Publications

2014

1. Self-Assembly of Amyloid Fibrils That Display Active Enzymes

   Zhou X., Entwistle A., Zhang H., Jackson A. P., Mason T. O., Shimanovich U., Knowles T. P. J., Smith A. T., Sawyer E. B., Perrett S. et al. (2014) ChemCatChem. 6, 7, p. 1961-1968  Abstract

   Enzyme immobilization is an important strategy to enhance the stability and recoverability of enzymes and to facilitate the separation of enzymes from reaction products. However, enzyme purification followed by separate chemical steps to allow immobilization on a solid support reduces the efficiency and yield of the active enzyme. Here we describe polypeptide constructs that self-assemble spontaneously into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We measured the steady-state kinetic parameters for the appended enzymes in situ within fibrils and compare these with the identical protein constructs in solution. Finally, we demonstrated that the fibrils can be recycled and reused in functional assays both in conventional batch processes and in a continuous-flow microreactor.

   [All authors]
2. Gene silencing by siRNA nanoparticles synthesized via sonochemical method

   Shimanovich U., Munder A., Loureiro A., Azoia N. G., Gomes A., Cavaco-Paulo A., Gedanken A. & Gruzman A. (2014) Journal of Nanomedicine and Nanotechnology. 5, 3, 1000204.  Abstract

   The knowledge that small RNAs can affect gene expression has had a tremendous impact on basic and applied research, and gene silencing is currently one of the most promising new approaches for disease therapy. However, RNAs cannot easily penetrate cell membranes, therefore RNA delivery become one of the major challenges for gene silencing technology. In the current paper we discuss a general approach for converting siRNA molecules into a dense siRNA nanoparticles using environmentally friendly sonochemical method. The RNA nanoparticulation enhance its gene-silencing activity in vascular bovine endothelial as well as in cancer 293T/GFP-Puro cell lines without causing any toxic effect. We show that ultrasonic waves do not lead to RNA degradation or any changes in its chemical structure. Moreover, sonochemically produced siRNA nanoparticles have been shown to be resistant to a variety of environmental stresses including pH levels, enzymes and temperatures, hence solving problem of the short half-life of the RNA molecules. As the siRNA nanoparticles are biocompatibile and biodegradabile, and their RNA release properties may be controlled within limits, sonochemical formation of siRNA nanoparticles represent a new promising approach for generation of functional bionano materials.
3. Design of Novel BSA/Hyaluronic Acid Nanodispersions for Transdermal Pharma Purposes

   Martins M., Azoia N. G., Shimanovich U., Matama T., Gomes A. C., Silva C. & Cavaco-Paulo A. (2014) Molecular Pharmaceutics. 11, 5, p. 1479-1488  Abstract

   A novel transdermal hyaluronic acid (HA) conjugated with bovine serum albumin (BSA) was developed in the form of solid-in-oil (S/O) nanodispersion (129.7 nm mean diameter). Ex vivo skin penetration analysis by fluorescence and confocal observation of histological skin sections revealed the ability of BSA/HA nanodispersions to cross the stratum corneum and penetrate into the dermis. Furthermore, no significant toxicity was found in fibroblast and keratinocyte cells in vitro. These results proved the potential of the developed nanodispersion for transdermal delivery of hyaluronic acid constituting a high value to biopharmaceutical and cosmetics industries.
4. Pre-miRNA expressing plasmid delivery for anti-cancer therapy

   Benisvy-Aharonovich E., Shimanovich U., Kronfeld N., Giladi N., Bier A., Kazimirsky G., Gedanken A. & Brodie C. (2014) MedChemComm. 5, 4, p. 459-462  Abstract

   The preparation of Au NP complexes of the premiRNA-145/GFP expressing plasmid is reported; the latter is successfully delivered to glioma cells and the transcripted miRNA-145 efficiently decreases the expression of its target gene, connective tissue growth factor (CTGF).
5. Nanoscale spatially resolved infrared spectra from single microdroplets

   Mueller T., Ruggeri F. S., Kulik A. J., Shimanovich U., Mason T. O., Knowles T. P. J. & Dietler G. (2014) Lab on a Chip. 14, 7, p. 1315-1319  Abstract

   Droplet microfluidics has emerged as a powerful platform allowing a large number of individual reactions to be carried out in spatially distinct microcompartments. Due to their small size, however, the spectroscopic characterisation of species encapsulated in such systems remains challenging. In this paper, we demonstrate the acquisition of infrared spectra from single microdroplets containing aggregation-prone proteins. To this effect, droplets are generated in a microfluidic flow-focussing device and subsequently deposited in a square array onto a ZnSe prism using a micro stamp. After drying, the solutes present in the droplets are illuminated locally by an infrared laser through the prism, and their thermal expansion upon absorption of infrared radiation is measured with an atomic force microscopy tip, granting nanoscale resolution. Using this approach, we resolve structural differences in the amide bands of the spectra of monomeric and aggregated lysozyme from single microdroplets with picolitre volume.
6. Protein micro- and nano-capsules for biomedical applications

   Shimanovich U., Bernardes G. J. L., Knowles T. P. J. & Cavaco-Paulo A. (2014) Chemical Society Reviews. 43, 5, p. 1361-1371  Abstract

   Micro- and nano-scale systems have emerged as important tools for developing clinically useful drug delivery systems. In this tutorial review, we discuss the exploitation of biomacromolecules for this purpose, focusing on proteins, polypeptides, nucleic acids and polysaccharides and mixtures thereof as potential building blocks for novel drug delivery systems. We focus on the mechanisms of formation of micro-and nano-scale protein-based capsules and shells, as well as on the functionalization of such structures for use in targeted delivery of bioactive materials. We summarise existing methods for protein-based capsule synthesis and functionalization and highlight future challenges and opportunities for delivery strategies based on biomacromolecules.
7. Sonochemically-induced spectral shift as a probe of green fluorescent protein release from nano capsules

   Shimanovich U., Munder A., Azoia N. G., Cavaco-Paulo A., Gruzman A., Knowles T. P. J. & Gedanken A. (2014) RSC Advances. 4, 20, p. 10303-10309  Abstract

   Encapsulation in the form of micro and nanocapsules is an attractive route for controlling the delivery and release of active proteins and peptides. Many approaches exist to probe the morphology of such capsules as well as their mechanisms of formation. By contrast, the release of proteins from such components in a complex biological environment has been challenging to probe directly. In this paper we show that the spectral differences between green fluorescent protein (GFP) in capsules and in its free form can be used to monitor in situ the release of the protein from the confinement of capsules. These findings represent a new route towards engineering the spectral characteristics of GFP through physical rather than chemical means. We demonstrate the use of GFP protein capsules for monitoring in real time the release of protein in live cells by exposing rat L6 myotubes to protein capsules. The GFP spheres with a blue fluorescent signal dissociate inside the L6 myotubes to individual GFP molecules with a change in fluorescent signal from blue to green. These sensitive spectral characteristics enabled us to resolve the dissociation of capsules inside the cells in both time and space. We discuss the implications of our results for quantifying parameters crucial for the delivery of proteins in biological environments.