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It is probably not well known that 4000 years B.C. Sumerians were able to prepare many medicaments and illness was considered a sort of divine punishment while healing was the consequent purification, a viewpoint that have heavily affected the western world up to the modern age. The scientific darkness permeating Europe after the end of the Roman Empire favoured the survival of this concept while the golden age of the Arab Science (9th -13th centuries) and Renaissance represented the seeds of a paramount change. However, it is only at the end of the 19th century that, under a rigorous experimental Galilean approach, the real origins of many diseases were discovered. As a matter of fact, the modern age of drug delivery started after the 2nd world war and the first example of mathematical modelling in this field dates back to 1961. The clear affirmation of mathematical modelling in the biopharmaceutical field took place in the last twenty years of the 20th century thanks to valuable researchers such as Peppas and Langer. The third millennium opened with new important challenges such as the simultaneous modelling of drug release, Adsorption, Distribution Metabolism and Elimination (ADME), processes that rule drug fate in vivo. Thus, the aim of this chapter is to describe and to comment the most recent advances in the mathematical modelling of drug release and ADME processes in the light of Paracelsus’ belief of the human body as a chemical plant.

The shear modulus G of the sputum obtained by expectoration from cystic fibrosis patients is fundamental to determine the mesh size of the polymeric network pervading the sputum, a parameter related to lung functionality. The Akaike criterion revealed that in 55.2 % of the examined samples, the best approach (among those considered) to determine G relied on the mechanical spectrum fitting by the generalised Maxwell model with relaxation times scaled by a factor 10. Thus, this approach was adopted to evaluate the mesh size distribution combining G knowledge with the determination of the average magnetic relaxation time (T2m) of sputum. As G and T2m determination can be negatively affected by sputum contamination by saliva, whose presence increases T2m and depresses G, we developed a proper “decontamination” procedure to obtain more reliable T2m and G estimations (necessary in 21 % of the samples). This procedure allowed to strengthen the T2m correlation with lung functionality evaluated by FEV1 (normalised air volume emitted in the first second during a spirometry test). This work is licensed under a Creative Commons Attribution 4.0 International License.

Chronic lung inflammation is the major pathophysiological trait in respiratory diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). There is a need for novel anti-inflammatory approaches due to ineffectiveness and/or side effects of current therapies. A central player in the regulation of the transcriptional response of lung epithelial and immune cells to various pro-inflammatory mediators in chronic lung disease is the nuclear factor (NF)-kappaB (NF-κB). Our group recently identified a non-pathogenic commensal of the lung microbiota, i.e. Rothia mucilaginosa, that inhibits inflammation by interfering with NF-κB pathway activation in in vitro and in vivo models of lung inflammation. Therefore, we hypothesized that the lung microbiota may contain other bacteria with anti-inflammatory properties. We isolated a collection of microbiota members, including aerobic and anaerobic species, from CF patient sputum samples and evaluated their ability to reduce lipopolysaccharide (LPS)-triggered NF-κB-pathway activation in an in vivo-like three-dimensional (3-D) alveolar epithelial cell model (A549 luciferase NF-κB-reporter). Species were tested at varying multiplicity of infection (MOI) to determine the minimal effective dose, and their cytotoxicity was evaluated using an LDH assay. We demonstrate that various lung microbiota members inhibit NF-κB pathway activation, and that the minimal effective dose strongly varies between anti-inflammatory species. Few commensal species exerted cytotoxic effects. This study shows that members of the lung microbiota may influence inflammation, which could offer new avenues for treatment.

The open border between non-living and living matter, suggested by increasingly emerging fields of nanoscience interfaced to biological systems, requires a detailed knowledge of nanomaterials properties. An account of the wide spectrum of phenomena, belonging to physical chemistry of interfaces, materials science, solid state physics at the nanoscale and bioelectrochemistry, thus is acquainted for a comprehensive application of carbon nanotubes interphased with neuron cells. This review points out a number of conceptual tools to further address the ongoing advances in coupling neuronal networks with (carbon) nanotube meshworks, and to deepen the basic issues that govern a biological cell or tissue interacting with a nanomaterial. Emphasis is given here to the properties and roles of carbon nanotube systems at relevant spatiotemporal scales of individual molecules, junctions and molecular layers, as well as to the point of view of a condensed matter or materials scientist. Carbon nanotube interactions with blood-brain barrier, drug delivery, biocompatibility and functionalization issues are also regarded.

