Background Safe and rational development of nanomaterials for clinical translation requires the assessment of potential biocompatibility. Autophagy, a critical homeostatic pathway intrinsically linked to cellular health and inflammation, has been shown to be affected by nanomaterials. It is, therefore, important to be able to assess possible interactions of nanomaterials with autophagic processes. Results CEM (T cell), Raji (B lymphocyte), and THP-1 (human monocyte) cell lines were subject to treatment with rapamycin and chloroquine, known to affect the autophagic process, in order to evaluate cell line-specific responses. Flow cytometric quantification of a fluorescent autophagic vacuole stain showed that maximum observable effects (105%, 446%, and 149% of negative controls) were achieved at different exposure durations (8, 6, and 24 h for CEM, Raji, and THP-1, respectively). THP-1 was subsequently utilised as a model to assess the autophagic impact of a small library of nanomaterials. Association was observed between hydrodynamic size and autophagic impact (r 2 = 0.11, p = 0.004). An ELISA for p62 confirmed the greatest impact by 10 nm silver nanoparticles, abolishing p62, with 50 nm silica and 180 nm polystyrene also lowering p62 to a significant degree (50%, 74%, and 55%, respectively, p < 0.05). Conclusions This data further supports the potential for a variety of nanomaterials to interfere with autophagic processes which, in turn, may result in altered cellular function and viability. The association of particle size with impact on autophagy now warrants further investigation. Graphic abstract

We describe rheological protocols to study layered and three-dimensional (3D)-printed gels. Our methods allow us to measure the properties at different depths and determine the contribution of each layer to the resulting combined properties of the gels. We show that there are differences when using different measuring systems for rheological measurement, which directly affects the resulting properties being measured. These methods allow us to measure the gel properties after printing, rather than having to rely on the assumption that there is no change in properties from a preprinted gel. We show that the rheological properties of fluorenylmethoxycarbonyl-diphenylalanine (FmocFF) gels are heavily influenced by the printing process.

Purpose The question of whether mesenchymal stromal cells (MSCs) home to injured kidneys remains a contested issue. To try and understand the basis for contradictory findings reported in the literature, our purpose here was to investigate whether MSC homing capacity is influenced by administration route, the type of injury model used, and/or the presence of exogenous macrophages. Procedures To assess the viability, whole-body biodistribution, and intra-renal biodistribution of MSCs, we used a multimodal imaging strategy comprising bioluminescence and magnetic resonance imaging. The effect of administration route (venous or arterial) on the ability of MSCs to home to injured renal tissue, and persist there, was assessed in a glomerular injury model (induced by the nephrotoxicant, Adriamycin) and a tubular injury model induced by ischaemia-reperfusion injury (IRI). Exogenous macrophages were used as a positive control because these cells are known to home to injured mouse kidneys. To assess whether the homing capacity of MSCs can be influenced by the presence of exogenous macrophages, we used a dual-bioluminescence strategy that allowed the whole-body biodistribution of the two cell types to be monitored simultaneously in individual animals. Results Following intravenous administration, no MSCs were detected in the kidneys, irrespective of whether the mice had been subjected to renal injury. After arterial administrationviathe left cardiac ventricle, MSCs transiently populated the kidneys, but no preferential homing or persistence was observed in injured renal tissue after unilateral IRI. An exception was when MSCs were co-administered with exogenous macrophages; here, we observed some homing of MSCs to the injured kidney. Conclusions Our findings strongly suggest that MSCs do not home to injured kidneys.

Investigation of the potential for nanomaterials to generate immunogenic effects is a key aspect of a robust preclinical evaluation. In combination with physicochemical characterization, such assessments also provide context for how material attributes influence biological outcomes. Furthermore, appropriate models for these assessments allow accurate in vitro to in vivo extrapolation, which is vital for the mechanistic understanding of nanomaterial action. Here we have assessed the immunogenic impact of a small panel of commercially available and in-house prepared nanomaterials on primary human peripheral blood mononuclear cells (PBMCs). A diethylaminoethyl-dextran (DEAE-dex) functionalized superparamagnetic iron oxide nanoparticle (SPION) generated detectable quantities of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and IL-10, the only tested material to do so. The human leukemia monocytic cell line THP-1 was used to assess the potential for the nanomaterial panel to affect cellular oxidation-reduction (REDOX) via measurement of reactive oxygen species and reduced glutathione. Negatively charged sulfonate-functionalized polystyrene nanoparticles demonstrated a size-related trend for the inhibition of caspase-1, which was not observed for amine-functionalized polystyrene of similar sizes. Silica nanoparticles (310 nm) resulted in a 93% increase in proliferation compared to the untreated control (p < 0.01). No other nanomaterial treatments resulted in significant change from that of unstimulated PBMCs. Responses to the nanomaterials in the assays described demonstrate the utility of primary cells as ex vivo models for nanomaterial biological impact.

