Development of functionalized poly(lactide) films with chitosan via SI-SARA ATRP as scaffolds for neuronal cell growth.

Polylactic acid (PLA), a polymer derived from renewable resources, is gaining increasing attention in the development of biomedical devices due to its cost-effectiveness, low immunogenicity, and biodegradability. However, its inherent hydrophobicity remains a problem, leading to poor cell adhesion features. On this basis, the aim of this work was to develop a method for functionalizing the surface of PLA films with a biopolymer, chitosan (CH), which was proved to be a material with intrinsic cell adhesive properties, but whose mechanical properties are insufficient to be used alone. The combination of the two polymers, PLA as a bulk scaffold and CH as a coating, could be a promising combination to develop a scaffold for cell growth. The modification of PLA films involved several steps: aminolysis followed by bromination to graft amino and then bromide groups, poly(glycidyl methacrylate) (PGMA) grafting by surface-initiated supplemental activator and reducing agent atom transfer radical polymerization (SI-SARA ATRP) and finally the CH grafting. To prove the effective adhesive properties, conjugated and non-conjugated films were tested in vitro as substrates for neuronal cell growth using differentiated neurons from human induced pluripotent stem cells. The results demonstrated enhanced cell growth in the presence of CH.

Biodegradable Defined Shaped Printed Polymer Microcapsules for Drug Delivery.

This work describes the preparation and characterization of printed biodegradable polymer (polylactic acid) capsules made in two different shapes: pyramid and rectangular capsules about 1 and 11 µm in size. Obtained core-shell capsules are described in terms of their morphology, loading efficiency, cargo release profile, cell cytotoxicity, and cell uptake. Both types of capsules showed monodisperse size and shape distribution and were found to provide sufficient stability to encapsulate small water-soluble molecules and to retain them for several days and ability for intracellular delivery. Capsules of 1 µm size can be internalized by HeLa cells without causing any toxicity effect. Printed capsules show unique characteristics compared with other drug delivery systems such as a wide range of possible cargoes, triggered release mechanism, and highly controllable shape and size.

Paclitaxel-containing nano-engineered polymeric capsules towards cancer therapy.

Paclitaxel is one of the anticancer agents most often used in clinical oncology practice for the treatment of ovarian, breast and non-small cell lung cancers. Nanoengineered polymeric capsules (NPCs) represent a new and very effective tool for the encapsulation and smart release of different compounds. In present work capsules were fabricated by means of the layer-by-layer assembly of oppositely charged polyelectrolytes onto colloidal particles, followed by removal of the cores at low pH to obtain hollow microcapsules. Paclitaxel was loaded into the capsule. As tumors exhibit a lower extracellular pH than normal tissues, the property of NPCs to open the pores in their shell at slightly acidic pH values could be used for the triggered release of paclitaxel within a tumor microenvironment. For the characterization of NPCs, quartz crystal microbalance was used to monitor the process of shell growth on planar supports. The effective encapsulation of paclitaxel was then demonstrated by atomic force microscopy and micro-Raman spectroscopy, whereas its release was characterized by Uv-vis spectroscopy. Finally the biological activity of encapsulated paclitaxel against human breast cancer cells was assessed.

Encapsulated functionalized stereocomplex PLA particles: An effective system to support mucolytic enzymes.

In this work, the preparation of a novel enzyme carrier based on a polymer multicomponent system was assessed. Indeed, the design of the above system considered several issues that are the need of applying a biodegradable polymer carrier, characterized by a nanometric dimension, thus suitable to diffuse into the dense mucus structure, with functionalities capable of interacting/reacting with enzymes but resistant to enzymatic degradation. The particles were prepared from solutions containing equimolar amount of high-molecular-weight poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA) and by applying the nanoprecipitation method. Dynamic Light Scattering (DLS) measurements allowed to establish the optimal preparation conditions to obtain polymer particles characterized by diameters lower than 1 µm, which dimensions were confirmed by Field Emission Scanning Electron Microscope (FE-SEM) analysis. In order to produce surface functionalization, necessary for anchoring enzymes, the stereocomplexed particles, whose structuration was confirmed by Differential Scanning Calorimetry (DSC) measurements, underwent an amminolysis reaction by using a diamine as reactant. The treated particles were characterized by means of FE-SEM, Fourier-Transform Infrared Spectroscopy (FTIR), DLS and zeta potential measurements and their characteristics were compared with those of the neat PLLA/PDLA particles. The degree of functionalization turned out to depend on the applied conditions, it increasing by enhancing the reaction time. The activity of enzymes, i.e. papain and alginate lyase, anchored to the particles, was evaluated by Quartz Crystal Microbalance (QCM) and UV measurements. Moreover, with the aim at exploiting the material for an inhalation administration, a method to encapsulate the enzyme-particles systems was assessed. Conversely to free enzymes, the developed systems were found to be capable of diminishing the viscosity of two hydrogels, ad hoc prepared and based on the main constituents of the real mucus.

