Post-coupling strategy enables true receptor-targeted nanoparticles

A key goal of our research is the targeted delivery of functional biopharmaceutical agents of interest, such as small interfering RNA (siRNA), to selected cells by means of receptor-mediated nanoparticle technologies. Recently, we described how pH-triggered, PEGylated siRNA-nanoparticles (pH triggered siRNA-ABC nanoparticles) were able to mediate the passive targeting of siRNA to liver cells in vivo. In addition, PEGylated siRNA nanoparticles enabled for long-term circulation (LTC siRNA-ABC nanoparticles, LEsiRNA nanoparticles) were shown to do the same to tumour cells in vivo. Further gains in the efficiency of siRNA delivery are expected to require active targeting with nanoparticles targeted for delivery and cellular uptake by means of attached biological ligands. Here we report on the development of a new synthetic chemistry and a bioconjugation methodology that allows for the controlled formulation of PEGylated nanoparticles which surface-present integrin-targeting peptides unambiguously and so enable integrin receptor-mediated cellular uptake. Furthermore, we present delivery data that provide a clear preliminary demonstration of physical principles that we propose should underpin successful, bonefide receptor-mediated targeted delivery of therapeutic and/or imaging agents to cells

Manipulating the Barrier Function of a Cell Monolayer Using a High-power Miniature Ultrasonic Transducer

Ultrasound (US) and cavitation agents such as microbubbles (MBs) have been demonstrated to decrease the barrier function of endothelial and epithelial layers. However, in vitro experiments that study this effect are often hindered by the inability to deliver buoyant contrast agents in proximity to cell monolayers in order to adequately control the decrease in barrier function whilst insonating a sufficiently large tissue area. We have addressed this by adapting a cell culture system and fabricating a bespoke high-power miniature unfocused US transducer. The setup was used to control the drop in barrier function and to determine how varying the mechanical index (MI) and the duty cycle affected the barrier function. It was found that buffer solution alone and buffer + MBs did not decrease the transepithelial electrical resistance (TEER) of the cell monolayer. Buffer + US decreased the TEER by ~40%, with 10% TEER recovery 9 min after switching US off. Buffer + MBs + US decreased the TEER by 80%, with little or no recovery following treatment. In the presence of MBs, the barrier function was decreased by a duty cycle = [1% - 50%] and by an MI = [0.25 - 0.5], without any recovery following treatment. Detectable amounts of fluorescent dextran were delivered across the Caco-2 monolayer only by a combination of US + MBs. These results suggest that our adapted setup and custom-built miniature transducer permits control of the decrease in barrier function for further therapeutic investigations

Characterising the chemical and physical properties of phase-change nanodroplets

Phase-change nanodroplets have attracted increasing interest in recent years as ultrasound theranostic nanoparticles. They are smaller compared to microbubbles and they may distribute better in tissues (e.g. in tumours). They are composed of a stabilising shell and a perfluorocarbon core. Nanodroplets can vaporise into echogenic microbubbles forming cavitation nuclei when exposed to ultrasound. Their perfluorocarbon core phase-change is responsible for the acoustic droplet vaporisation. However, methods to quantify the perfluorocarbon core in nanodroplets are lacking. This is an important feature that can help explain nanodroplet phase change characteristics. In this study, we fabricated nanodroplets using lipids shell and perfluorocarbons. To assess the amount of perfluorocarbon in the core we used two methods, 19F-NMR and FTIR. To assess the cavitation after vaporisation we used an ultrasound transducer (1.1MHz) and a high-speed camera. The 19F-NMR based method showed that the fluorine signal correlated accurately with the perfluorocarbon concentration. Using this correlation, we were able to quantify the perfluorocarbon core of nanodroplets. This method was used to assess the content of the perfluorocarbon of the nanodroplets in solutions over time. It was found that perfluoropentane nanodroplets lost their content faster and at higher ratio compared to perfluorohexane nanodroplets. The high-speed camera showed that these nanodroplets have similar cavitation with commercial microbubbles. Nanodroplet characterisation should include perfluorocarbon concentration assessment as critical information for their development

Ultrasound mediated delivery of quantum dots from a proof of concept capsule endoscope to the gastrointestinal wall

