On the way back from 3D to 2D: Chitosan promotes adhesion and development of neuronal networks onto culture supports.

Most in vitro functional and morphological studies for developing nervous system have been performed using traditional monolayer cultures onto supports modified by extracellular matrix components or synthetic biopolymers. These biomolecules act as adhesion factors essential for neuronal growth and differentiation. In this study, the use of chitosan as adhesion factor was investigated. Primary rat neurons and neurons differentiated from human induced pluripotent stem cells were cultured onto chitosan and standard adhesion factors modified supports. The initiation, elongation and branching of neuritic processes, synaptogenesis and electrophysiological behavior were studied. The biopolymers affected neurites outgrowth in a time dependent manner; in particular, chitosan promoted neuronal polarity in both cell cultures. These results indicate chitosan as a valid adhesion factor alternative to the standard ones, with the advantage that it can be used both in 2D and 3D cultures, acting as a bridge between these in vitro models.

Lipoperoxide Nanoemulsion as Adjuvant in Cisplatin Cancer Therapy: In Vitro Study on Human Colon Adenocarcinoma DLD-1 Cells.

Cisplatin is a first-choice chemotherapeutic agent used to treat solid tumors even though the onset of multi-drug resistance and the time-dose side-effects impair its mono-therapeutic application. Therefore, new drug-delivery approaches, based on nanomedicine strategies, are needed to enhance its therapeutic potential in favor of a dose-reduction of cisplatin. Polyunsaturated fatty acids and their metabolism-derived intermediates, as well as lipid peroxidation end-products, are used as adjuvants to improve the effectiveness of chemotherapy. Lipid hydroperoxides, derived from the oxidation of edible oils, can contribute to cell death, generating breakdown products (e.g., reactive aldehydes). In this regard, the aim of this present study was to evaluate an invitro combinatory strategy between a lecithin-based nanoemulsion system of K600, a patented mixture of peroxidated oil and peroxidated cholesterol, and cisplatin on DLD1 human adenocarcinoma cells. Our findings showed that nanoemulsions, acting in synergy with cisplatin, improve cisplatin bioactivity, in terms of enhancing its anti-cancer activity, towards DLD1 cells. Indeed, this combination approach, whilst maintaining cisplatin at low concentrations, induces a significant reduction in DLD1 cell viability, an increase in pro-apoptotic markers, and genotoxic damage. Therefore, K600 nanoemulsions as an efficient targeted delivery system of cisplatin allow for the reduction in the chemotherapeutic agent doses.

Alginate microbeads with internal microvoids for the sustained release of drugs.

The process of Ca2+ mediated gelation of alginate and the fabrication of nanoengineered polyelectrolyte capsules were combined for the preparation of alginate microbeads characterized by the presence of well-defined drug loaded microvoids in their volume. The obtained engineered alginate microbeads are described in terms of their morphology, loading efficiency and release characteristics. It was found that the generation of microvoids in the volume of alginate microbeads could be a promising approach for the creation of microstructured and biocompatible hydrogels, prospectively having highly tunable properties in terms of loading and releasing characteristics. In particular, it was found that the developed system was able to limit drug leakage during the gelation process and to control the initial burst release of small hydrophilic drug molecules, such as doxorubicin hydrochloride. Finally, the cytocompatibility of the developed microhydrogels was assessed on MCF-7 human breast cancer cells as well as their ability to sustain the release of the model drug during time.

Multicompartment Hydrogels for the Local Delivery of Chemotherapic Drugs.

Over 85% of human cancers are solid tumors. The effectiveness of anticancer therapy in solid tumors depends on adequate delivery of the therapeutic agent to tumor cells. Inadequate delivery would result in residual tumor cells, which in turn would lead to regrowth of tumors and possibly development of resistant cells. The most prominent option, for now, is the local delivery of chemotherapic drugs into the cavity resection of the tumor. However, the burst release of massive concentrations of the drugs usually boosts the side effects of chemotherapy. Aiming to block the burst release a new drug delivery system (DDS) for the local delivery of Doxorubicin (DOX) was designed and tested, combining different materials and techniques. Following a bottom-up approach, porous spherical calcium carbonate (CaCO3) microspheres, with high loading properties, were loaded with DOX and Layer by Layer (LbL) assembled by biocompatible and biodegradable polyelectrolytes, dextran sodium sulfate (DSS) and polyarginine (PARG). Then, a protocol for the fabrication of alginate (Alg) hydrogels associated with LbL coated drug loaded CaCO3 microspheres were developed by combining internal and external gelation. Therefore, injectable multicompartment hydrogels (MCH) for the local and sustained delivery of chemotherapeutic drugs, with the ability to block the burst release, were developed and characterized.