The aim of this theoretical paper is to develop a mathematical model for describing the dissolution process, in a finite liquid environment, of an ensemble of poly-dispersed drug particles, in form of sphere, cylinder and parallelepiped that can undergo solubility reduction due to phase transition induced by dissolution. The main result of this work consists in its simplicity as, whatever the particular particles size distribution, only two ordinary differential equations are needed to describe the dissolution process. This, in turn, reflects in a very powerful and agile theoretical tool that can be easily implemented in electronic sheets, a widespread tool among the research community. Another model advantage lies on the possibility of determining its parameters by means of common independent techniques thus enabling the evaluation of the importance of solid wettability on the dissolution process.

The human immune cell response against bacterial biofilms is a crucial, but still poorly investigated area of research. Herein, we aim to establish an in vitro host cell-biofilm interaction model suitable to investigate the peripheral blood mononuclear cell (PBMC) response to Pseudomonas aeruginosa biofilms. P. aeruginosa biofilms were obtained by incubating bacteria in complete RPMI 1640 medium with 10% human plasma for 24 h. PBMC obtained from healthy donors were added to preformed P. aeruginosa biofilms. Following a further 24 h incubation, we assessed (i) PBMC viability and activation; (ii) cytokine profiles in the supernatants; and (iii) CFU counts of biofilm forming bacteria. Cell-death was

In primary ciliary dyskinesia (PCD) patients, Pseudomonas aeruginosa is a major opportunistic pathogen, frequently involved in chronic infections of the lower airways. Infections by this bacterial species correlates with a worsening clinical prognosis and recalcitrance to currently available therapeutics. The antimicrobial peptide, lin-SB056-1, in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA), was previously demonstrated to be bactericidal against P. aeruginosa in an artificial sputum medium. The purpose of this study was to validate the anti-P. aeruginosa activity of such a combination in PCD sputum and to evaluate the in vitro anti-virulence effects of EDTA. In combination with EDTA, lin-SB056-1 was able to significantly reduce the load of endogenous P. aeruginosa ex vivo in the sputum of PCD patients. In addition, EDTA markedly reduced the production of relevant bacterial virulence factors (e.g., pyocyanin, proteases, LasA) in vitro by two representative mucoid strains of P. aeruginosa isolated from the sputum of PCD patients. These results indicate that the lin-SB056-1/EDTA combination may exert a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting a therapeutic potential against chronic airway infections sustained by this bacterium.

The ability of many anti-microbial peptides (AMPs) to modulate the host immune response has highlighted their possible therapeutic use to reduce uncontrolled inflammation during chronic infections. In the present study, we examined the anti-inflammatory potential of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K, which were previously found to have anti-microbial activity against Pseudomonas aeruginosa in in vivo-like models mimicking the challenging environment of chronically infected lungs (i.e., artificial sputum medium and 3-D lung mucosa model). The dendrimeric derivative exerted a stronger anti-inflammatory activity than its monomeric counterpart towards lung epithelial- and macrophage-cell lines stimulated with P. aeruginosa lipopolysaccharide (LPS), based on a marked decrease (up to 80%) in the LPS-induced production of different pro-inflammatory cytokines (i.e., IL-1&beta, IL-6 and IL-8). Accordingly, (lin-SB056-1)2-K exhibited a stronger LPS-binding affinity than its monomeric counterpart, thereby suggesting a role of peptide/LPS neutralizing interactions in the observed anti-inflammatory effect. Along with the anti-bacterial and anti-biofilm properties, the anti-inflammatory activity of (lin-SB056-1)2-K broadens its therapeutic potential in the context of chronic (biofilm-associated) infections.

In primary ciliary dyskinesia (PCD) patients, Pseudomonas aeruginosa is a major opportunistic pathogen, frequently involved in chronic infections of the lower airways. Infections by this bacterial species correlates with a worsening clinical prognosis and recalcitrance to currently available therapeutics. The antimicrobial peptide, lin-SB056-1, in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA), was previously demonstrated to be bactericidal against P. aeruginosa in an artificial sputum medium. The purpose of this study was to validate the anti-P. aeruginosa activity of such a combination in PCD sputum and to evaluate the in vitro anti-virulence effects of EDTA. In combination with EDTA, lin-SB056-1 was able to significantly reduce the load of endogenous P. aeruginosa ex vivo in the sputum of PCD patients. In addition, EDTA markedly reduced the production of relevant bacterial virulence factors (e.g., pyocyanin, proteases, LasA) in vitro by two representative mucoid strains of P. aeruginosa isolated from the sputum of PCD patients. These results indicate that the lin-SB056-1/EDTA combination may exert a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting a therapeutic potential against chronic airway infections sustained by this bacterium.