Biologic drugs, defined as therapeutic agents produced from or containing components of a living organism, are of growing importance to the pharmaceutical industry. Though oral delivery of medicine is convenient, biologics require invasive injections because of their poor bioavailability via oral routes. Delivery of biologics to the small intestine using electronic delivery with devices that are similar to capsule endoscopes is a promising means of overcoming this limitation and does not require reformulation of the therapeutic agent. The efficacy of such capsule devices for drug delivery could be further improved by increasing the permeability of the intestinal tract lining with an integrated ultrasound transducer to increase uptake. This paper describes a novel proof of concept capsule device capable of electronic application of focused ultrasound and delivery of therapeutic agents. Fluorescent markers, which were chosen as a model drug, were used to demonstrate in vivo delivery in the porcine small intestine with this capsule. We show that the fluorescent markers can penetrate the mucus layer of the small intestine at low acoustic powers when combining microbubbles with focused ultrasound during in vivo experiments using porcine models. This study illustrates how such a device could be potentially used for gastrointestinal drug delivery and the challenges to be overcome before focused ultrasound and microbubbles could be used with this device for the oral delivery of biologic therapeutics

Effect of phase-change nanodroplets and ultrasound on blood-brain barrier permeability in vitro

Phase-change nanodroplets (PCND;NDs) are emulsions with a perfluorocarbon (PFC) core that undergo acoustic vaporisation as a response to ultrasound (US). Nanodroplets change to microbubbles and cavitate while under the effect of US. This cavitation can apply forces on cell connections in biological barrier membranes, such as the blood–brain barrier (BBB), and trigger a transient and reversible increased permeability to molecules and matter. This study aims to present the preparation of lipid-based NDs and investigate their effects on the brain endothelial cell barrier in vitro. The NDs were prepared using the thin-film hydration method, followed by the PFC addition. They were characterised for size, cavitation (using a high-speed camera), and PFC encapsulation (using FTIR). The bEnd.3 (mouse brain endothelial) cells were seeded onto transwell inserts. Fluorescein with NDs and/or microbubbles were applied on the bEND3 cells and the effect of US on fluorescein permeability was measured. The Live/Dead assay was used to assess the BBB integrity after the treatments. Size and PFC content analysis indicated that the NDs were stable while stored. High-speed camera imaging confirmed that the NDs cavitate after US exposure of 0.12 MPa. The BBB cell model experiments revealed a 4-fold increase in cell membrane permeation after the combined application of US and NDs. The Live/Dead assay results indicated damage to the BBB membrane integrity, but this damage was less when compared to the one caused by microbubbles. This in vitro study shows that nanodroplets have the potential to cause BBB opening in a similar manner to microbubbles. Both cavitation agents caused damage on the endothelial cells. It appears that NDs cause less cell damage compared to microbubbles

An atom efficient, single-source precursor route to plasmonic CuS nanocrystals

The synthesis of colloidal semiconductor nanocrystals (NCs) from single-source precursors offers simplified manufacturing processes at the cost of reduced atom efficiency. Self-capping routes have the potential to maximise this efficiency although investigation has so far been limited to organic solvents. Here we present the synthesis of copper sulfide NCs via the decomposition of a copper dithiocarbamate complex in water. Nanocrystalline covellite particles were prepared without the need for additional capping ligand and exhibited a hollow nanosphere morphology. Mass spectrometry of the water-stable NCs indicated the presence of a number of surface ligands, including a small amine fragment of the single-source precursor (SSP) complex. A broad plasmon resonance in the near-infrared (NIR) at 990 nm was also observed and the photothermal effect of this demonstrated. Cytotoxicity experiments indicated cell viability remained above 95% for NC concentrations up to 1 mg mL(−1), indicating high biocompatibility

Cytotoxicity of polycations: Relationship of molecular weight and the hydrolytic theory of the mechanism of toxicity

The mechanism of polycation cytotoxicity and the relationship to polymer molecular weight is poorly understood. To gain an insight into this important phenomenon a range of newly synthesised uniform (near monodisperse) linear polyethylenimines, commercially available poly(L-lysine)s and two commonly used PEI -based transfectants (broad 22 kDa linear and 25 kDa branched) were tested for their cytotoxicity against the A549 human lung carcinoma cell line. Cell membrane damage assays (LDH release) and cell viability assays (MTT) showed a strong relationship to dose and polymer molecular weight, and increasing incubation times revealed that even supposedly "non-toxic" low molecular weight polymers still damage cell membranes. The newly proposed mechanism of cell membrane damage is acid catalysed hydrolysis of lipidic phosphoester bonds, which was supported by observations of the hydrolysis of DOPC liposomes. Crown Copyright (C) 2017 Published by Elsevier B.V. All rights reserved