Functionalized Microbubbles and Their Application in Ultrasonography Functional Molecular Imaging • Describes a heterogeneous family of noninvasive techniques, suitable both for basic science and clinical settings • Requires molecular specific contrast agents to obtain real-time images • Provides a cheap, portable and minimally invasive system Application of MBs Contrast-Enhanced Ultrasound (CEUS) – Primarily applied in clinical use • in echocardiography (“echo contrast agents”) • in hepatology for liver cancer diagnostics, additional to the MRI, CT • for blood-pool radiology (plaque vulnerability) • destruction of tumor vascular endothelium – Only small volumes of contrast agents required for intravenous injection Ultrasound (US) I • Most often used in terms of molecular imaging • ADVANTAGES: – real time – able to visualize motion (convenient for needle biopsies and tissue ablations) – safe, portable, commercially available Ultrasound (US) II • DISADVANTAGES: – US propagation attenuated by tissues (bone, fat) – practical experience and sufficient training of sonographer is required to reveal adequate results – rather small field of view (20-30cm) – two-dimensional images only Ultrasound Contrast Agents • The “must have”: – non-toxic natural or synthetic biodegradable materials – easily detected in small amounts – stable in the bloodstream – cheap, easy to usage, storable Types of US Contrast Agents What are microbubbles? • Basicaly like liposomes BUT: • Lipid monolayer e.g. phosphatidylcholine • Polar head – water solution • Non-polar chain – gas perfluorobutane, SF6 or liquid decafluoropentane Quotation: Dijmans, et al., Eur J Echocardiogr (2004) 245-256 lipid monolayer Microbubbles (MBs) • Purely intravascular contrast particles composed of a thin membrane surrounding a hollow space filled with gas Schematic Model of a MB The MB Shell • lipid, protein, polymer • stabilizes microbubbles to enhance the circulation time in blood after injection • should only slightly limit the MB vibration in the US field generated by echo imaging systems The Filling Gas • usually filled with a hydrophobic high-molecular gas (SF6, perfluorocarbons) Fundamental Techniques for MB Preparation • Sonication • Shear Mixing • Coaxical electrohydrodynamic atomisation • Microfluidic processing using a T- junction 1. Sonication • most commonly used method for MB preparation • use of high intensity US to produce a suspension of gas MBs in a liquid containing a suitable surfactant or polymer solution which adsorbs on the MB surface and forms a stabilizing coating • DISADVANTAGE: produces relatively broad size MB suspensions → any large MBs must be filtered and/or removed before the injection + low productivity of MBs Preparation of MB by mixing • Put liposome suspension into hermetic vial flask • Fill the flask with gas ( sulphur hexafluoride ) • Mix intensively in 3M ESPE CapMix for 30 seconds ( f=2000 ) 3. Coaxical electrohydrodynamic atomization Schematic of the experimental set-up for coaxical electrohydrodynamic microbubbling (Farook et al. 2007). 4. Microfluidic Processing Using T-Junction T-junction processing: a fluid flow through the junction; b schematic of the experimental apparatus used; c bubbles approaching the device exit; d bubble formation at the junction; e optical micrograph of bubbles after collection (Stride & Edirisinghe 2009). Methods for MB Characterization • Microscopy: optical, confocal, TEM, and SEM • Static Light Scattering • Cell Counter • Flow Cytometry Specific Contributions of the Research Concerned Optical and TEM microscopy (A) Optical microscopy of DPPC/1% DOGSNTA-Ni MB with carboxy-fluorescein-PE (1%). (B) The same MB as in (A) observed by epifluorescence microscopy. (C) TEM of MB. (D) TEM detailed picture of MB. (Lukáč et al. 2011) Laser Confocal Microscopy C. Transmission and Scanning Electron Microscopy (C) TEM of MB. (D) TEM detailed picture of MB. (E) SEM picture of lyophilized microbubbles. (F) Residual liposome embedded in matrix (white arrow). Static light scattering Static Light Scattering (HORIBA-LA300) of MB sample (c(DPPC) = 10 mg/ml) NUMBER 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 size [m] q0* Flow cytometry Histogram of FSC measurement of particle size standards and the MB sample. 