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.

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.

Development of a piezoelectric immunosensor for matrix metalloproteinase-1 detection.

Monoclonal antibodies were immobilized onto the surface of quartz crystals for the development of a piezoelectric biosensor by means of the Layer by Layer self assembly technique (LBL). The immobilization of immunoglobulins specific to the matrix metalloproteinase-1 (MMP-1) was investigated. To this purpose multilayered ultra-thin films composed by precursor layers of cationic poly(dimethyldiallylammonium) chloride and anionic poly(styrenesulfonate) followed by a monolayer of antibodies were assembled by LBL. A quartz crystal microbalance was used to monitor and optimize the assembly process and to test the immunological activity of the deposited antibody molecules. Atomic force microscopy was used to characterize the surface roughness of the multilayers before and after the deposition of the immunoglobulins. The obtained results confirmed the successful deposition of the proposed immunosensor and demonstrated its high potential for the measurement of analytes of clinical interest.

Human osteoblast-like cells response to nanofunctionalized surfaces for tissue engineering.

Cells are sensitive both to the micro/nanotopographic and chemical features of their surrounding environment. The engineering of the surface properties of biomaterials is then critical to develop bioactive devices with which to elicit appropriate cellular responses. To this regard, the layer by layer (LBL) self assembly technique represents a simple and versatile method to modify surface properties by the deposition of ultrathin films with specific and predetermined properties. In this work biomimetic coatings containing fibronectin, an adhesive glycoprotein of the extracellular matrix, were assembled by means of the LBL technique, and tested for the growth of MG63 human osteoblast-like cells, in order to evaluate their potential for the treatment of materials employed in bone-tissue engineering. As a first step the assembly process was optimized by quartz crystal microbalance measurements and subsequently was repeated on nickel/titanium, silicon and glass samples. The results obtained from the investigation of cell response to the modified surfaces, put in evidence that the deposited nanostructured ultrathin films are effective in promoting cell proliferation. Our results show the high potential of the developed bioactive coatings for the engineering of biomimetic implants and for the optimization of their integration with the surrounding tissues.

Nanostructured thin films for the development of piezoelectric immunosensors.

Monoclonal antibodies were immobilized onto the surface of quartz crystals for the development of piezoelectric biosensors by means of the Layer by Layer self assembly technique (LBL). Specifically, the immobilization of immunoglobulins specific to the human cytokine Transforming Growth Factor Beta1 and to taxol was investigated. To this purpose multilayered ultra-thin films composed by precursor layers of cationic poly(dimethyldiallylammonium) chloride and anionic poly(styrenesulfonate) followed by a monolayer of antibodies were assembled by LBL. A quartz crystal microbalance was used to monitor and optimize the assembly process and to test the immunological activity of the deposited antibody molecules. Atomic force microscopy was used to characterize the surface roughness of the multilayers before and after the deposition of the immunoglobulins. The results confirmed the successful deposition of the proposed immunosensors and demonstrated their high potential for the measurement of analytes of clinical interest.

Nanofunctionalisation for the treatment of peripheral nervous system injuries.

A construct based on the electrostatic layer-by-layer self assembly technique has been fabricated, to be used as a tailored device to encourage nerve regeneration. A multilayered nanocoating composed of three precursor bilayers of cationic poly(dimethyldiallylammonium) chloride (PDDA) and anionic poly(styrenesulfonate) (PSS), followed by bilayers of poly-D-lysine (PDL) and antibody specific to transforming growth factor 1 (anti-TGF-1), has been deposited on HYAFF 11. The assembly process has been monitored by quartz crystal microbalance (QCM) for its characterisation and then it has been used on HYAFF 11. Structural studies of the resulting multilayers confirmed stepwise deposition of anti-TGF-1, with an average layer thickness of 2.2+/-0.2 nm and an average surface density of 0.36+/-0.03 mug cm(-2). Scanning electron microscopy has been used to characterise multilayer uniformity. Finally, the immunological activity of the multilayered structure has been assessed. The results show that anti-TGF-1 can be included in its active form in a predetermined multilayered structure onto HYAFF 11 with quantitative control of layer thickness and weight, providing a high tool with great potential in tissue engineering.