In vitro and ex vivo evaluation of the biological performance of sclerosing foams.

Since the first reports on foam sclerotherapy, multiple studies have been conducted to determine the physical properties and behavior of foams, but relatively little is known about their biological effects on the endothelial cells lining the vessel wall. Moreover, a systematic comparison of the biological performance of foams produced with different methods has not been carried out yet. Herein, a 2D in vitro method was developed to compare efficacy of commercially available polidocanol injectable foam (PEM, Varithena) and physician-compounded foams (PCFs). Endothelial cell attachment upon treatment with foam was quantified as an indicator of therapeutic efficacy, and was correlated with foam physical characteristics and administration conditions. An ex vivo method was also developed to establish the disruption and permeabilisation of the endothelium caused by sclerosing agents. It relied on the quantitation of extravasated bovine serum albumin conjugated to Evans Blue, as an indicator of endothelial permeability. In our series of comparisons, PEM presented a greater overall efficacy compared to PCFs, across the different biological models, which was attributed to its drainage dynamics and gas formulation. This is consistent with earlier studies that indicated superior physical cohesiveness of PEM compared to PCFs.

Physical Vein Models to Quantify the Flow Performance of Sclerosing Foams.

Foam sclerotherapy is clinically employed to treat varicose veins. It involves intravenous injection of foamed surfactant agents causing endothelial wall damage and vessel shrinkage, leading to subsequent neovascularization. Foam production methods used clinically include manual techniques, such as the Double Syringe System (DSS) and Tessari (TSS) methods. Pre-clinical in-vitro studies are conducted to characterize the performance of sclerosing agents; however, the experimental models used often do not replicate physiologically relevant physical and biological conditions. In this study, physical vein models (PVMs) were developed and employed for the first time to characterize the flow behavior of sclerosing foams. PVMs were fabricated in polydimethylsiloxane (PDMS) by replica molding, and were designed to mimic qualitative geometrical characteristics of veins. Foam behavior was investigated as a function of different physical variables, namely (i) geometry of the vein model (i.e., physiological vs. varicose vein), (ii) foam production technique, and (iii) flow rate of a blood surrogate. The experimental set-up consisted of a PVM positioned on an inclined platform, a syringe pump to control the flow rate of a blood substitute, and a pressure transducer. The static pressure of the blood surrogate at the PVM inlet was measured upon foam administration. The recorded pressure-time curves were analyzed to quantify metrics of foam behavior, with a particular focus on foam expansion and degradation dynamics. Results showed that DSS and TSS foams had similar expansion rate in the physiological PVM, whilst DSS foam had lower expansion rate in the varicose PVM compared to TSS foam. The degradation rate of DSS foam was lower than TSS foam, in both model architectures. Moreover, the background flow rate had a significant effect on foam behavior, enhancing foam displacement rate in both types of PVM.

Analysis of the Diffusion Process by pH Indicator in Microfluidic Chips for Liposome Production.

In recent years, the development of nano- and micro-particles has attracted considerable interest from researchers and enterprises, because of the potential utility of such particles as drug delivery vehicles. Amongst the different techniques employed for the production of nanoparticles, microfluidic-based methods have proven to be the most effective for controlling particle size and dispersity, and for achieving high encapsulation efficiency of bioactive compounds. In this study, we specifically focus on the production of liposomes, spherical vesicles formed by a lipid bilayer encapsulating an aqueous core. The formation of liposomes in microfluidic devices is often governed by diffusive mass transfer of chemical species at the liquid interface between a solvent (i.e., alcohol) and a non-solvent (i.e., water). In this work, we developed a new approach for the analysis of mixing processes within microfluidic devices. The method relies on the use of a pH indicator, and we demonstrate its utility by characterizing the transfer of ethanol and water within two different microfluidic architectures. Our approach represents an effective route to experimentally characterize diffusion and advection processes governing the formation of vesicular/micellar systems in microfluidics, and can also be employed to validate the results of numerical modelling.

Liposomal Systems as Nanocarriers for the Antiviral Agent Ivermectin.

RNA virus infections can lead to the onset of severe diseases such as fever with haemorrhage, multiorgan failure, and mortality. The emergence and reemergence of RNA viruses continue to pose a significant public health threat worldwide with particular attention to the increasing incidence of flaviviruses, among others Dengue, West Nile Virus, and Yellow Fever viruses. Development of new and potent antivirals is thus urgently needed. Ivermectin, an already known antihelminthic drug, has shown potent effects in vitro on Flavivirus helicase, with EC50 values in the subnanomolar range for Yellow Fever and submicromolar EC50 for Dengue Fever, Japanese encephalitis, and tick-borne encephalitis viruses. However ivermectin is hampered in its application by pharmacokinetic problems (little solubility and high cytotoxicity). To overcome such problems we engineered different compositions of liposomes as ivermectin carriers characterizing and testing them on several cell lines for cytotoxicity. The engineered liposomes were less cytotoxic than ivermectin alone and they showed a significant increase of the antiviral activity in all the Dengue stains tested (1, 2, and S221). In the current study ivermectin is confirmed to be an effective potential antiviral and liposomes, as drug carriers, are shown to modulate the drug activity. All together the results represent a promising starting point for future improvement of ivermectin as antiviral and its delivery.

Liposome production by microfluidics: potential and limiting factors.

This paper provides an analysis of microfluidic techniques for the production of nanoscale lipid-based vesicular systems. In particular we focus on the key issues associated with the microfluidic production of liposomes. These include, but are not limited to, the role of lipid formulation, lipid concentration, residual amount of solvent, production method (including microchannel architecture), and drug loading in determining liposome characteristics. Furthermore, we propose microfluidic architectures for the mass production of liposomes with a view to potential industrial translation of this technology.

Suramin inhibits chikungunya virus replication through multiple mechanisms.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe and often persistent arthritis. In recent years, millions of people have been infected with this virus for which registered antivirals are still lacking. Using our recently established in vitro assay, we discovered that the approved anti-parasitic drug suramin inhibits CHIKV RNA synthesis (IC50 of ∼5µM). The compound inhibited replication of various CHIKV isolates in cell culture with an EC50 of ∼80µM (CC50>5mM) and was also active against Sindbis virus and Semliki Forest virus. In vitro studies hinted that suramin interferes with (re)initiation of RNA synthesis, whereas time-of-addition studies suggested it to also interfere with a post-attachment early step in infection, possibly entry. CHIKV (nsP4) mutants resistant against favipiravir or ribavirin, which target the viral RNA polymerase, did not exhibit cross-resistance to suramin, suggesting a different mode of action. The assessment of the activity of a variety of suramin-related compounds in cell culture and the in vitro assay for RNA synthesis provided more insight into the moieties required for antiviral activity. The antiviral effect of suramin-containing liposomes was also analyzed. Its approved status makes it worthwhile to explore the use of suramin to prevent and/or treat CHIKV infections.