Matteo Gazzarri

Chitosan nanoparticles loaded with the antimicrobial peptide temporin B exert a long-term antibacterial activity in vitro against clinical isolates of Staphylococcus epidermidis

Nowadays, the alarming rise in multidrug-resistant microorganisms urgently demands for suitable alternatives to current antibiotics. In this regard, antimicrobial peptides (AMPs) have received growing interest due to their broad spectrum of activities, potent antimicrobial properties, unique mechanisms of action, and low tendency to induce resistance. However, their pharmaceutical development is hampered by potential toxicity, relatively low stability and manufacturing costs. In the present study, we tested the hypothesis that the encapsulation of the frog-skin derived AMP temporin B (TB) into chitosan nanoparticles (CS-NPs) could increase peptide’s antibacterial activity, while reducing its toxic potential. TB-loaded CS-NPs with good dimensional features were prepared, based on the ionotropic gelation between CS and sodium tripolyphosphate. The encapsulation efficiency of TB in the formulation was up to 75%. Release kinetic studies highlighted a linear release of the peptide from the nanocarrier, in the adopted experimental conditions. Interestingly, the encapsulation of TB in CS-NPs demonstrated to reduce significantly the peptide’s cytotoxicity against mammalian cells. Additionally, the nanocarrier evidenced a sustained antibacterial action against various strains of Staphylococcus epidermidis for at least 4 days, with up to 4-log reduction in the number of viable bacteria compared to plain CS-NPs at the end of the observational period. Of note, the antimicrobial evaluation tests demonstrated that while the intrinsic antimicrobial activity of CS ensured a “burst” effect, the gradual release of TB further reduced the viable bacterial count, preventing the regrowth of the residual cells and ensuring a long-lasting antibacterial effect. The developed nanocarrier is eligible for the administration of several AMPs of therapeutic interest with physical–chemical characteristics analog to those of TB.

A new hydroxyapatite-based biocomposite for bone replacement

Since the 1970s, various types of ceramic, glass and glass-ceramic materials have been proposed and used to replace damaged bone in many clinical applications. Among them, hydroxyapatite (HA) has been successfully employed thanks to its excellent biocompatibility. On the other hand, the bioactivity of HA and its reactivity with bone can be improved through the addition of proper amounts of bioactive glasses, thus obtaining HA-based composites. Unfortunately, high temperature treatments (1200°C÷1300°C) are usually required in order to sinter these systems, causing the bioactive glass to crystallize into a glass-ceramic and hence inhibiting the bioactivity of the resulting composite. In the present study novel HA-based composites are realized and discussed. The samples can be sintered at a relatively low temperature (800 °C), thanks to the employment of a new glass (BG_Ca) with a reduced tendency to crystallize compared to the widely used 45S5 Bioglass®. The rich glassy phase, which can be preserved during the thermal treatment, has excellent effects in terms of in vitro bioactivity; moreover, compared to composites based on 45S5 Bioglass® having the same HA/glass proportions, the samples based on BG_Ca displayed an earlier response in terms of cell proliferation.

Additive manufacturing of star poly(ε-caprolactone) wet-spun scaffolds for bone tissue engineering applications

Three-dimensional fibrous scaffolds made of a three-arm star poly(ε-caprolactone) were developed by employing a novel computer-aided wet-spinning apparatus to precisely control the deposition pattern of an extruded polymeric solution as a filament into a coagulation bath. Star poly(ε-caprolactone)/hydroxyapatite composite scaffolds composed of fibres with a porous morphology both in the outer surface and in the cross section were successfully produced with a layer-by-layer approach achieving good reproducibility of the internal architecture and external shape. Changes in processing parameters were used to fabricate scaffolds with different architectural parameters in terms of average pore size in the xy-axes (from 190 to 297 µm) and in the z-axis (from 54 to 126 µm) and porosity (in the range of 20%–60%). Based on the mechanical characterization, processing variations and hydroxyapatite loading have an influence on scaffold compression properties. Cell cultures, using a murine pre-osteoblast cell line, had good cell responses in terms of proliferation and osteoblastic differentiation. Thus, this technique appears to be an effective method for producing customized polymeric scaffolds for bone tissue engineering applications.

Preparation, physical-chemical and biological characterization of chitosan nanoparticles loaded with lysozyme.

