Andrea Civra

Encapsulation of Acyclovir in new carboxylated cyclodextrin-based nanosponges improves the agent's antiviral efficacy

Cyclodextrin-based nanosponges (NS) are solid nanoparticles, obtained from the cross-linking of cyclodextrins that have been proposed as delivery systems for many types of drugs. Various NS derivatives are currently under investigation in order that their properties might be tuned for different applications. In this work, new carboxylated cyclodextrin-based nanosponges (Carb-NS) carrying carboxylic groups within their structure were purposely designed as novel Acyclovir carriers. TEM measurements revealed their spherical shape and size of about 400 nm. The behaviour of Carb-NS, with respect to the incorporation and delivery of Acyclovir, was compared to that of NS, previously investigated as a drug carrier. DSC, XRPD and FTIR analyses were used to investigate the two NS formulations. The results confirm the incorporation of the drug into the NS structure and NS-Acyclovir interactions. The Acyclovir loading into Carb-NS was higher than that obtained using NS, reaching about 70% (w/w). In vitro release studies showed the release kinetics of Acyclovir from Carb-NS to be prolonged in comparison with those observed with NS, with no initial burst effect. The NS uptake into cells was evaluated using fluorescent Carb-NS and revealed the nanoparticle internalisation. Enhanced antiviral activity against a clinical isolate of HSV-1 was obtained using Acyclovir loaded in Carb-NS.

Preparation and characterization of dextran nanobubbles for oxygen delivery

Dextran nanobubbles were prepared with a dextran shell and a perfluoropentan core in which oxygen was stored. To increase the stability polyvinylpirrolidone was also added to the formulation as stabilizing agent. Rhodamine B was used as fluorescent marker to obtain fluorescent nanobubbles. The nanobubble formulations showed sizes of about 500nm, a negative surface charge and a good capacity of loading oxygen, no hemolytic activity or toxic effect on cell lines. The fluorescent labelled nanobubbles could be internalized in Vero cells. Oxygen-filled nanobubbles were able to release oxygen in different hypoxic solutions at different time after their preparation in in vitro experiments. The oxygen release kinetics could be enhanced after nanobubble insonation with ultrasound at 2.5MHz. The oxygen-filled nanobubble formulations might be proposed for therapeutic applications in various diseases.

New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization.

Background The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Methods and results Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Conclusion Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

Enhanced antiviral activity of Acyclovir loaded into β-cyclodextrin-poly(4-acryloylmorpholine) conjugate nanoparticles

Novel polymeric nanoparticles based on a beta-cyclodextrin-poly(4-acryloylmorpholine) mono-conjugate (beta-CD-PACM), a tadpole-shaped polymer in which the beta-CD ring is the hydrophilic head and the PACM chain the amphiphilic tail, were prepared by the solvent injection technique. Acyclovir-loaded nanoparticles were prepared from inclusion complexes of Acyclovir with beta-CD-PACM. Both unloaded and drug-loaded nanoparticles were characterized in terms of particle size distribution, morphology, zeta potential, drug loading and in vitro drug release rate. The antiviral activity of Acyclovir loaded into beta-CD-PACM nanoparticles against two clinical isolates of HSV-1 was evaluated and found to be remarkably superior compared with that of both the free drug and a soluble beta-CD-PACM complex reported in a previous paper. Fluorescent nanoparticles loaded with coumarin 6 were also prepared in order to investigate the nanoparticle cell uptake by confocal laser microscopy. It was found that the nanoparticles are internalized in cells and locate in the perinuclear compartment.

Identification of a Dendrimeric Heparan Sulfate-Binding Peptide That Inhibits Infectivity of Genital Types of Human Papillomaviruses

ABSTRACT Peptide dendrimers consist of a peptidyl branching core and/or covalently attached surface functional units. They show a variety of biological properties, including antiviral activity. In this study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was evaluated for in vitro activity against human papillomaviruses (HPVs). The peptide dendrimer SB105-A10 was found to be a potent inhibitor of genital HPV types (i.e., types 16, 18, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 2.8 and 4.2 μg/ml (0.59 and 0.88 μM), and no evidence of cytotoxicity was observed. SB105-A10 interacts with immobilized heparin and with heparan sulfates exposed on the cell surface, most likely preventing virus attachment. The findings from this study indicate SB105-A10 to be a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.