This work represents a detailed investigation into the phase and morphological behavior of synergistic dual-stimuli-responsive poly(N-isopropylacrylamide) nanogels, a material that is of considerable interest as a matrix for in situ forming implants. Nanogels were synthesized with four different diameters (65, 160, 310, and 450 nm) as monodispersed particles. These different samples were then prepared and characterized as both dilute (0.1 wt %) and concentrated dispersions (2–22 wt %). In the dilute form, all of the nanogels had the same response to the triggers of the physiological temperature and ionic strength. In water, the nanogels would deswell when heated above 32 °C, while they would aggregate if heated above this temperature at the physiological ionic strength. In the concentrated form, the nanogels exhibited a wide range of morphological changes, with liquid, swollen gel, shrunken gel, and aggregate structures all possible. The occurrence of these structures was dependent on many factors such as the temperature, ionic strength of the solvent, size and ζ-potential of the nanogel, and dispersion concentration. We explored these factors in detail with techniques such as visual studies, rheology, effective volume fraction, and shape factor measurement. The different-sized nanogels displayed differing phase and morphological behavior, but generally higher concentrations of the nanogels (>7 wt %) yielded gels in water with the transitions depending on the temperature. The smallest nanogel (65 nm diameter) exhibited the most unique behavior; it did not form a swollen gel at any concentration tested. Shape factor measurement for the nanogel samples showed that two of the larger three samples (160 and 310 nm) had core–shell structures with denser core cross-linking, while the smallest nanogel sample displayed a homogeneous cross-linked structure. We hypothesize that the smallest nanogels are able to undergo more extensive interpenetration compared to the larger nanogels, which meant that the smallest nanogel was not able to form a swollen gel. In the presence of salt at 12 wt %, all of the nanogels formed aggregates when heated above 35 °C due to the screening of the electrostatic stabilization by the salt. This work revealed unique behavior of the smallest nanogel with a homogeneous cross-linked structure; its phase and morphological behavior were unlike a particle dispersion, rather these were more similar to those of a branched polymer solution. In total, these findings can be used to provide information about the design of poly(N-isopropylacrylamide) nanogel dispersions for different applications where highly specific spatiotemporal control of morphology is required, for example, in the formation of in situ forming implants or for pore blocking behavior.

Nanoparticle contrast agents are useful tools to label stem cells and monitor the in vivo bio-distribution of labeled cells in pre-clinical models of disease. In this context, understanding the in vivo fate of the particles after injection of labelled cells is important for their eventual clinical use as well as for the interpretation of imaging results. We examined how the formulation of superparamagnetic iron oxide nanoparticles (SPIONs) impacts the labelling efficiency, magnetic characteristics and fate of the particles by comparing individual SPIONs with polyelectrolyte multilayer capsules containing SPIONs. At low labelling concentration, encapsulated SPIONs served as an efficient labelling agent for stem cells. The bio-distribution after intra-cardiac injection of labelled cells was monitored longitudinally by MRI and as an endpoint by inductively coupled plasma-optical emission spectrometry. The results suggest that, after being released from labelled cells after cell death, both formulations of particles are initially stored in liver and spleen and are not completely cleared from these organs 2 weeks post-injection.

