Laura Pastorino

Nanoengineered polymeric S-layers based capsules with targeting activity.

Nanostructured polymeric capsules are regarded as highly promising systems with different potential applications ranging from drug delivery, biosensing and artificial cells. To fully exploit this potential, it is required to produce bio-activated stable and biocompatible capsules. To this purpose, in present work we proposed the combination of the layer-by-layer self assembly method with bacterial S-layer technology to fabricate stable and biocompatible polymeric capsules having a well defined arrangement of functional groups allowing the covalent attachment of antibody molecules. Hollow microcapsules were obtained by the layer-by-layer self assembly of oppositely charged polyelectrolytes onto colloidal particles, followed by removal of the cores at acidic pH. S-layers were crystallized onto the shell of the obtained capsules. Quartz crystal microbalance was used to characterize the crystallization process onto planar surfaces. S-layer containing capsules were investigated by atomic force microscopy. Immunoenzymatic tests were performed to assess the effective modification of the S-layer with antibody molecules both on planar surfaces and on hollow capsules. Fluorescent microscopy was employed to visualize the presence of the antibody molecules onto the capsule shell and immunological tests used to assess the bioactivity of the immobilized antibodies. Finally, the in vitro cytotoxicity of fabricated S-layer containing capsules was studied. The obtained results demonstrated the possibility to fabricate bio-activated S-layer containing capsules with improved features in terms of biocompatibility.

Collagen containing microcapsules: smart containers for disease controlled therapy.

The protein collagen is the major component of connective tissue and it is involved in many biological functions. Its degradation is at the basis of different pathological processes. The up-regulated expression of matrix metalloproteinases and the down-regulated expression of their inhibitors are the causes for such degradation. The aim of this work was to evaluate the possibility to fabricate collagen based containers for drug encapsulation and release by cellular demand by the action of matrix metalloproteinases. In present work collagen type I based microcapsules were fabricated by means of the layer-by-layer assembly of oppositely charged collagen and poly (stirene sulfonate) onto colloidal particles, followed by removal of the cores to obtain hollow microcapsules. The process of shell growth on planar supports was monitored by quartz crystal microbalance. X-ray reflectivity measurements were carried out at the solid/water interface to study the interaction of matrix metalloproteinase 1 with LbL films of collagen. The morphology of hollow capsules was characterized by scanning electron microscopy, and compared to that of capsules exposed to the matrix metalloproteinase 1. Finally the matrix metalloproteinase 1 mediated permeability of capsules variation was studied by Confocal Laser Scanning Microscopy. The results demonstrated the possibility to fabricate a drug delivery system where the release of the drug is dependent on the biochemistry of the pathological state.

Adhesion and Proliferation of Osteoblast-Like Cells on Anodic Porous Alumina Substrates With Different Morphology

We have fabricated nanoporous alumina surfaces by means of anodization in oxalic acid in different conditions and used them as the substrates for the growth of cells from a human osteoblast-like cell line. The rough nanoporous alumina substrates have been compared both with smooth standard Petri dishes used as the control and with commercial substrates of similar material. The viability of the cells has been assessed at different culture times of 4, 11, 18, and 25 days in vitro. It turned out that the porous side of the galvanostatically fabricated alumina performed similar to the control and better than the commercial porous alumina, whereas the potentiostatically fabricated porous alumina performed better than all the other substrates at all times, and in particular at the two shortest time periods of 4 and 11 days in vitro. The best performance of the substrates is associated with intermediate surface roughness and feature spacing.

Oriented collagen nanocoatings for tissue engineering

Collagens are among the most widely present and important proteins composing the human total body, providing strength and structural stability to various tissues, from skin to bone. In this paper, we report an innovative approach to bioactivate planar surfaces with oriented collagen molecules to promote cells proliferation and alignment. The Langmuir-Blodgett technique was used to form a stable collagen film at the air-water interface and the Langmuir-Schaefer deposition was adopted to transfer it to the support surface. The deposition process was monitored by estimating the mass of the protein layers after each deposition step. Collagen films were then structurally characterized by atomic force, scanning electron and fluorescent microscopies. Finally, collagen films were functionally tested in vitro. To this aim, 3T3 cells were seeded onto the silicon supports either modified or not (control) by collagen film deposition. Cells adhesion and proliferation on collagen films were found to be greater than those on control both after 1 (p<0.05) and 7 days culture. Moreover, the functionalization of the substrate surface triggered a parallel orientation of cells when cultured on it. In conclusion, these data demonstrated that the Langmuir-Schaefer technique can be successfully used for the deposition of oriented collagen films for tissue engineering applications.

