tracers in NMR and imaging-NMR experiments, are detectable under appropriate conditions in standard transmission electron microscopy, and have demonstrated their potential in sensing a number of reversible molecular interactions, such as protein-protein, DNA-DNA, protein-small molecule, and enzyme reactions. In particular, we considered the pathway to design and prepare nanotracers by covalently binding food-related bioactive proteins to dextran-coated iron oxide magnetic nanoparticles (NP). The particles, with a diameter of 70-90 nm, were synthesized by chemical co-precipitation from an aqueous solution of Fe3+, Mn2+, and Zn2+ chloride, and then coated with dextran. Proteins considered in our studies included food allergens (betalactoglobulin), antibodies to food proteins (anti-gliadin IgGs), and enzymes. Trypsin was immobilized since it provides ease of quantification, and because of its usefulness in interactomics studies. RNAse was also conjugated to NP and used as structural probe by extending to NP conjugates some physico-chemical methodologies already used for characterization of the free enzyme. NP were modified either by epoxydation, followed by amination and succinylation, or by carboxymethylation in order to introduce on dextran carboxyl groups that were then converted by carbodiimide into reactive esters, allowing the subsequent coupling with different specific food proteins added in the reaction mixture. The successful coupling to the NP of all the proteins used in this project was assessed and measured by a number of different procedures. Microscopic, immunological, enzymatic, and physico-chemical approaches were used, whereas spectrofluorimetric techniques have proven not to be useful due to the high scattering and absorbance of the iron-oxide based particles that prevented us from obtaining reliable results. Size and morphology of both unmodified and functionalized NP were characterized by transmission electron microscopy. These measurements showed that aggregation phenomena were induced by both dextran activation approaches and, in particular, by the various centrifugation steps required in the functionalization process, although fresh unmodified NP also tend to form aggregates. However, the procedure based on carboxymethylation resulted in the lowest extent of particle aggregation. Dot blotting with specific antibodies, followed by immunoenzymatic detection of the bound antibodies, demonstrated the actual presence of betalactoglobulin (BLG) on the conjugated NP. Competitive ELISA tests performed by using conjugates between carboxymethylated NP and BLG led us to measure a coupling yield of 20 μg protein per mg NP. This indicates that each NP, assuming an average mass of 1 MDa for a particle of the given composition and diameter, binds one bioactive protein molecule. The resulting conjugates were then used to address the issue of allergen recognition and subsequent uptake by cell model systems. In particular, human monocytes were incubated with unmodified and conjugated NP and then separated by MACS® technology, that uses a magnetic field strong enough to retain cells that integrate even the smallest amount of magnetic material. We could therefore study the uptake of both kinds of particles by this peculiar cell line: BLG-conjugated NP are endocytosed in higher amounts and faster with respect to non-conjugated NP. Indeed, the presence of bioactive BLG favors particle endocytosis, although a certain extent of particle internalization was assessed and may be due to non specific interactions between the NP and cell membranes. Both kinds of NP were also used for assessing via different microscopy techniques (optical, fluorescence and electron microscopy) the uptake by Triticum durum sprouts, and the particle effects on model animal cell systems. Tests aimed at assessing the citotoxicity of unmodified and conjugated NP showed that the viability of both differentiated HT-29 and Caco-2 cells after various times of incubation with the NP was close to that of control untreated cells, indicating the full biocompatibility of these particles with these in vitro intestinal cellular models. This result was confirmed by both the cell proliferation rate and the levels of apoptotic markers that were unaffected by 24 hours incubation with the same NP. Trans-Epithelial Electric Resistance measurements were performed on Caco-2 cells differentiated towards an epithelial structure, and demonstrated that incubation with unmodified and conjugated NP increases the TEER. This reveals a direct effect on the paracellular permeability of intestinal cells by the NP that might act as protective agents against foreign molecules that are potentially dangerous for the integrity of the epithelial barrier made by the cellular tight junctions. The same immobilization approaches described above were used to develop magnetic nanotracers that may be used in interactomics studies for assessing molecular recognition events occurring in the complex machineries involved in cofactor and protein assembly. We immobilized commercial anti-gliadin IgG to the NP, and we subsequently performed, as test of protein immobilization, dot-blotting and immunoprecipitation experiments in the presence of gliadins that showed that anti-gliadin IgG coupled to the NP were able to recognize bioactive protein epitopes after the binding procedure. The coupling ratio of both our functionalization approaches was also estimated by conjugating analytical-grade trypsin to the dextran-coated NP, and by evaluating the immobilized enzyme activity on synthetic (benzoyl-L-arginine p-nitroanilide) and complex substrates (caseins). Although yields were in all cases quite low, they compare well with those reported for equivalent procedures reported in the literature. Most relevant is the fact that carboxymethylation resulted in a higher protein/particle ratio that was comparable to that obtained by ELISA in the case of BLG. Moreover, NP-trypsin conjugates were used in interactomics experiments and succeeded in recovering a trypsin inhibitor from a protein solution paving the way to their use as nanosized probes for the development of innovative analytical food-related tools. Possible changes in the thermal stability of the NP-bound RNase were investigated by DSC experiments that demonstrated that the presence of NP did not alter the denaturation curve of RNase, although the amount of bound RNase was too low to detect a differential heat flow after washing away unbound protein. These studies pave the way to the use of the conjugates between magnetic NP and bioactive proteins as biological tracers in order to monitor via appropriate techniques the intracellular and/or intratissutal path of specific proteins (with particular reference to food allergens, to food-derived compounds of known toxicity, as well as to protein-derived materials that could act as nutraceuticals), and to elucidate specific complex cell machineries that involve specific interactions between proteins and other biomolecules (with a special focus on systems that have been previously studied in our lab).