RELATING QUANTUM DOT ASSOCIATION WITH HUMAN ENDOTHELIAL CELLS WITH THEIR CYTOTOXIC EFFECTS

INTRODUCTION Advances in the field of nanotechnology have enabled researchers to pursue biomedical applications of nanoparticles. Quantum dots are commonly used fluorescent probes because they are brighter and less prone to photobleaching than other fluorophores [1]. However, despite the advantages, potential for toxicity must be acknowledged. Quantum dots are commonly made with toxic metal elements, which can cause oxidative stress [2]. Cadmium ions have been shown to disrupt mitochondria activity, leading to cell death [2]. Quantum dots have been shown to attach to the cell membrane as well as be internalized through endocytic mechanisms [3]. In this study, we aim to quantify quantum dot association and compare results from cytotoxicity assays for identical conditions, relating cellular association with cytotoxicity. METHODS Human Umbilical Vein Endothelial Cells (HUVECs) and Human Micro-vascular Endothelial Cells (HMVECs) were cultured in static conditions in 8-well chamber slides then exposed to amino-PEG quantum dots at a concentration of 0.2nM to 200nM. After exposure for 24 hours, the cells were washed, fixed, and stained. Z-stacks were obtained using an Olympus Fluoview FV1000 confocal microscope. Images were analyzed using ImageJ software to quantify mean fluorescence intensity within the defined region of interest, selected from the boundaries of stained cell membranes. Statistical analysis using one-way Analysis of Variance (ANOVA) and post-hoc Tukey HSD test was performed. Finally, Vialight assay was used to test cell viability after exposure to quantum dots under the same experimental conditions used for association experiments. RESULTS Exposure to different concentrations of quantum dots results in significant changes in the observed fluorescence intensity per area. Non-linear dependence of cellular association of quantum dots on exposure concentration was observed. A representative example of mean fluorescence intensity of quantum dots associated with HUVECs is shown in Figure 1.A significant decrease in the viability of HUVECs was observed on exposure to quantum dots (30-50% cell viability relative to 100% for non-exposed cells). However, no significant difference in cell viability was observed between 0.2nM to 200nM concentrations. DISCUSSION AND CONCLUSIONS Nanoparticle association studies play a vital role in predicting cell viability in nanoparticle cytotoxicity studies. The non-linear trend observed suggests that for the range of concentrations examined, cellular association does not increase linearly with exposure concentration, and that cytotoxicity can be related to association, rather than just to exposure concentration. This experiment provides an approach to advance future studies relating cellular association to cytotoxicity

RELATING CELLULAR ASSOCIATION WITH LIPOSOME CYTOTOXICITY IN HUMAN ENDOTHELIAL CELLS

INTRODUCTION Interactions with the endothelium play a key role in the behaviour of intravenously administered nanoparticle drug carriers[1]. Hence, quantifying cellular association (membrane adhesion and cell internalization) of liposomes with endothelial cells is an effective screening method of biocompatibility and success of new drug carriers. Current methods are inaccurate as concentration does not necessarily equate to local cellular association. The focus of this experiment is to quantify the cellular association between liposomes and two types of human endothelial cells and compare the associations with cells’ cytotoxic response. Cellular association of liposomes as well as cell viability were quantified on cellular level at different concentrations of liposomes. METHODS Two different types of cells, Human Umbilical Vein Endothelial cell, which is a common cell type used in vitro studies, and Human MicroVascular Cell, which is more accurate representation of in vivo, were used[2]. HUVEC and HMVEC were cultured and passaged onto chamber slides using standard cell culture techniques. The confluent cells were exposed to fluorescent liposomes with hydrodynamic diameter of 90.4 nm at concentrations ranging from 0.08nM to 8nM for 24 hours, membrane stained with CellMask Deep Red and fixed with paraformaldehyde, following same protocols for both types of cells. Cell viability on exposure to the same concentration range of liposomes was determined using Vialight assay using manufacturer protocols. Z-stacks of the treated cells were obtained using Olympus Fluoview FV1000 confocal microscope. Region of interest, limited by cell membranes, was set using the membrane stain channel using ImageJ. The region of interest was superimposed onto the fluorescent liposome channel to determine exclusively the fluorescence of cell adhered and cell internalized liposomes RESULTS Compared to HUVECs, higher cellular association of liposomes was observed for HMVC as shown in Figure 1.While cellular association of liposomes increased with concentration, cell viability was in the range of 85 to nearly 100% for the concentration range of 0.08-4 nM with no significant difference. Only at 8 nM, cell viability decreased significantly to approximately 62 %. DISCUSSION AND CONCLUSIONS Liposome cellular association provide insight into the cytotoxicity and the endothelial cytotoxicity of the liposomes at low concentration of 8nM raises cautions on documented innocuous properties of liposomes. Cytotoxicity and cellular association upon comparison showed exponential relationship. Because the cytotoxicity and cellular association relationship is exponential, slight over-administration can cause severe toxicity. 8nM is lower than concentration of current intravenous liposome-based drug doxorubicin[3]. High toxicity and exponential relationship raise caution on the importance of proper safe dosage