Inhibition of pathogenic non-enveloped viruses by 25-hydroxycholesterol and 27-hydroxycholesterol

Recent studies reported a broad but selective antiviral activity of 25-hydroxycholesterol (25HC) against enveloped viruses, being apparently inactive against non-enveloped viruses. Here we show that 25HC is endowed with a marked antiviral activity against three pathogenic non-enveloped viruses, i.e. human papillomavirus-16 (HPV-16), human rotavirus (HRoV), and human rhinovirus (HRhV), thus significantly expanding its broad antiviral spectrum, so far recognized to be limited to viruses with envelope. Moreover, here we disclose the remarkable antiviral activity of another oxysterol of physiological origin, i.e. 27-hydroxycholesterol (27HC), against HPV-16, HRoV and HRhV. We have also identified a much weaker antiviral activity of other oxysterols of pathophysiological relevance, i.e 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol. These findings suggest that appropriate modulation of endogenous production of oxysterols might be a primary host strategy to counteract a broad panel of viral infections. Moreover, 25HC and 27HC could be considered for new therapeutic strategies against HPV-16, HRoV and HRhV.

Inhibition of Human Respiratory Syncytial Virus Infectivity by a Dendrimeric Heparan Sulfate-Binding Peptide

ABSTRACT Respiratory syncytial virus (RSV) interacts with cell surface heparan sulfate proteoglycans (HSPGs) to initiate infection. The interaction of RSV with HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In the present study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was screened with the aim of identifying peptides able to bind HSPGs and thus block RSV attachment and infectivity. Of the compounds identified, the dendrimer SB105-A10 was the most potent inhibitor of RSV infectivity, with 50% inhibitory concentrations (IC50s) of 0.35 μM and 0.25 μM measured in Hep-2 and A549 cells, respectively. SB105-A10 was found to bind to both cell types via HSPGs, suggesting that its antiviral activity is indeed exerted by competing with RSV for binding to cell surface HSPGs. SB105-A10 prevented RSV infection when added before the viral inoculum, in line with its proposed HSPG-binding mechanism of action; moreover, antiviral activity was also exhibited when SB105-A10 was added postinfection, as it was able to reduce the cell-to-cell spread of the virus. The antiviral potential of SB105-A10 was further assessed using human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. SB105-A10 strongly reduced RSV infectivity in this model and exhibited no signs of cytotoxicity or proinflammatory effects. Together, these features render SB105-A10 an attractive candidate for further development as a RSV inhibitor to be administered by aerosol delivery.

Highly Sulfated K5 Escherichia coli Polysaccharide Derivatives Inhibit Respiratory Syncytial Virus Infectivity in Cell Lines and Human Tracheal-Bronchial Histocultures

ABSTRACT Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The interaction between RSV and HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was used to generate a collection of sulfated K5 derivatives with a backbone structure that mimics the heparin/heparan sulfate biosynthetic precursor. The screening of a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation revealed the highly sulfated K5 derivatives K5-N,OS(H) and K5-OS(H) to be inhibitors of RSV. Their 50% inhibitory concentrations were between 1.07 nM and 3.81 nM in two different cell lines, and no evidence of cytotoxicity was observed. Inhibition of RSV infection was maintained in binding and attachment assays but not in preattachment assays. Moreover, antiviral activity was also evident when the K5 derivatives were added postinfection, both in cell-to-cell spread and viral yield reduction assays. Finally, both K5-N,OS(H) and K5-OS(H) prevented RSV infection in human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. Together, these features put K5-N,OS(H) and K5-OS(H) forward as attractive candidates for further development as RSV inhibitors.

Auto-associative heparin nanoassemblies: a biomimetic platform against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV.

A new, simple and green method was developed for the manufacturing of heparin nanoassemblies active against the heparan sulfate-dependent viruses HSV-1, HSV-2, HPV-16 and RSV. These nanoassemblies were obtained by the auto-association of O-palmitoyl-heparin and α-cyclodextrin in water. The synthesized O-palmitoyl-heparin derivatives mixed with α-cyclodextrin resulted in the formation of crystalline hexagonal nanoassemblies as observed by transmission electron microscopy. The nanoassembly mean hydrodynamic diameters were modulated from 340 to 659 nm depending on the type and the initial concentration of O-palmitoyl-heparin or α-cyclodextrin. The antiviral activity of the nanoassemblies was not affected by the concentration of the components. However, the method of the synthesis of O-palmitoyl-heparin affected the antiviral activity of the formulations. We showed that reduced antiviral activity is correlated with lower sulfation degree and anticoagulant activity.

Oxysterols: An emerging class of broad spectrum antiviral effectors.

Oxysterols are a family of cholesterol oxidation derivatives that contain an additional hydroxyl, epoxide or ketone group in the sterol nucleus and/or a hydroxyl group in the side chain. The majority of oxysterols in the blood are of endogenous origin, derived from cholesterol via either enzymatic or non-enzymatic mechanisms. A large number of reports demonstrate multiple physiological roles of specific oxysterols. One such role is the inhibition of viral replication. This biochemical/biological property was first characterised against a number of viruses endowed with an external lipid membrane (enveloped viruses), although antiviral activity has since been observed in relation to several non-enveloped viruses. In the present paper, we review the recent findings about the broad antiviral activity of oxysterols against enveloped and non-enveloped human viral pathogens, and provide an overview of their putative antiviral mechnism(s).