A commercially available chitosan (CS) was employed in the formulation of nanoparticles loaded with lysozyme (LZ) as antimicrobial protein drug model. Due to the variability of commercially available batches of chitosans and to the strict dependence of their physical and biological properties to the molecular weight (Mw) and deacetylation degree (DD) of the material, the CS was fully characterized resulting in weight-average molecular weight of 108,120g/mol and DD of 92%. LZ-loaded nanoparticles (LZ-NPs) of 150nm diameter were prepared by inotropic gelation. The nanoparticles were effectively preserving the antibacterial activity of the loaded enzyme, which was slowly released over 3 weeks in vitro and remained active toward Staphylococcus epidermidis up to 5 days of incubation. Beyond the intrinsic antibacterial activity of CS and LZ, the LZ-NPs evidenced a sustained antibacterial activity that resulted in about 2 log reduction of the number of viable S. epidermidis compared to plain CS nanoparticles. Furthermore, the LZ-NPs showed a full in vitro cytocompatibility toward murine fibroblasts and, in addition to the potential antimicrobial applications of the developed system, the proposed study could serve as an optimal model for development of CS nanoparticles carrying antimicrobial peptides for biomedical applications.

Fibrous star poly(ε-caprolactone) melt-electrospun scaffolds for wound healing applications

Polymeric fibrous scaffolds based on the biocompatible and biodegradable three-arm-branched star poly(ε-caprolactone) (Mw = 189,000 g/mol) were prepared by a melt electrospinning technique. The possibility of processing polymers without the use of organic solvents is one of the main advantages over solution electrospinning. Scaffolds were biologically tested for their ability of supporting skin tissue regeneration. For this purpose, mouse embryo fibroblast (BALB/3T3 clone A31) and human keratinocyte (HaCaT) cell lines were selected as models, and seeded onto the polymeric supports both as single and co-culture. Cell viability, proliferation, and collagen production were assessed by WST-1 assay and Direct Red 80 dye, respectively. Cell morphology and colonization of the supports were evaluated by scanning electron microscopy and confocal laser scanning microscopy. Results highlighted that the star poly(ε-caprolactone) scaffolds were able to promote collagen production by fibroblasts. In co-culture studies, scaffolds supported adhesion, proliferation, and spatial organization of both cell lines. By virtue of the observed results, the developed polymeric scaffolds appeared suitable as biodegradable and biocompatible three-dimensional supports for skin tissue regeneration in wound healing dressing.

Additive manufacturing of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] scaffolds for engineered bone development

A wide range of poly(hydroxyalkanoate)s (PHAs), a class of biodegradable polyesters produced by various bacteria grown under unbalanced conditions, have been proposed for the fabrication of tissue‐engineering scaffolds. In this study, the manufacture of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] (or PHBHHx) scaffolds, by means of an additive manufacturing technique based on a computer‐controlled wet‐spinning system, was investigated. By optimizing the processing parameters, three‐dimensional scaffolds with different internal architectures were fabricated, based on a layer‐by‐layer approach. The resulting scaffolds were characterized by scanning electron microscopy, which showed good control over the fibre alignment and a fully interconnected porous network, with porosity in the range 79–88%, fibre diameter 47–76 µm and pore size 123–789 µm. Moreover, the resulting fibres presented an internal porosity connected to the external fibre surface as a consequence of the phase‐inversion process governing the solidification of the polymer solution. Scaffold compressive modulus and yield stress and strain could be varied in a certain range by changing the architectural parameters. Cell‐culture experiments employing the MC3T3‐E1 murine pre‐osteoblast cell line showed good cell proliferation after 21 days of culture. The PHBHHx scaffolds demonstrated promising results in terms of cell differentiation towards an osteoblast phenotype. Copyright

Bioactive polymeric materials for targeted administration of active agents: synthesis and evaluation.

Bioerodible polymers displaying both stealth and targeting properties for the preparation of nanosystems for targeted and controlled delivery of fibrinolytic drugs to the thrombus were prepared by straightforward synthetic routes and characterized. Poly[(maleic anhydride)-alt-(butylvinyl ether)]s were synthesized in the presence of dodecyl mercaptan as chain transfer agent allowing for the preparation of copolymers with tunable molecular weight. 2-Methoxyethanol hemiesters containing antiopsonizing molecules of poly(ethylene glycol) were prepared and further biofunctionalized with a Fab fragment by a two-step reaction. In vitro biocompatibility investigation of the prepared materials supported their suitability for biomedical applications.