Antimicrobial peptides (AMPs) are promising templates for the development of novel antibiofilm drugs. Despite the large number of studies on screening and optimization of AMPs, only a few of these evaluated the antibiofilm activity in physiologically relevant model systems. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predict the clinical potential of novel AMP-based antimicrobials. In the present study, we examined the antibiofilm activity of the semisynthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)(2)-K against Pseudomonas aeruginosa in an in vivo-like three-dimensional (3-D) lung epithelial cell model and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels). Although moderately active when tested alone, lin-SB056-1 was effective in reducing P aeruginosa biofilm formation in association with 3-D lung epithelial cells in combination with the chelating agent EDTA. The dimeric derivative (Iin-SB056-1)(2)-K demonstrated an enhanced biofilm-inhibitory activity as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death. Biofilm inhibition by (lin-SB056-1)(2)-K was reported both for the reference strain PAO1 and cystic fibrosis lung isolates of P aeruginosa. In addition, using fluorescence microscopy, a significant decrease in biofilm-like structures associated with 3-D cells was observed after peptide exposure. Interestingly, effectiveness of (lin-SB056-1)(2)-K was also demonstrated in the wound model with a reduction of up to 1-Log unit in biofilm formation by P aeruginosa PAO1 and wound isolates. Overall, combination treatment and peptide dendrimerization emerged as promising strategies to improve the efficacy of AMPs, especially under challenging host-mimicking conditions. Furthermore, the results of the present study underlined the importance of evaluating the biological properties of novel AMPs in in vivo-like model systems representative of specific infectious sites in order to make a more realistic prediction of their therapeutic success, and avoid the inclusion of unpromising peptides in animal studies and clinical trials.

Combining a tissue engineering scaffold made of a load-bearing polymer with a hydrogel represents a powerful approach to enhancing the functionalities of the resulting biphasic construct, such as its mechanical properties or ability to support cellular colonization. This research activity was aimed at the development of biphasic scaffolds through the combination of an additively manufactured poly(epsilon-caprolactone) (PCL) fiber construct and a chitosan/poly(gamma-glutamic acid) polyelectrolyte complex hydrogel. By investigating a set of layered structures made of PCL or PCL/hydroxyapatite composite, biphasic scaffold prototypes with good integration of the two phases at the macroscale andmicroscale were developed. The biphasic constructs were able to absorb cell culturemedium up to 10-fold of their weight, and the combination of the two phases had a significant influence on compressive mechanical properties compared with hydrogel or PCL scaffold alone. In addition, due to the presence of chitosan in the hydrogel phase, biphasic scaffolds exerted a broad-spectrum antibacterial activity. The developed biphasic systems appear well suited for application in periodontal bone regenerative approaches in which a biodegradable porous structure providing mechanical stability and a hydrogel phase functioning as absorbing depot of endogenous proteins are simultaneously required. (C) 2016 Society of Chemical Industry

Silver nanoparticles (AgNPs) have been intensively investigated in virtue of their optical and antimicrobial properties, although their applications have been limited due to inherent toxicity and to the need of employing harsh chemical reagents for the synthesis. In this work, ulvan, a sulfated polysaccharide extracted from green algae belonging to Ulva armoricana sp., was for the first time investigated and identified as reducing and stabilizing agent for AgNPs synthesis by using milder conditions than those conventionally adopted by chemical methods. The synthesized AgNPs were thoroughly characterized to highlight the structure and the role exerted by ulvan in their synthesis and stabilization. The formation of AgNPs stabilized by a thick ulvan shell was assessed by UV–vis, XRD, TEM, DLS and zeta potential analyses. The developed Ulvan based AgNps showed an IC50 in the range of 10 μg/ml in Balb/3T3 mouse embryo fibroblasts and antimicrobial activity toward both Gram + and Gram − bacteria.

