Correlation of biocapping agents with cytotoxic effects of silver nanoparticles on human tumor cells

10.1039/C3RA41346BRSC Advances314329-1433

EBV-gp350 Confers B-Cell Tropism to Tailored Exosomes and Is a Neo-Antigen in Normal and Malignant B Cells—A New Option for the Treatment of B-CLL

gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies

Phosphatidylserine Targets Single-Walled Carbon Nanotubes to Professional Phagocytes In Vitro and In Vivo

Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid “eat-me” signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells

Hypoxic enhancement of exosome release by breast cancer cells

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citedBackground Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling. Methods Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant. Results Exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1α siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions. Conclusions These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1α. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.HK was recipient of a Flinders University Unibooks Honours Scholarship and the work was funded by the Flinders Medical Centre Research Foundation, the Lyn Wrigley Breast Cancer Research and Development Fund, and the Flinders Medical Centre Clinicians Special Purpose Fund

Interactions of engineered and endogenous nanoparticles with cells in the immune system

Nanotechnology is a fast developing area, which refers to research and technology development at the nanometer scale, ranging from 0.1-100 nm. The properties of nanomaterials offer the ability to interact with complex biological functions, implying enormous opportunities for novel applications within medicine. However, there is little information available concerning the potential toxicity of nanoparticles and what influence such particles have on the immune system, e.g. on dendritic cells (DCs). DCs are the most efficient antigen presenting cells, having a capacity to initiate and direct immune responses against foreign material. The aim of this thesis was to study effects of differently sized and shaped nanomaterials in the interaction with primary human monocyte derived DCs (MDDCs), thereby obtaining an insight on what impact these materials have on the immune system and their potential use in medical applications. In addition, we wanted to determine if endogenous nanoparticles (exosomes), produced by various cells, are natural targeting vehicles. We show that conventionally produced gold nanoparticles had a maturing effect on human MDDCs, but this was found to be a result of lipopolysaccharide (LPS) contamination. By modification of the production process, clean particles were obtained, which had practically no effect on phenotype or cytokine production of MDDCs. These findings emphasize the importance of retaining high purity during the production of nanoparticles, since possible contaminants may interfere with the assessment of nanoparticles biological effects and result in hazardous particles. To investigate whether various shapes of gold nanoparticles affect MDDC differently, a novel method was developed for the preparation of gold nanorods with high aspect ratios (ARs) based on a self-seeded surfactant-mediated protocol. The biocompatibility of these high AR gold nanorods, with potential use in thermal therapy, was compared with spherical gold nanoparticles. Both materials had no or minor effects on MDDC s viability and phenotype, thus shape did not seem to affect the biocompatibility of gold. To determine whether the size of the particle is important for its biocompatibility, the impact of mesoporous silica nano- (270 nm) and microparticles (2.5 μm) was compared. Size- and concentration-dependent effects were seen where the smaller particles and lower concentrations affected MDDCs to a minor degree compared to the larger particles and higher concentrations, both in terms of viability, uptake, and immune regulatory markers. Thus, the larger particles have promising features to serve as an immune-stimulant, with possible T cell modulatory properties, while the smaller particles are more suitable for neutral drug delivery systems. Finally, we evaluated whether exosomes of different origins are selectively targetingdifferent immune cells. Results revealed that exosomes derived from human MDDCs and breast milk preferably associated with monocytes, whereas exosomes from an Epstein-Barr virus (EBV) transformed B cell line (LCL1) selectively targeted B cells. The interaction between LCL1-derived exosomes and peripheral blood B cells was dependent on CD21 on B cells and the exosome associated EBV glycoprotein gp350. This finding suggests that exosome-based vaccines can be engineered for specific B-cell targeting by inducing gp350 expression. To summarize, the work included in this thesis has contributed to the understanding of how particles of various materials, shapes and sizes affect and interact with human MDDCs. The knowledge gained is of importance for the further development of the studied materials within various medical applications. This thesis also highlights the potential use of endogenous nanoparticles, exosomes, as targeting delivery vehicles in medical applications