Ivan Roitt

Glucose-Coated Gold Nanoparticles Transfer across Human Brain Endothelium and Enter Astrocytes In Vitro

The blood-brain barrier prevents the entry of many therapeutic agents into the brain. Various nanocarriers have been developed to help agents to cross this barrier, but they all have limitations, with regard to tissue-selectivity and their ability to cross the endothelium. This study investigated the potential for 4 nm coated gold nanoparticles to act as selective carriers across human brain endothelium and subsequently to enter astrocytes. The transfer rate of glucose-coated gold nanoparticles across primary human brain endothelium was at least three times faster than across non-brain endothelia. Movement of these nanoparticles occurred across the apical and basal plasma membranes via the cytosol with relatively little vesicular or paracellular migration; antibiotics that interfere with vesicular transport did not block migration. The transfer rate was also dependent on the surface coating of the nanoparticle and incubation temperature. Using a novel 3-dimensional co-culture system, which includes primary human astrocytes and a brain endothelial cell line hCMEC/D3, we demonstrated that the glucose-coated nanoparticles traverse the endothelium, move through the extracellular matrix and localize in astrocytes. The movement of the nanoparticles through the matrix was >10 µm/hour and they appeared in the nuclei of the astrocytes in considerable numbers. These nanoparticles have the correct properties for efficient and selective carriers of therapeutic agents across the blood-brain barrier.

The influence of ligand organization on the rate of uptake of gold nanoparticles by colorectal cancer cells

We have explored the uptake of different hydrophilic mono- and dual-ligand gold nanoparticles in colorectal cancer cells in vitro and find that the rate of uptake is dependent on the structural organization of the ligands on the surface of the particles rather than their charge or chemical properties. Gold nanoparticles with 50%PEG-NH(2)/50% glucose are taken up eighteen fold faster than nanoparticles carrying only PEG-NH(2) or glucose. Glutathione-coated gold particles are by far the most efficiently internalized; however, glucose-glutathione dual-ligand nanoparticles are taken up at a thirty fold reduced rate. We found furthermore that the rates are influenced by the cell density and concentration of glucose in the growth medium. Rather than being internalized through a conventional receptor-mediated mechanism the particles appear to be taken up by the cells via an energy-independent diffusion across the cell membrane through pre-existing pores or openings in the lipid bi-layer created by ligands on the gold nanoparticles.

Lipoxin A4 selectively programs the profile of m2 tumor‐associated macrophages which favour control of tumor progression

In tumor microenvironments, the macrophage population is heterogeneous, but some macrophages can acquire tumor‐promoting characteristics. These tumor‐associated macrophages (TAM) exhibit an M2‐like profile, with deficient production of NO and ROS, characteristics of pro‐inflammatory M1 cytotoxic macrophages. Lipoxins (LX) and 15‐epi‐lipoxins are lipid mediators which can induce certain features of M2 macrophages in mononuclear cells, but their effects on TAM remain to be elucidated. This study tested the hypothesis that ATL‐1, a synthetic analogue of 15‐epi‐lipoxin A4, could modulate TAM activity profile. We show that human macrophages (MΦ) differentiated into TAM‐like cells after incubation with conditioned medium from MV3, a human melanoma lineage cell. Contrasting with the effects observed in other M2 subsets and M1 profile macrophages, ATL‐1 selectively decreased M2 surface markers in TAM, suggesting unique behavior of this particular M2 subset. Importantly, these results were replicated by the natural lipoxins LXA4 and the aspirin induced 15‐epi‐LXA4 (ATL). In parallel, ATL‐1 stimulated TAM to produce NO by increasing the iNOS/arginase ratio and activated NADPH oxidase, triggering ROS production. These alterations in TAM profile induced by ATL‐1 led to loss of the anti‐apoptotic effects of TAM on melanoma cells and increased their cytotoxic properties. Finally, ATL‐1 was found to inhibit tumor progression in a murine model in vivo, which was accompanied by alterations in TAM profile and diminished angiogenesis. Together, the results show an unexpected effect of lipoxin, which induces in TAM a change from an M2‐ to an M1‐like profile, thereby triggering tumor cell apoptosis and down‐modulating the tumor progression.

Detection of the tau protein in human serum by a sensitive four-electrode electrochemical biosensor

This study presents a novel approach based on a four-electrode electrochemical biosensor for the detection of tau protein - one of the possible markers for the prediction of Alzheimers disease (AD). The biosensor is based on the formation of stable antibody-antigen complexes on gold microband electrodes covered with a layer of a self-assembled monolayer and protein G. Antibodies were immobilized on the gold electrode surface in an optimal orientation by protein G interaction. Electrochemical impedance spectroscopy was used to analyze impedance change, which revealed a linear response with increasing tau concentrations. The assay is fast (<1h for incubation and measurement) and very sensitive. The limit of quantification for the full-length 2N4R tau protein is 0.03pM, a value unaltered when the assay was processed in bovine serum albumin or human serum. This technology could be adapted for the detection of other biomarkers to provide a multiple assay to identify AD progression in a point of care setting.

Emerging applications of nanotechnology for diagnosis and therapy of disease: a review

Nanotechnology is of increasing interest in the fields of medicine and physiology over recent years. Its application could considerably improve disease detection and therapy, and although the potential is considerable, there are still many challenges that need to be addressed before it is accepted in routine clinical use. This review focuses on emerging applications that nanotechnology could enhance or provide new approaches in diagnoses and therapy. The main focus of recent research centres on targeted therapies and enhancing imaging; however, the introduction of nanomaterial into the human body must be controlled, as there are many issues with possible toxicity and long-term effects. Despite these issues, the potential for nanotechnology to provide new methods of combating cancer and other disease conditions is considerable. There are still key challenges for researchers in this field, including the means of delivery and targeting in the body to provide effective treatment for specific disease conditions. Nanoparticles are difficult to measure due to their size and physical properties; hence there is still a great need to improve physiological measurement methods in the field to ascertain how effective their use is in the human subject. This review is a brief snapshot into the fast changing research field of measurement and physiological links to nanoparticle use and its potential in the future.

Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations.

An investigation of the impedance properties of gold nanoparticles

Over recent years there has been rapid growth in the research being carried out on nanoparticles. In the field of medical imaging, this interest has focussed primarily on the potential for drug delivery and using nanoparticles as a contrast agent, e.g. super-paramagnetic iron-oxide (SPIO) particles in MRI. More recently gold nanoparticles have been used in radiotherapy treatment of tumours to provide dose enhancement by increasing the efficacy of the radiation absorption. Nanoparticles coated with molecules such as glucose or cancer-specific antibodies can be directed towards specific cancer cells in vivo. Such targeting combined with the properties of nanoparticles shows great promise for localised therapy of tumours while leaving neighbouring healthy tissue unaffected. However, on the nanoparticle scale of sub-100nm the weighting of various factors and inter-atomic interactions which determine the bulk properties of a material changes. Many properties of the bulk material no longer hold. As such, each aspect of nanoparticle behaviour must be investigated afresh to explore the full extent of their potential. The property of nanoparticles we wish to explore and characterise is impedance. Bulk gold is well known to be highly conductive. If this were to remain the case on the nanoscale, it could be highly effective as a contrast agent for electrical impedance tomography, particularly when combined with tumour targeting.