Persister cells (PCs) are a subset of dormant, phenotypic variants of regular bacteria, highly tolerant to antibiotics. Generation of PCs in vivo may account for the recalcitrance of most chronic infections to antimicrobial treatment and demands for the identification of new antimicrobial agents able to target such cells. The present study explored the possibility to obtain in vitro PCs of Pseudomonas aeruginosa and Staphylococcus aureus at high efficiency through chemical treatment, and to test their susceptibility to structurally different antimicrobial peptides (AMPs) and two clinically used peptide-based antibiotics, colistin and daptomycin. The main mechanism of action of these molecules (i.e., membrane-perturbing activity) renders them potential candidates to act against dormant cells. Exposure of stationary-phase cultures to optimized concentrations of the uncoupling agent cyanide m-chlorophenylhydrazone (CCCP) was able to generate at high efficiency PCs exhibiting an antibiotic-tolerant phenotype toward different classes of antibiotics. The metabolic profile of CCCP-treated bacteria was investigated by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry. CCCP-pretreated bacteria of both bacterial species underwent a substantial decrease in heat production and oxidoreductase activity, as compared to the untreated controls. After CCCP removal, induced persisters showed a delay in heat production that correlated with a lag phase before resumption of normal growth. The metabolic reactivation of bacteria coincided with their reversion to an antibiotic-sensitive phenotype. Interestingly, PCs generated by CCCP treatment resulted highly sensitive to three different membrane-targeting AMPs at levels comparable to those of CCCP-untreated bacteria. Colistin was also highly active against PCs of P. aeruginosa, while daptomycin killed PCs of S. aureus only at concentrations 32 to 64-fold higher than those of the tested AMPs. In conclusion, CCCP treatment was demonstrated to be a suitable method to generate in vitro PCs of medically important bacterial species at high efficiency. Importantly, unlike conventional antibiotics, structurally different AMPs were able to eradicate PCs suggesting that such molecules might represent valid templates for the development of new antimicrobials active against persisters.

Pseudomonas aeruginosa is a major cause of chronic lung infections in cystic fibrosis (CF) patients. The ability of the bacterium to form biofilms and the presence of a thick and stagnant mucus in the airways of CF patients largely contribute to antibiotic therapy failure and demand for new antimicrobial agents able to act in the CF environment. The present study investigated the anti-P. aeruginosa activity of lin-SB056-1, a recently described semi-synthetic antimicrobial peptide, used alone and in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA). Bactericidal assays were carried out in standard culture conditions and in an artificial sputum medium (ASM) closely resembling the CF environment. Peptide’s structure and interaction with large unilamellar vesicles in media with different ionic strengths were also investigated through infrared spectroscopy. Lin-SB056-1 demonstrated fast and strong bactericidal activity against both mucoid and non-mucoid strains of P. aeruginosa in planktonic form and, in combination with EDTA, caused significant reduction of the biomass of P. aeruginosa mature biofilms. In ASM, the peptide/EDTA combination exerted a strong bactericidal effect and inhibited the formation of biofilm-like structures of P. aeruginosa. Overall, the results obtained highlight the potential of the lin-SB056-1/EDTA combination for the treatment of P. aeruginosa lung infections in CF patients.

The frog skin-derived peptide Temporin 1Tb (TB) has gained increasing attention as novel antimicrobial agent for the treatment of antibiotic-resistant and/or biofilm-mediated infections. Nevertheless, such a peptide possesses a preferential spectrum of action against Gram-positive bacteria. In order to improve the therapeutic potential of TB, the present study evaluated the antibacterial and antibiofilm activities of two TB analogs against medically relevant bacterial species. Of the two analogs, TB_KKG6A has been previously described in the literature, while TB_L1FK is a new analog designed by us through statistical-based computational strategies. Both TB analogs displayed a faster and stronger bactericidal activity than the parental peptide, especially against Gram-negative bacteria in planktonic form. Differently from the parental peptide, TB_KKG6A and TB_L1FK were able to inhibit the formation of Staphylococcus aureus biofilms by more than 50% at 12 μM, while only TB_KKG6A prevented the formation of Pseudomonas aeruginosa biofilms at 24 μM. A marked antibiofilm activity against preformed biofilms of both bacterial species was observed for the two TB analogs when used in combination with EDTA. Analysis of synergism at the cellular level suggested that the antibiofilm activity exerted by the peptide-EDTA combinations against mature biofilms might be due mainly to a disaggregating effect on the extracellular matrix in the case of S. aureus, and to a direct activity on biofilm-embedded cells in the case of P. aeruginosa. Both analogs displayed a low hemolytic effect at the active concentrations and, overall, TB_L1FK resulted less cytotoxic toward mammalian cells. Collectively, the results obtained demonstrated that subtle changes in the primary sequence of TB may provide TB analogs that, used alone or in combination with adjuvant molecules such as EDTA, exhibit promising features against both planktonic and biofilm cells of medically relevant bacteria.