Fabrication of alginate modified brushite cement impregnated with antibiotic: Mechanical, thermal, and biological characterizations.

Treatment of postsurgical infections, associated with orthopedic surgeries, has been a major concern for orthopedics. Several strategies including systematic and local administration of antibiotics have been proposed to this regard. The present work focused on fabricating alginate (Alg) modified brushite (Bru) cements, which could address osteogeneration and local antibiotic demands. To find the proper method of drug incorporation, Gentamicin sulfate (Gen) was loaded into the samples in the form of solution or powder. Several characterization tests including compression test, morphology, cytotoxicity, and cell adhesion assays were carried out to determine the proper concentration of Alg as a modifier of the Bru cement. The results indicated that addition of 1 wt% Alg led to superior mechanical and biological properties of the cement. Moreover, Alg addition changed the morphology of the cement from plate and needle-like structures to petal-like structure. Fourier transform infrared spectroscopy results confirmed the successful loading of Gen on the cements, specifically when Gen solution was used, and X-Ray Diffractometer result indicated that Gen caused a decrease in crystalline size. Furthermore, thermal analysis revealed that Gen-loaded sample had more stable structure as the transformation temperature slightly shifted to a higher one. The stability study confirmed the chemical stability and adequate mechanical performance of the cements within 1 month of soaking time. Finally, the addition of Alg has a positive impact on the release behavior at low concentration of Gen solution so that 20% decrease within 2 weeks of release experiment was remarkably detected.

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.

Stereocomplex poly(lactic acid) nanocoated chitosan microparticles for the sustained release of hydrophilic drugs.

In this work, novel chitosan based microparticles were developed by the layer-by-layer deposition of poly(lactic acid) stereocomplex films on their surface in the view of controlling the release of encapsulated hydrophilic drugs. As first step, the quartz crystal microbalance technique was used to monitor the step-by-step deposition of the stereocomplex layers onto chitosan by evaluating the deposited mass for each layer. Chitosan microparticles, with a size ranging between 40 and 90µm, were then produced by an aerodynamically-assisted jetting technique and covered by a poly(lactic acid) stereocomplex shell. Infrared spectroscopy, wide X-ray diffraction, field emission scanning electron microscopy and contact angle measurements were used to verify the effective poly(lactic acid) adsorption onto chitosan microparticles and the stereocomplex formation. Finally, the release of a hydrophilic local anesthetic, procaine hydrochloride, from uncoated and stereocomplex-nanocoated microparticles was preliminary evaluated over a period of 15days.

Functionalized biocompatible polyelectrolyte multilayers for drug delivery: in situ investigation of mechanical properties by dissipative quartz crystal microbalance.

Nanostructured polymeric capsules have been applied in different fields, and specifically are regarded as promising for smart drug delivery applications. The physical-chemical and mechanical properties, and thus the permeability of the polyelectrolyte multilayer shell, play an important role in efficient delivery. Quartz crystal microbalance working in liquid has been used for the characterization of the buildup process and of the viscoelastic properties of biocompatible multilayers and of their functionalization by S-layer proteins. Optical and scanning electron microscopy have been used for the morphological characterization of nanostructured capsules obtained at physiological conditions by the assembly of the characterized multilayers onto spherical cores and by their subsequent removal. The proposed functionalized biocompatible capsules can be regarded as promising candidates for smart drug delivery applications.

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.

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.

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.

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.

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.

Complex catalytic colloids on the basis of firefly luciferase as optical nanosensor platform.

In the present work the layer-by-layer nano-assembly technique was used for the development of complex catalytic microparticles on the basis of firefly luciferase (FL). FL films containing 1, 2, or 3 monolayers were assembled on silver electrode QCM-resonators and on 520-nm diameter sulfonated polystyrene latex by alternate adsorption of FL and polycations using electrostatic interactions for the interlayer interaction. The assembly process was studied with quartz crystal microbalance, UV-vis spectroscopy, and microelectrophoresis (surface potential). Structural studies of the resulting multilayers confirmed stepwise deposition of FL and cationic poly(dimethyldiallyl ammonium chloride) with a bilayer thickness of 14 nm; a systematic shift of the surface potential from +28 mV for poly(dimethyldiallyl ammonium chloride) to -14 mV for luciferase outermost layer was established. The functionality and stability of the biocolloids were demonstrated by monitoring the intensity of the light emission. Factors influencing the light emitted upon catalytic activity of FL such as the number of luciferase layers in the film and polyion layer at the outermost layer were studied.