A Comparative Study of Ultrasmall Calcium Carbonate Nanoparticles for Targeting and Imaging Atherosclerotic Plaque.

Atherosclerosis is a complex disease that can lead to life-threatening events, such as myocardial infarction and ischemic stroke. Despite the severity of this disease, diagnosing plaque vulnerability remains challenging due to the lack of effective diagnostic tools. Conventional diagnostic protocols lack specificity and fail to predict the type of atherosclerotic lesion and the risk of plaque rupture. To address this issue, technologies are emerging, such as noninvasive medical imaging of atherosclerotic plaque with customized nanotechnological solutions. Modulating the biological interactions and contrast of nanoparticles in various imaging techniques, including magnetic resonance imaging, is possible through the careful design of their physicochemical properties. However, few examples of comparative studies between nanoparticles targeting different hallmarks of atherosclerosis exist to provide information about the plaque development stage. Our work demonstrates that Gd (III)-doped amorphous calcium carbonate nanoparticles are an effective tool for these comparative studies due to their high magnetic resonance contrast and physicochemical properties. In an animal model of atherosclerosis, we compare the imaging performance of three types of nanoparticles: bare amorphous calcium carbonate and those functionalized with the ligands alendronate (for microcalcification targeting) and trimannose (for inflammation targeting). Our study provides useful insights into ligand-mediated targeted imaging of atherosclerosis through a combination of in vivo imaging, ex vivo tissue analysis, and in vitro targeting experiments.

Assessing the role of surface glycans of extracellular vesicles on cellular uptake.

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. However, there is ambiguity around the fundamental mechanisms by which these effects are transduced, particularly in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Lectin microarray technology was employed to define the surface glycosylation patterns of EVs. Glycosidases PNGase F and neuraminidase which cleave N-glycans and terminal sialic acids, respectively, were used to analyze the relevance of these modifications to EV surface glycans on the uptake of fluorescently labelled EVs by a panel of cells representing a variety of tissues. Flow cytometry revealed an increase in affinity for EVs modified by both glycosidase treatments. High-content screening exhibited a broader range of responses with different cell types preferring different vesicle glycosylation states. We also found differences in vesicle charge after treatment with glycosidases. We conclude that glycans are key players in the tuning of EV uptake, through charge-based effects, direct glycan recognition or both, supporting glycoengineering as a toolkit for therapy development.

Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives.

It is now acknowledged that extracellular vesicles (EVs) are important effectors in a vast number of biological processes through intercellular transfer of biomolecules. Increasing research efforts in the EV field have yielded an appreciation for the potential role of glycans in EV function. Indeed, recent reports show that the presence of glycoconjugates is involved in EV biogenesis, in cellular recognition and in the efficient uptake of EVs by recipient cells. It is clear that a full understanding of EV biology will require researchers to focus also on EV glycosylation through glycomics approaches. This review outlines the major glycomics techniques that have been applied to EVs in the context of the recent findings. Beyond understanding the mechanisms by which EVs mediate their physiological functions, glycosylation also provides opportunities by which to engineer EVs for therapeutic and diagnostic purposes. Studies characterising the glycan composition of EVs have highlighted glycome changes in various disease states, thus indicating potential for EV glycans as diagnostic markers. Meanwhile, glycans have been targeted as molecular handles for affinity-based isolation in both research and clinical contexts. An overview of current strategies to exploit EV glycosylation and a discussion of the implications of recent findings for the burgeoning EV industry follows the below review of glycomics and its application to EV biology.

Lectin-Array Blotting.

Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc.

Microarray-based identification of lectins for the purification of human urinary extracellular vesicles directly from urine samples.

As cellular-derived vesicles largely maintain the biomolecule composition of their original tissue, exosomes, which are found in nearly all body fluids, have enormous potential as clinical disease markers. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. We employed lectin arrays to identify potential lectins as probes for affinity-based isolation of exosomes from the urinary matrix. We found three lectins that showed specific interactions to vesicles and no (or only residual) interaction with matrix proteins. Based on these findings a bead-based method for lectin-based isolation of exosomes from urine was developed as a sample preparation step for exosome-based biomarker research.

A practical total synthesis of hapalosin, a 12-membered cyclic depsipeptide with multidrug resistance-reversing activity, by employing improved segment coupling and macrolactonization.

A practical total synthesis of hapalosin, a compound with multidrug resistance-reversing activity, has been carried out using an unprecedented macrolactonization strategy. One of the features of the new approach is the straightforward and fully stereocontrolled access to the key gamma-amino beta-hydroxy carboxylic acid subunit via an efficient acetate aldol addition reaction with N-methyl alpha-aminoaldehydes, which relies on a camphor-derived chiral lithium acetate enolate reagent. The scope of this aldol reaction is investigated and its potential application to the synthesis of other structurally related, biologically relevant compounds illustrated. Remarkably, the chiral tether in the resulting gamma-amino aldol adducts sterically protect the carbonyl group, thus avoiding intramolecular cyclization during the amino group deprotection and the subsequent segment coupling event. After successful segment coupling and smooth, clean release of the chiral auxiliary, a new macrolactonization protocol, based on the principle of double activation of both reactive sites, is applied, which leads to the 12-membered macrolactone hapalosin in unprecedented chemical efficiency.