Microstructured chitosan/poly(γ-glutamic acid) polyelectrolyte complex hydrogels by computer-aided wet-spinning for biomedical three-dimensional scaffolds

The application of additive manufacturing principles to hydrogel processing represents a powerful route to develop porous three-dimensional constructs with a variety of potential biomedical applications, such as scaffolds for tissue engineering and three-dimensional in vitro tissue models. The aim of this study was to develop novel porous hydrogels based on a microstructured polyelectrolyte complex between chitosan and poly(γ-glutamic acid) by applying a computer-aided wet-spinning technique. The developed fabrication process was used to build up three-dimensional porous hydrogels by collecting microstructured layers made of chitosan/poly(γ-glutamic acid) on top of the other. Microstructured polyelectrolyte complex hydrogels were characterized and compared to chitosan/poly(γ-glutamic acid) porous hydrogels with similar composition prepared by conventional freeze-drying technique. Fourier transform infrared analysis confirmed the formation of an electrostatic interaction between the two processed polymers in all the developed chitosan/poly(γ-glutamic acid) hydrogels. The composition of the porous constructs as well as the employed processing techniques had a significant influence on the resulting swelling, thermal, and mechanical properties. In particular, the combination of the ionic interaction between chitosan/poly(γ-glutamic acid) and the defined internal microarchitecture of microstructured polyelectrolyte complex hydrogels provided a significant improvement of the compressive mechanical properties. Preliminary in vitro biological investigations revealed that microstructured polyelectrolyte complex hydrogels were suitable for the adhesion and proliferation of Balb/3T3 clone A31 mouse embryo fibroblasts. The encouraging results in terms of cytocompatibility and stability of the microstructure in aqueous solutions due to the ionic crosslinking suggest the investigation of the developed microstructured polyelectrolyte complex hydrogels as suitable scaffolds for three-dimensional cells’ culture.

Mg- and/or Sr-doped tricalcium phosphate/bioactive glass composites: Synthesis, microstructure and biological responsiveness

Presently, there is an increasing interest towards the composites of calcium phosphates, especially β-tricalcium phosphate (TCP), and bioactive glasses. In the present contribution, the recently developed BG_Ca/Mix glass has been used because its low tendency to crystallize allows to sinter the composites at relatively low temperature (i.e. 850°C), thus minimizing the glass devitrification and the interaction with TCP. A further improvement is the introduction of lab-produced TCP powders doped with specific ions instead of non-doped commercial powders, since the biological properties of materials for bone replacement can be modulated by doping them with certain metallic ions, such as Mg and Sr. Therefore, novel binary composites have been produced by sintering the BG_Ca/Mix glass with the addition of pure, Mg-substituted, Sr-substituted or Mg/Sr bisubstituted TCP powders. After an accurate characterization of the starting TCP powders and of the obtained samples, the composites have been used as three-dimensional supports for the culture of mouse calvaria-derived pre-osteoblastic cells. The samples supported cell adhesion and proliferation and induced promising mechanisms of differentiation towards an osteoblastic phenotype. In particular, the Mg/Sr bi-doped samples seemed to better promote the differentiation process thus suggesting a combined stimulatory effect of Mg(2+) and Sr(2+) ions.

A Novel Method for the Preparation of Retinoic Acid-Loaded Nanoparticles

The goal of present work was to investigate the use of bioerodible polymeric nanoparticles as carriers of retinoic acid (RA), which is known to induce differentiation of several cell lines into neurons. A novel method, named “Colloidal-Coating”, has been developed for the preparation of nanoparticles based on a copolymer of maleic anhydride and butyl vinyl ether (VAM41) loaded with RA. Nanoparticles with an average diameter size of 70 nm and good morphology were prepared. The activity of the encapsulated RA was evaluated on SK-N-SH human neuroblastoma cells, which are known to undergo inhibition of proliferation and neuronal differentiation upon treatment with RA. The activity of RA was not affected by the encapsulation and purification processes.