In search of new antimicrobials with anti-biofilm potential, in the present study activity of the frog-skin derived antimicrobial peptide temporin 1Tb (TB) against Staphylococcus epidermidis biofilms was investigated. A striking ability of TB to kill both forming and mature S. epidermidis biofilms was observed, especially when the peptide was combined with cysteine or EDTA, respectively. Kinetics studies demonstrated that the combination TB/EDTA was active against mature biofilms already after 2-4-h exposure. A double 4-h exposure of biofilms to TB/EDTA further increased the therapeutic potential of the same combination. Of note, TB/EDTA was able to eradicate S. epidermidis biofilms formed in vitro on silicone catheters. At eradicating concentrations, TB/EDTA did not cause hemolysis of human erythrocytes. The results shed light on the anti-biofilm properties of TB and suggest a possible application of the peptide in the lock therapy of catheters infected with S. epidermidis.

Background Multiple Sclerosis (MS) is a widespread progressive neurologic disease with consequent impairments in daily activities. Disorders of balance are frequent and equilibrium tests are potentially useful to quantify disability and to verify treatment effectiveness. The fair sensitivity of the widely used not-perturbed tests to detect balance disturbances in MS patients have prompted the development of mechatronic systems capable to impose known equilibrium perturbations, in order to challenge the balance control and, consequently, to better assess the level of impairment. We sought to clarify whether the proposed perturbed-test is capable to discriminate healthy subjects from patients with MS, even in mild or in the absence of clinically evident balance disturbances. Methods We assessed balance performances of 17 adults with MS and 13 age-matched healthy controls (HC) using both perturbed (PT) and not-perturbed (NPT) postural tests by means of a 3 Degree Of Freedom (DOF) rotational mechatronic platform. Participants stood barefoot on the platform in standing position and their center of pressure (CoP) was gathered by using a pressure matrix. Each trial lasted 30 s and was carried out with and without visual stimuli. Several postural indices were computed for each trial. Correlations between postural indices and clinical scales were analyzed. Results No significant differences were found between groups for all indices when subjects performed NPTs. Conversely, significant differences in postural indices between MS and HC emerged during PTs. Additionally, PTs revealed significant differences between patients without any cerebellar impairment (cerebellar EDSS subscore equal to 0) and HC. The discrimination capability of PTs was confirmed by the ROC analysis. No significant change of the selected metrics occurred in HC when NPTs were performed with eyes closed, while indices presented a significant worsening in MS subjects. Conclusions Not-perturbed tests showed lower sensitivity than perturbed ones in the identification of equilibrium impairments in minimally disabled MS patients. However, not-perturbed tests allow to better evaluate the influence of visual flow disturbances on balance control in MS. In conclusion, our findings proved that the use of the novel tests based on a 3DOF mechatronic device represents an effective tool to investigate early balance disturbances in MS.

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 analogue to those of TB.

Posturography is a fundamental tool to investigate the systems involved in the postural control either in static and in dynamic conditions. In the present paper we decided, by using sinusoidal perturbations of base support around the anteroposterior axis, to analyze the equilibrium parameters widely used in posture control studies - i.e. Compensatory Postural Adjustments (CPAs) and Anticipatory Postural Adjustments (APAs) - in the perspective of defining a range of equilibrium parameters for healthy adult subjects. Fourteen subjects (9 males and 5 females) with no experience of the experimental procedure participated to the experiments. Sine and step movements of the moving base were imposed through a 3-DOF (Degrees Of Freedom) robotic system and both standard and here proposed novel indices were calculated for the evaluation of equilibrium performance. The results suggest that the length of the Center Of Pressure (COP) trajectory is proportional to the frequency of the oscillations of the platform in the sine perturbation trials, while the range of motion of COP is not influenced. Moreover, we found the absence of asymmetries in the healthy subjects when they performed the session with step perturbations.

Linear poly(1,4-piperazinediyl-1-oxotrimethylene) (1), in which amido and tertiary amino groups regularly alternate along the macromolecular chain, the isomeric poly[1-(1-piperazinyl-carbonyl)ethylene] (8) for polyvinylic structure and its N-methylated derivative, poly{1-[(4-methyl-1-piperazinyl)carbonyl]ethylene} (3), were prepared and characterized. Their protonation behaviour was investigated in aqueous solution by potentiometric and calorimetric techniques. In all cases the basicity constants were found to decrease linearly with α (the degree of protonation), whereas the enthalpy changes show a different behaviour toward protonation. The thermodynamic values indicate the important role of hydrophobic interactions.