Methods for sonochemical manufacture of proteinaceous ultrasound contrast agents

Laboratory procedures for the sonochemical manufacture of proteinaceous contrast agents are reviewed and documented. The objective was to develop a standard protocol to enable ultrasound contrast researchers to develop consistent agents. Proteinaceous agents continue to be in clincal use and are the subject of renewed interest owing to their potential as a platform for antibody-targeted diagnostics. The agents are prepared by sonication. Protocols for agent preparation are scattered throughout literature with methodologies varying between laboratories. While new imaging technologies improve agent visualisation, there are limited correlations between contrast agent manufacturing methods and in vitro performance. The present experimental study has determined that successful production of a stable shell coating the bubble is a result of precise attention to denaturation of protein prior to sonication. The resulting shell coats the bubble and greatly slows down gas dissolution that would otherwise result in rapid disappearance of the agent or poor shelf life. The fluid-dynamical process by which the agent is formed is discussed. Issues of technique such as the location of the sonotrode tip, sonication time and power, and and the container size are detailed. Finally, procedures for quantitative characterisation of the agent by a range of instruments are discussed. In conclusion, a complete and consistent protocol from the initial biochemical source to the agent characterisation can be produced

Micro bubble flow detachment in a model ultrasound contrast agent system

The in-vitro detachment of targeted micro-bubbles in both static and continuous flow conditions was investigated to determine if micro-bubbles can be clinically used as targeted ultrasound contrast agents. Ultrasound contrast agents are micro-bubbles coated in a lipid, protein or polymer shell. On intravenous injection they dramatically enhance blood vessels in ultrasound imaging. Targeted micro-bubbles are now being developed, which would adhere to and thus identify microscopic disease tissues. The present work is based on a novel form of protein shelled micro-bubble whose performance has never been assessed. Reynolds number and shear stress are both thought to affect the detachment of micro-bubbles. In particular the links binding bubbles to their targets can easily be broken by fluid flow forces. Pairs of molecules modelling the link between the bubble and target were selected (BSA, Anti-BSA and Streptavidin, Biotin) based on their affinity for each other and their ease of use. Micro-bubbles were prepared and purified. These bubbles were then attached to Petri dishes that had been prepared with the relevant pair and had been blocked to stop any non-specific binding. These dishes were then placed in a flow cell where shear stresses applicable to in-vivo conditions were applied. Flow rates were varied. Data were captured using a microscope with an attached video camera. The images were then processed to quantify the detachment. Preliminary results show that protein based micro-bubbles can be successfully targeted and suggest that they should remain on target under the shear stresses present in human blood vessels

Surfactant protein-A nanobody-conjugated liposomes loaded with methylprednisolone increase lung-targeting specificity and therapeutic effect for acute lung injury

The advent of nanomedicine requires novel delivery vehicles to actively target their site of action. Here, we demonstrate the development of lung-targeting drug-loaded liposomes and their efficacy, specificity and safety. Our study focuses on glucocorticoids methylprednisolone (MPS), a commonly used drug to treat lung injuries. The steroidal molecule was loaded into functionalized nano-sterically stabilized unilamellar liposomes (NSSLs). Targeting functionality was performed through conjugation of surfactant protein A (SPANb) nanobodies to form MPS–NSSLs–SPANb. MPS–NSSLs–SPANb exhibited good size distribution, morphology, and encapsulation efficiency. Animal experiments demonstrated the high specificity of MPS–NSSLs–SPANb to the lung. Treatment with MPS–NSSLs–SPANb reduced the levels of TNF-α, IL-8, and TGF-β1 in rat bronchoalveolar lavage fluid and the expression of NK-κB in the lung tissues, thereby alleviating lung injuries and increasing rat survival. The nanobody functionalized nanoparticles demonstrate superior performance to treat lung injury when compared to that of antibody functionalized systems

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/β receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK–STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100–1000-fold reduced potency compared with rmIFNα1 and rmIFNβ. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.Full Tex