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.

3D Porous Gelatin/PVA Hydrogel as Meniscus Substitute Using Alginate Micro-Particles as Porogens.

One of the current major challenges in orthopedic surgery is the treatment of meniscal lesions. Some of the main issues include mechanical consistency of meniscal implants, besides their fixation methods and integration with the host tissues. To tackle these aspects we realized a micro-porous, gelatin/polyvinyl alcohol (PVA)-based hydrogel to approach the high percentage of water present in the native meniscal tissue, recapitulating its biomechanical features, and, at the same time, realizing a porous implant, permissive to cell infiltration and tissue integration. In particular, we adopted aerodynamically-assisted jetting technology to realize sodium alginate micro-particles with controlled dimensions to be used as porogens. The porous hydrogels were realized through freezing-thawing cycles, followed by alginate particles leaching. Composite hydrogels showed a high porosity (74%) and an open porous structure, while preserving the elasticity behavior (E = 0.25 MPa) and high water content, typical of PVA-based hydrogels. The ex vivo animal model validation proved that the addition of gelatin, combined with the micro-porosity of the hydrogel, enhanced implant integration with the host tissue, allowing penetration of host cells within the construct boundaries. Altogether, these results show that the combined use of a water-insoluble micro-porogen and gelatin, as a bioactive agent, allowed the realization of a porous composite PVA-based hydrogel to be envisaged as a potential meniscal substitute.

Soft chitosan microbeads scaffold for 3D functional neuronal networks.

The availability of 3D biomimetic in vitro neuronal networks of mammalian neurons represents a pivotal step for the development of brain-on-a-chip experimental models to study neuronal (dys)functions and particularly neuronal connectivity. The use of hydrogel-based scaffolds for 3D cell cultures has been extensively studied in the last years. However, limited work on biomimetic 3D neuronal cultures has been carried out to date. In this respect, here we investigated the use of a widely popular polysaccharide, chitosan (CHI), for the fabrication of a microbead based 3D scaffold to be coupled to primary neuronal cells. CHI microbeads were characterized by optical and atomic force microscopies. The cell/scaffold interaction was deeply characterized by transmission electron microscopy and by immunocytochemistry using confocal microscopy. Finally, a preliminary electrophysiological characterization by micro-electrode arrays was carried out.

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.

Fabrication and characterization of novel multilayered structures by stereocomplexion of poly(D-lactic acid)/poly(L-lactic acid) and self-assembly of polyelectrolytes.

The enantiomers poly(D-lactic acid) (PDLA) and poly(L-lactic acid) (PLLA) were alternately adsorbed directly on calcium carbonate (CaCO3) templates and on poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) multilayer precursors in order to fabricate a novel layer-by-layer (LBL) assembly. A single layer of poly(L-lysine) (PLL) was used as a linker between the (PDLA/PLLA) n stereocomplex and the cores with and without the polymeric (PSS/PAH) n /PLL multilayer precursor (PEM). Nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC) were used to characterize the chemical composition and molecular weight of poly(lactic acid) polymers. Both multilayer structures, with and without polymeric precursor, were firstly fabricated and characterized on planar supports. A quartz crystal microbalance (QCM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and ellipsometry were used to evaluate the thickness and mass of the multilayers. Then, hollow, spherical microcapsules were obtained by the removal of the CaCO3 sacrificial template. The chemical composition of the obtained microcapsules was confirmed by differential scanning calorimetry (DSC) and wide X-ray diffraction (WXRD) analyses. The microcapsule morphology was evaluated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) measurements. The experimental results confirm the successful fabrication of this innovative system, and its full biocompatibility makes it worthy of further characterization as a promising drug carrier for sustained release.

Peroxidated olive oil nanoemulsion for cancer targeted therapy.

A reactive oxygen species-mediated targeting system has been used to selectively kill cancer cells. Two different cell lines, normal and cancer cells, have been cultured and treated with a peroxide olive oil (K600) in simple solution and in form of nanoemulsion (N-K600). Preliminary results of both treatments have been compared.

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.

Multilayered polyelectrolyte microcapsules: interaction with the enzyme cytochrome C oxidase.

Cell-sized polyelectrolyte capsules functionalized with a redox-driven proton pump protein were assembled for the first time. The interaction of polyelectrolyte microcapsules, fabricated by electrostatic layer-by-layer assembly, with cytochrome c oxidase molecules was investigated. We found that the cytochrome c oxidase retained its functionality, that the functionalized microcapsules interacting with cytochrome c oxidase were permeable and that the permeability characteristics of the microcapsule shell depend on the shell components. This work provides a significant input towards the fabrication of an integrated device made of biological components and based on specific biomolecular functions and properties.