2,04 1 3 6 10 15 MBs in the US Field • to produce effective backscattering, MBs must be insonated by their characteristic resonant frequency • increasing of the US frequency leads to the explosion of the MB, accompanied by a ‘shock wave’ (Quaia & Bartollota, 2005; Leighton 1997; www.escardio.org). Perforation of Cells by MB Shrapnels Penetration of macromolecules into cells by sonoporation Blue – cell nucleus, red – propidium iodide, green – fluorescein labelled dextrane )MW40 kDa) Functionalized MBs – passive targeting - non-specific accumulation of MBs at the target site – active targeting - modification of the contrast shell to allow selective binding to cellular epitopes or other receptors of interest Functionalized MBs for Targeted Delivery Biotin-Avidin Coupling • most often used non-covalent targeting technique via a hydrophobic anchor inserted into the MB monolayer shell, or via avidin-biotin sandwich (Klibanov 2005) DISADVANTAGE: Avidin/streptavidin can act as foreign protein - can cause immunogenic and allergic reactions Coupling of viruses to microbubbles Functionalized MBs • histidine-metallochelating lipid complex - very low immunogenicity, high affinity binding units for histidine tagged molecules esp. recombinant proteins • reversible character of the bond Lukáč et al. 2011 Laser Confocal Microscopy CEUS Ultrasound contrast images of the mouse heart in a long axis view. (A)Without MB. (B) After the intravenous administration of DPPC/1% DOGS-NTA-Ni MB. (C) After the intravenous administration of commercial MB by Bracco. c: left ventricle cavity; a: left ventricle anterior wall; p: left ventricle posterior wall (Lukáč et al. 2011) Microbubbles in the Blood Stream • due to their size (comparable with erythrocytes), microbubbles behave as pure blood agents → capable of penetrating into small capillaries and releasing drug and genes under the action of US after reaching the area of interest SOURCE: news.sciencemag.org Infucon To Prevent the MB Flotation • = a special aim when constructing the device ‘Infucon’ • prevents the tendency of MBs to float and accumulate directly under the surface • Kauerová et al. 2012 Infusion pump with gas Magnetic stirrer Inlet capillaryOutlet capillary Cannula Infucon cell Int. J. Pharmaceutics IF 3.5 Ultrasonographic imaging of the rabbit heart: A) control B) and C) after the application of MBbased contrast agents (Kauerová et al. 2012) ‘Infucon’ Results • A prototypic device for continuous infusion of MBs for clinical use was introduced, reducing the negative impact of intravenous application of MBs on their physicalchemical features • Commercialisation of the prototype is udner development Clinical Use of MB Targeting • visualization of inflammation or transplant rejection • imaging of the angiogenesis and atherosclerosis • Doppler studies (improvement of signal-to-noise ration in microcirculation) • drug and gene delivery How it works Quotation: Skyba et al., Circulation (1998) 290-293 and Unger et al., Echocardiography (2001) 355-361 www.sciencedirect.com (Linh et al. 2010) Animal testing Image of aorta abdominalis in vivo after the injection fo microbubbles.before the injection of microbubbles. Ultrasound diagnosis of experimental embolisation in rabbits Vlasin et al. 2013, Canadian Journal of Veterinary Medicine To the team of Dr. Vlašín Advantages • Compared to a control group, the application of MBs as US contrast medium decreased the time to clot identification ten times • The transparency of the image is increased when MBs are applied (2/3 of images - the highest degree of transparency) Vet. Can. J. Results • Standard DPPC MBs were produced and characterized before the application into the rabbit model • The contrast enhanced sonography using was performed, revealing clear and easily recognizable images of the clots in thromboembolism • The time to clot identification decreased ten times Sonothrombolysis Sonothrombolysis Revascularization of graft islets (day 30). Red: human insulin; green: CD31 - Platelet endothelial cell adhesion molecule ; blue: DAPI; arrow: vessel in islets Conclusion • Delivery of therapeutic agents (DNA/RNA constructs, proteins, chemotherapeutic agents) • Stable in blood circulation; US imaging. • Visualization, size and count characterization by diferent techniques • Size 2 – 8 µm, count 108 MBs/ml • Developed DPPC microbubbles can be modified by addition of: DOGS-Ni lipid for His-Tag binding (metalochellating binding) Contribution of the Grant Projects • KAN200520703 The Use of Ultrasound in Nanomedicine (GAAV, finished successfully in 2011) • KAN200100801 Bioactive Biocompatible Surfaces and New Nanostructure Composites for Medicine and Pharmacology (GAAV, finished successfully in 2012) • GAP304/10/1951 Nanoliposomes for Development of Recombinant Vaccines and Targeted Immunotherapeutics (GAČ, till 2013) • GAP503/12/G147 Centre of Excellence for Nanotoxicology (GAČR, till 2018) • FNUSA-ICRC no. CZ.1.05/1.1.00.02.0123 from the European Regional Development Fund to M.V.