Alessandro Poma

Analytical methods for determination of mycotoxins: An update (2009–2014)

Mycotoxins are a problematic and toxic group of small organic molecules that are produced as secondary metabolites by several fungal species that colonise crops. They lead to contamination at both the field and postharvest stages of food production with a considerable range of foodstuffs affected, from coffee and cereals, to dried fruit and spices. With wide ranging structural diversity of mycotoxins, severe toxic effects caused by these molecules and their high chemical stability the requirement for robust and effective detection methods is clear. This paper builds on our previous review and summarises the most recent advances in this field, in the years 2009-2014 inclusive. This review summarises traditional methods such as chromatographic and immunochemical techniques, as well as newer approaches such as biosensors, and optical techniques which are becoming more prevalent. A section on sampling and sample treatment has been prepared to highlight the importance of this step in the analytical methods. We close with a look at emerging technologies that will bring effective and rapid analysis out of the laboratory and into the field.

Biocompatible pH-responsive nanoparticles with a core-anchored multilayer shell of triblock copolymers for enhanced cancer therapy

Drug nanocarriers are synthesised via a facile self-assembly approach using gold nanoparticles (Au NPs) as a structural core. The nanocarriers feature a multilayer shell of POEGMA-PDPA-PMPC triblock copolymers with a chain-end thiol functional group for anchoring to the Au NP surface. This water-soluble triblock copolymer was synthesised via atom transfer radical polymerisation (ATRP) from a bi-functional initiator containing a disulphide bridge. The resultant nanocarriers exhibit high biocompatibility plus excellent colloidal stability and antifouling capability in bio-media (50% PBS/FBS). Encapsulation and release of a hydrophobic drug can be effectively triggered by a pH-stimulus. Meanwhile drug-loaded nanocarriers show enhanced efficacy towards cancer cells compared to plain drug.

Influence of Surface‐Imprinted Nanoparticles on Trypsin Activity

Here, the modulation of enzyme activity is presented by protein-imprinted nanoparticles produced using a solid-phase approach. Using trypsin as target, binding of the nanoparticles to the enzyme results in its inhibition or in stabilization, depending on the orientation of the immobilized enzyme used during imprinting.

Comparison of metal free polymer–dye conjugation strategies in protic solvents

Polymer–dye conjugations are one of the common examples for polymer modifications. They represent a crucial step for imaging in optical microscopy or for tracing and marking macromolecules. In this work, we present a comparison between quick and efficient conjugation reactions that do not rely on the use of a metal catalyst, which could contaminate the resulting polymer and hinder its use for subsequent biological applications. Moreover, since the conjugation strategy needs to be compatible with the solvent system used to solubilise the polymer, we focussed our attention on assessing these conjugation approaches for their feasibility with a sample polymer that is only soluble in protic solvents. The methods studied include the ring-strain promoted azide–alkyne click reaction, thiol/maleimide conjugation as well as the 1,2,4-triazoline-3,5-dione (TAD) with a diene moiety, one of the latest click chemistries available.

162 Polymersomes Functionalized with HSP70 – Novel, Synthetic Cardioprotective Nanovesicles

Background Exosomes -– nano-sized, lipidvesicles released by cells into the blood – can protect the myocardium against ischaemia/reperfusion (IR) injury.1 This cardioprotection is mediated by heat shock protein 70 (HSP70) on the exosome surface interacting with Toll-like receptor 4 (TLR-4) on cardiomyocytes and activating intracellular protective signalling kinases.1 Polymersomes are synthetic nanovesicles with a structural similarity to exosomes, and the capacity to function as drug delivery vehicles. Aim The aim of this project was to develop polymersomes functionalized with HSP70 peptides as “synthetic exosomes” with a potential therapeutic application against IR injury. Methods POEGMA-PDPA polymersomes were synthesised from hydrophilic poly[oligo (ethylene glycol) methacrylate] and poly[2-(diisopropylamino)ethyl methacrylate] blocks, and covalently functionalized with either KSTGKANKITITNDKGRLSK (“KST”) or TKDNNLLGRFELSG (“TKD”) peptides from HSP70. They were analysed using Nanosight LM10-HS nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). Adult rat ventricular cardiomyocytes were pre-treated with polymersomes, then subjected to simulated IR. Percentage cell death was assessed using a vital dye and fluorescent microscopy. Results In line with previously published data, pre-incubation with recombinant HSP70 protected cardiomyocytes from simulated IR injury, significantly reducing cell death from 74+-4% to 44+-1% (P < 0.001) with maximal protection observed at 1 ng/ml HSP70 equivalent to molar concentration of 14.3 pM. Cytoprotection was blocked in the presence of TAK-242, an inhibitor of TLR4 (83+-3%). The average size of polymersomes was ~70 nm (DLS) or 80–90 nm (NTA), and they expressed ~145 peptides per polymersome. Pre-incubation with KST- or TKD- functionalized polymersomes reduced death of cardiomyocytes exposed to simulated IR from 62+-3% to 38+-4% or 42+-4% respectively (P < 0.001). Significant protection was observed even at 108 particles /ml, representing a concentration of 0.17 pM particles, or 0.025 pM of HSP70 peptide. No protection was recorded with non-functionalized polymersomes. Conclusion Polymersomes with HSP70-derived peptide sequences are non-toxic to cardiomyocytes and powerfully cardioprotective in a cell model of acute IR injury. Future ex vivo and in vivo experiments are required for pre-clinical assessment of these novel nanoparticles, before potential translational application. Reference Vicencio et al. Plasma exosomes protect the myocardium from ischemia-reperfusion injury, J Am Coll Cardiol. 2015;65(2015):1525–36