EFFECT OF PEG COATING ON NANOPARTICLE DIFFUSION THROUGH TUMOUR EXTRACELLULAR MATRIX

INTRODUCTION Nanoparticle drug delivery systems have the potential to improve current cancer treatments through encapsulating cytotoxic agents and delivering them to specific sites in the body. One such class of particle, liposomes, has already found some commercial success [1]. Liposomes are vesicles composed of a lipid bi-layer surrounding an aqueous solution. Poly(ethylene) glycol (PEG) surface coating is commonly used to improve the hydrophilicity of liposomes, thereby increasing their stability in aqueous solutions. Furthermore, PEG limits the binding of blood antigens, which minimizes opsonisation and phagocytosis, extending circulation time in the blood stream. When applied to the surface of liposomes at lower molecular weights and surface densities, PEG adopts a “mushroom” conformation, in which adjacent chains of PEG do not interact laterally, therefore portions of the bi-layer remain exposed [2]. However, at higher molecular weights and surface densities, the “brush” conformation is adopted; where lateral interactions occur between neighbouring PEG strands and provide complete coverage of the lipid bi-layer [2]. This study will investigate the effect of varying PEG molecular weight and surface density on liposome transport through tumour extracellular matrix. METHODS Seven different formulations of liposomes were synthesized using a modification of the lipid extrusion method described in [1]. Molecular weight and surface density values were chosen to include both PEG conformations. The Type I collagen hydrogel was prepared with a collagen concentration of 2.5mg/mL. Confocal Microscopy was used to track the liposome transport into the gels via the bilayer incorporated Rhodamine dye. While simple collagen hydrogels may not capture all of the complexity of native tumour ECM, they allow for more carefully controlled conditions than in vivo models. Images were taken every 30 minutes until the 900 minute mark. RESULTS As shown in Figure 1, the liposomes with a lower PEG loading (DOPC, 5, 10% PEG 1000, 5, 10% PEG 2000), all accumulated at the interface of the hydrogel, and had identical diffusion coefficients. The 5% and 10% PEG 5000 however, accumulated significantly less and therefore had a much greater diffusion coefficient.DISCUSSION AND CONCLUSIONS The liposomes with low PEG surface density, and DOPC control liposomes shown in Figure 1, are all within the “mushroom” conformation of PEG [2] and therefore would all have exposed bilayer which is not shielded by the PEG strands. The formulations that penetrated deeply were notably only higher PEG surface densities (5 and 10% PEG 5000) which literature suggests would have been in the “brush” conformation [2]. This suggests that the high PEG surface densities sterically shielded the liposomes, and reduced the electrostatic interactions between the hydrogels and the liposomes, allowing increased diffusion

