Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies

Reducing protein corona formation and enhancing colloidal stability of gold nanoparticles by capping with silica monolayers

A study demonstrated the reducing of protein corona (PC) formation and enhancing colloidal stability of gold nanoparticles (Au NP) by capping with silica monolayers. Au NP surface was needed to achieve silica monolayer formation. In a first attempt to have a high density ligand coverage around NS-1, an excess of 3-mercaptopropyltrimethoxysilane (MPTMS) was directly added to a Au NP hydrosol, but this resulted in immediate aggregation. The resulting trimethoxysilane moiety covering the surface of NS-3 was subsequently hydrolyzed by addition of 5 mM NaOH in a 2:1 methanol/water mixture, yielding a polymeric monolayer

Reversible control of protein corona formation on gold nanoparticles using host-guest interactions

When nanoparticles (NPs) are exposed to biological media, proteins are adsorbed, forming a so-called protein corona (PC). This cloud of protein aggregates hampers the targeting and transport capabilities of the NPs, thereby compromising their biomedical applications. Therefore, there is a high interest in the development of technologies that allow control over PC formation, as this would provide a handle to manipulate NPs in biological fluids. We present a strategy that enables the reversible disruption of the PC using external stimuli, thereby allowing a precise regulation of NP cellular uptake. The approach, demonstrated for gold nanoparticles (AuNPs), is based on a biorthogonal, supramolecular host-guest interactions between an anionic dye bound to the AuNP surface and a positively charged macromolecular cage. This supramolecular complex effectively behaves as a zwitterionic NP ligand, which is able not only to prevent PC formation but also to disrupt a previously formed hard corona. With this supramolecular stimulus, the cellular internalization of AuNPs can be enhanced by up to 30-fold in some cases, and even NP cellular uptake in phagocytic cells can be regulated. Additionally, we demonstrate that the conditional cell uptake of purposely designed gold nanorods can be used to selectively enhance photothermal cell death

Remodeling arteries: studying the mechanical properties of 3D-bioprinted hybrid photoresponsive materials.

3D-printed cell models are currently in the spotlight of medical research. Whilst significant advances have been made, there are still aspects that require attention to achieve more realistic models which faithfully represent the in vivo environment. In this work we describe the production of an artery model with cyclic expansive properties, capable of mimicking the different physical forces and stress factors that cells experience in physiological conditions. The artery wall components are reproduced using 3D printing of thermoresponsive polymers with inorganic nanoparticles (NPs) representing the outer tunica adventitia, smooth muscle cells embedded in extracellular matrix representing the tunica media, and finally a monolayer of endothelial cells as the tunica intima. Cyclic expansion can be induced thanks to the inclusion of photo-responsive plasmonic NPs embedded within the thermoresponsive ink composition, resulting in changes in the thermoresponsive polymer hydration state and hence volume, in a stimulated on-off manner. By changing the thermoresponsive polymer composition, the transition temperature and pulsatility can be efficiently tuned. We show the direct effect of cyclic expansion and contraction on the overlying cell layers by analyzing transcriptional changes in mechanoresponsive mesenchymal genes associated with such microenvironmental physical cues. The technique described herein involving stimuli-responsive 3D printed tissue constructs, also described as four- dimensional (4D) printing, offers a novel approach for the production of dynamic biomodels.Financial support is acknowledged from the MCIN/AEI/ 10.13039/501100011033 through grant # PID2019-108854RAI00. C. G. A. thanks to the Ministerio de Ciencia e Innovacio´n (MCIN) for a Juan de la Cierva Incorporacio´n Fellowship (IJC2019-040827-I). M. S.-A. is recipient of a Ramo´n y Cajal contract and a ‘‘Generacio´n de Conocimiento’’ grant from the Ministerio de Ciencia e Innovacio´n (RYC2020-029690-I and PID2021-128106NA-I00). MAdP is coordinator and PL of ‘‘AtheroConvergence’’ La Caixa Foundation Health Research consortium (HR20-00075). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S). We acknowledge ALBA for provision of synchrotron radiation facilities. We would like to thank Dr Marc Malfois for assistance in using BL11-NCD beamline, and Unai Cossı´o and Daniel Padro for help with image analysis.S

Gold Nanostar-Coated Polystyrene Beads as Multifunctional Nanoprobes for SERS Bioimaging

Hybrid colloidal nanocomposites comprising polystyrene beads and plasmonic gold nanostars are reported as multifunctional optical nanoprobes. Such self-assembled structures are excellent Raman enhancers for bioapplications as they feature plasmon modes in the near-infrared "first biological transparency window". In this proof of concept study, we used 4-mercaptobenzoic acid as a Raman-active molecule to optimize the density of gold nanostars on polystyrene beads, improving SERS performance and thereby allowing in vitro cell culture imaging. Interestingly, intermediate gold nanostar loadings were found to yield higher SERS response, which was confirmed by electromagnetic modeling. These engineered hybrid nanostructures notably improve the possibilities of using gold nanostars as SERS tags. Additionally, when fluorescently labeled polystyrene beads are used as colloidal carriers, the composite particles can be applied as promising tools for multimodal bioimaging

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on Bilayer properties, biodistribution, and immune responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses. © 2013 American Chemical Society

Hybrid Au–SiO₂ Core–Satellite Colloids as Switchable SERS Tags

Gold–silica self-assembled nanostructures are reported as multiplex surface enhanced Raman scattering (SERS) tags for bioimaging applications. These hybrid colloidal particles were obtained by heteroassembly of Au–SiO₂ Janus particles with smaller Au spheres and two Raman-active molecules that can be independently imaged by varying the wavelength of the excitation laser. The Janus structure of Au–SiO₂ not only directs the assembly but also provides physical separation of the Raman tags and colloidal stability thereby facilitating complete silica encapsulation. The bioimaging capabilities of this system are demonstrated through SERS mapping of individual cells

3D‐Printed Biocompatible Scaffolds with Built‐In Nanoplasmonic Sensors

3D printing strategies have acquired great relevance toward the design of 3D scaffolds with precise macroporous structures, for supported mammalian cell growth. Despite advances in 3D model designs, there is still a shortage of detection tools to precisely monitor in situ cell behavior in 3D, thereby allowing a better understanding of the progression of diseases or to test the efficacy of drugs in a more realistic microenvironment. Even if the number of available inks has exponentially increased, they do not necessarily offer the required functionalities to be used as internal sensors. Herein the potential of surface-enhanced Raman scattering (SERS) spectroscopy for the detection of biorelevant analytes within a plasmonic hydrogel-based, 3D-printed scaffold is demonstrated. Such SERS-active scaffolds allow for the 3D detection of model molecules, such as 4-mercaptobenzoic acid. Flexibility in the choice of plasmonic nanoparticles is demonstrated through the use of gold nanoparticles with different morphologies, gold nanorods showing the best balance between SERS enhancement and scaffold transparency. Detection of the biomarker adenosine is also demonstrated as a proof-of-concept toward the use of these plasmonic scaffolds for SERS sensing of cell-secreted molecules over extended periods of time.ISSN:1616-3028ISSN:1616-301