Specific Drug Delivery to Cancer Cells with Double-Imprinted Nanoparticles against Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, is over-expressed in many tumors, including almost half of triple-negative breast cancers. The latter belong to a very-aggressive and drug-resistant form of malignancy. Although humanized anti-EGFR antibodies can work efficiently against these cancers both as monotherapy and in combination with genotoxic drugs, instability and high production costs are some of their known drawbacks in clinical use. In addition, the development of antibodies to target membrane proteins is a very challenging task. Accordingly, the main focus of the present work is the design of supramolecular agents for the targeting of membrane proteins in cancer cells and, hence, more-specific drug delivery. These were produced using a novel double-imprinting approach based on the solid-phase method for preparation of molecularly imprinted polymer nanoparticles (nanoMIPs), which were loaded with doxorubicin and targeted toward a linear epitope of EGFR. Additionally, upon binding, doxorubicin-loaded anti-EGFR nanoMIPs elicited cytotoxicity and apoptosis only in those cells that over-expressed EGFR. Thus, this approach can provide a plausible alternative to conventional antibodies and sets up a new paradigm for the therapeutic application of this class of materials against clinically relevant targets. Furthermore, nanoMIPs can promote the development of cell imaging tools against difficult targets such as membrane proteins.

Targeting mononuclear phagocytes for eradicating intracellular parasites

Mononuclear phagocytes such as monocytes, tissue-specific macrophages and dendritic cells are primary actors in both innate and adaptive immunity, as well as tissue homoeostasis. They have key roles in a range of physiological and pathological processes, so any strategy targeting these cells will have wide-ranging impact. These phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favouring chronic or disseminated infection. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to both circulating monocytes and resident macrophages, using pH sensitive nanoscopic polymersomes made of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-co-poly(2-(di-isopropylamino)ethyl methacrylate) (PMPC-PDPA). Polymersome selectivity to mononuclear phagocytes is demonstrated and ascribed to the polymerised phosphorylcholine motifs affinity toward scavenger receptors. Finally, we demonstrate the successful exploitation of this targeting for the effective eradication of several intracellular bacteria, including the hazardous human pathogens Mycobacterium tuberculosis and Staphylococcus aureusMononuclear phagocytes such as monocytes, tissue-specific macrophages and dendritic cells are primary actors in both innate and adaptive immunity, as well as tissue homoeostasis. They have key roles in a range of physiological and pathological processes, so any strategy targeting these cells will have wide-ranging impact. These phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favouring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis, which is the most pandemic and one of the deadliest disease with one third of the world’s population infected, and 1.8 million deaths worldwide in 2015. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to both circulating monocytes and resident macrophages, using pH sensitive nanoscopic polymersomes made of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-co-poly(2-(di-isopropylamino)ethyl methacrylate) (PMPC-PDPA). Polymersome selectivity to mononuclear phagocytes is demonstrated and ascribed to the polymerised phosphorylcholine motifs affinity toward scavenger receptors. Finally, we demonstrate the successful exploitation of this targeting for the effective eradication of intracellular bacteria that cause tuberculosis Mycobacterium tuberculosis as well as other intracellular parasites including the Mycobacterium bovis, Mycobacterium marinum and the most common bacteria associated with antibiotic resistance, the Staphylococcus aureus.