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Impact Metrics

Virtually every evaluative task in the academy involves some sort of metric (Elkana et al. 1978; Espeland & Sauder 2016; Gingras 2016; Hix 2004; Jensenius et al. 2018; Muller 2018; Osterloh and Frey 2015; Todeschini & Baccini 2016; Van Noorden 2010; Wilsdon et al. 2015). One can decry this development, and inveigh against its abuses and its over-use (as many of the foregoing studies do). Yet, without metrics, we would be at pains to render judgments about scholars, published papers, applications (for grants, fellowships, and conferences), journals, academic presses, departments, universities, or subfields. Of course, we also undertake to judge these issues ourselves through a deliberative process that involves reading the work under evaluation. This is the traditional approach of peer review. No one would advocate a system of evaluation that is entirely metric-driven. Even so, reading is time-consuming and inherently subjective; it is, after all, the opinion of one reader (or several readers, if there is a panel of reviewers). It is also impossible to systematically compare these judgments. To be sure, one might also read, and assess, the work of other scholars, but this does not provide a systematic basis for comparison – unless, that is, a standard metric(s) of comparison is employed. Finally, judging scholars through peer review becomes logistically intractable when the task shifts from a single scholar to a large group of scholars or a large body of work, e.g., a journal, a department, a university, a subfield, or a discipline. It is impossible to read, and assess, a library of work

Making Research Data Accessible

This chapter argues that these benefits will accrue more quickly, and will be more significant and more enduring, if researchers make their data “meaningfully accessible.” Data are meaningfully accessible when they can be interpreted and analyzed by scholars far beyond those who generated them. Making data meaningfully accessible requires that scholars take the appropriate steps to prepare their data for sharing, and avail themselves of the increasingly sophisticated infrastructure for publishing and preserving research data. The better other researchers can understand shared data and the more researchers who can access them, the more those data will be re-used for secondary analysis, producing knowledge. Likewise, the richer an understanding an instructor and her students can gain of the shared data being used to teach and learn a particular research method, the more useful those data are for that pedagogical purpose. And the more a scholar who is evaluating the work of another can learn about the evidence that underpins its claims and conclusions, the better their ability to identify problems and biases in data generation and analysis, and the better informed and thus stronger an endorsement of the work they can offer

Developing Rapid Screening Tools for Predicting Nanomedicine Transport Limitations

Nanomedicines represent the future of medicine. Targeted therapies promise to increase treatment efficacy while simultaneously reducing side effects. However, despite two decades of dedicated research, this paradigm shift has found little clinical traction. Partly to blame is the multitude of off-target sinks and degrading factors that limit delivery efficiency. Rapid, cost effective, in vitro models may be able to screen novel nanomedicines for their susceptibility to these transport limitations. This thesis focuses on studying nanoparticle transport in two specific domains: endothelium interactions and extracellular matrix diffusion, utilizing in vitro platforms. Laser-scanning confocal microscopy and associated image analysis techniques allow fluorescently-labelled, cell-associated nanoparticles to be quantified. However, image analysis procedures lack standardization. Endothelial cells were exposed to fluorescent nanoparticles to investigate whether different image analysis techniques could impact particle quantification. Significant differences were found when fluorescence quantification and image normalization methods were varied, as well as when image projections were analysed. Fluid flow forces impact nanoparticle interactions with the endothelium. The association of quantum dots with human endothelial cells was studied after flow preconditioning in a parallel plate flow chamber at various flow magnitudes. The results were compared with distribution patterns of quantum dots in zebrafish embryo vasculature. It was found that quantum dots preferentially accumulate in lower flow vessels, and associate more with cells that have undergone lower flow preconditioning. A novel platform was developed to study the transport of gold nanoparticles in extracellular matrix. It was found that matrix density and particle diameter impact the matrix diffusion of particles. These results were supported by a tumour-bearing murine model and in silico predictions of particle behaviour. Characterization of these three models lead to a decision matrix to select nanoparticle properties based on patient-specific pathophysiology. The novel platform was further applied to understanding the effect of polyethylene glycol surface functionalization on liposome diffusion in extracellular matrix. It was found that polymer conformation is an important driver of particle-matrix interactions. Together this work provides new insights into nanoparticle transport limitations, showcases the predicative value of in vitro modelling of particle transport and offers new tools towards increasing the clinical translation of nanomedicines