Chitosan/dextran multilayer microcapsules for polyphenol co-delivery

Polysaccharide-based nanostructured polymeric microcapsules were fabricated by the electrostatic layer-by-layer self-assembly technique and used to encapsulate mixtures of four different polyphenols in order to achieve their controlled release. The real-time fabrication of the dextran/chitosan multilayer was monitored by quartz crystal microbalance with dissipation monitoring, and the morphology of the nanostructured polymeric capsules was characterized by scanning electron microscopy. The polyphenol encapsulation was obtained by reversible permeability variation of the capsule shell in ethanol:water mixtures. The loading efficiency in different water:ethanol mixtures and the release rate in acidic conditions were characterized by UV spectroscopy and HPLC. The higher loading efficiency was obtained with an ethanol:water 35:65 phenolic solution, equal to 42.0±0.6%, with a total release of 11.5±0.7 mg of total polyphenols per 11.3 μL of microcapsules after 240 min of incubation in acidic environment. The results suggest that polysaccharide-based capsules can be successfully used to encapsulate and release low water-soluble molecules, such as polyphenols.

Biocatalytic Langmuir–Blodgett assemblies based on penicillin G acylase

Abstract The recently developed ‘protective plate’ method offers the possibility to include protein layers into a Langmuir–Blodgett (LB) assembly without contact of protein molecules with the air–water interface thus avoiding their denaturation. In the present work, this technique was applied for the deposition of biocatalysts with active layers of penicillin G acylase (PGA), an enzyme widely used for medicine production. Easy selection of LB and adsorbed layers resulted in the creation of appropriate environments for the preservation of PGA functions. Two structures were tested regarding such performances as the enzymatic activity value and the level of PGA detachment in aqueous solutions. It was shown that they satisfy the requirements for biocatalytic applications. The enzymatic activity of PGA monolayer incorporated into the film reached 25–30% of the activity value of the equivalent amount of protein in the solution, which is a good result for an immobilized enzyme. Further modification of the deposition procedure resulted in increasing the effective activity per unit of the substrate surface due to adsorption of a thicker protein layer in one cycle. Probably, a three-dimensional frame-like structure was formed, which allowed the substrate molecules to penetrate into the film. The enzymatic activity of such films per unit of the substrate surface was 20–25 times higher than that of the assemblies with one adsorbed monolayer. Finally, the method is proposed of biocatalytic LB assembly deposition onto flexible supports of practically unlimited length without the exposure of protein layer to air medium.

Polyelectrolyte based molecular carriers: The role of self-assembled proteins in permeability properties

Polyelectrolyte capsules are seen as promising nanotechnology based drug delivery systems. In previous works, we have demonstrated the possibility to fabricate bio-activated surface layer containing capsules with improved features in terms of biocompatibility. In this study, we have characterized the permeability properties of such capsules towards low and high molecular weight molecules, including proteins. The results indicated that the presence of the surface layer strongly affects the permeability properties of the capsules in terms of loading capacity which was found to be higher compared to that of plain capsules. These properties make such systems interesting candidates as drug delivery platforms.

Langmuir–Blodgett films of lipase for biocatalysis

Abstract Lipase thin films were fabricated by means of the Langmuir–Blodgett (LB) technique and then structurally and functionally characterized by means of biophysical techniques toward biocatalytic applications. Protein films were studied at the air–water interface and then transferred onto a solid support by means of the horizontal lift method, namely, the Langmuir–Schaefer (LS) method. The biophysical studies were carried out mainly by means of Brewster Angle Microscopy (BAM), UV–Vis spectroscopy, quartz crystal microbalance (QCM) and circular dichroism (CD) allowed to analyze the main properties and features of such enzyme films. Finally, the functionality of lipase in the Langmuir–Blodgett films was checked with the monitoring of the enzymatic catalysis. The results indicated that the catalytic activity of the enzyme not only was preserved but also increased with respect the solution.

Investigation of integrin expression on the surface of osteoblast-like cells by atomic force microscopy.

The transforming growth factor beta1 (TGF-beta1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-beta1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalized with monoclonal antibodies specific to the beta1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-beta1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the beta1 integrin subunit was enhanced by TGF-beta1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-beta1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.

Self-assembly and recrystallization of bacterial S-layer proteins of Bacillus sphaericus and Bacillus thuringiensis on silicone, mica and quartz crystal supports

The Oriented architecture of macromolecules plays a critical role in many aspects of Nanobiotechnology such as in the development of biosensors. To this regard, S-layers which constitute the outermost cell envelope component of many prokaryotic organisms, represent unique self assembled systems with the capability to rearrange into monomolecular and oriented arrays. These properties can be exploited to promote their crystallization on surfaces (e.g. silicone) which is pivotal for the subsequent immobilization of macromolecules and development of new biosensors. In this work the crystallization of bacterial S-layers obtained from Bacillus thuringiensis and Bacillus sphaericus CCM2177 on silicone, mica and quartz crystal surfaces were investigated. The SDS page results of S-layers isolated from the above mentioned bacteria put in evidence that their molecular weight (MW) was around 120 KDa and, as reported in the literature, slightly higher for those extracted by Bacillus thuringiensis. In addition, results showed that S-layers isolated from Bacillus thuringiensis form large crystalline domains on mica after 5 min whereas those extracted from Bacillus sphaericus CCM 2177 form a compact monolayer on silicone after 2 h. Results in this work put in evidence the possibility to use these substrates for the fabrication of sensitive biosensors.