BackgroundCell-based regenerative medicine therapies are now frequently tested in clinical trials. In many conditions, cell therapies are administered systemically, but there is little understanding of their fate, and adverse events are often under-reported. Currently, it is only possible to assess safety and fate of cell therapies in preclinical studies, specifically by monitoring animals longitudinally using multi-modal imaging approaches. Here, using a suite of in vivo imaging modalities to explore the fate of a range of human and murine cells, we investigate how route of administration, cell type and host immune status affect the fate of administered cells.MethodsWe applied a unique imaging platform combining bioluminescence, optoacoustic and magnetic resonance imaging modalities to assess the safety of different human and murine cell types by following their biodistribution and persistence in mice following administration into the venous or arterial system.ResultsLongitudinal imaging analyses (i) suggested that the intra-arterial route may be more hazardous than intravenous administration for certain cell types, (ii) revealed that the potential of a mouse mesenchymal stem/stromal cell (MSC) line to form tumours depended on administration route and mouse strain and (iii) indicated that clinically tested human umbilical cord (hUC)-derived MSCs can transiently and unexpectedly proliferate when administered intravenously to mice.ConclusionsIn order to perform an adequate safety assessment of potential cell-based therapies, a thorough understanding of cell biodistribution and fate post administration is required. The non-invasive imaging platform used here can expose not only the general organ distribution of these therapies, but also a detailed view of their presence within different organs and, importantly, tumourigenic potential. Our observation that the hUC-MSCs but not the human bone marrow (hBM)-derived MSCs persisted for a period in some animals suggests that therapies with these cells should proceed with caution.

There is growing interest in the design of supramolecular structures that are photocatalytically active. Perylene bisimides can be self-assembled to produce structures for photocatalytic hydrogen evolution. Herein we explore the role of pH in controlling self-assembly and photocatalysis. It is shown that self-assembly, which occurs as the pH of the system is decreased, is required for hydrogen evolution to occur.

The number of clinical trials using cell-based therapies is increasing, as is the range of cell types being tested, but without a thorough understanding of cell fate and safety. Therefore, there is a pressing need for monitoring of cell fate in preclinical studies to identify potential hazards that might arise in patients. Utilising a unique imaging toolkit combining bioluminescence, optoacoustic and magnetic resonance imaging modalities, we assessed the safety of different cell types by following their biodistribution and persistence in mice. Our imaging studies suggest that the intra-arterial route is more hazardous than intravenous administration. Longitudinal imaging analysis over four weeks revealed that the potential of mouse mesenchymal stem/stromal cells (mMSCs) to form tumours, depended on administration route and mouse strain. Clinically tested human umbilical cord (hUC)-derived MSCs formed growths in 15% of animals that persisted for up to three weeks, indicating a potential tumourigenicity hazard that warrants further testing.

Hydrogels prepared from low molecular weight gelators (LMWGs) are formed as a result of hierarchical intermolecular interactions between gelators to form fibres, and then further interactions between the self-assembled fibres via physical entanglements, as well as potential branching points. These interactions can allow hydrogels to recover quickly after a high shear rate has been applied. There are currently limited design rules describing which types of morphology or rheological properties are required for a LMWG hydrogel to be used as an effective, printable gel. By preparing hydrogels with different types of fibrous network structures, we have been able to understand in more detail the morphological type which gives rise to a 3D-printable hydrogel using a range of techniques, including rheology, small angle scattering and microscopy.

Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.

SPIONs consisting predominantly of magnetite or maghemite display distinct chemical stability in solution but equivalent imaging properties when in cells.

BACKGROUND AIMS:Tracking cells during regenerative cytotherapy is crucial for monitoring their safety and efficacy. Macrophages are an emerging cell-based regenerative therapy for liver disease and can be readily labeled for medical imaging. A reliable, clinically applicable cell-tracking agent would be a powerful tool to study cell biodistribution. METHODS:Using a recently described chemical design, we set out to functionalize, optimize and characterize a new set of superparamagnetic iron oxide nanoparticles (SPIONs) to efficiently label macrophages for magnetic resonance imaging-based cell tracking in vivo. RESULTS:A series of cell health and iron uptake assays determined that positively charged SPIONs (+16.8 mV) could safely label macrophages more efficiently than the formerly approved ferumoxide (-6.7 mV; Endorem) and at least 10 times more efficiently than the clinically approved SPION ferumoxytol (-24.2 mV; Rienso). An optimal labeling time of 4 h at 25 µg/mL was demonstrated to label macrophages of mouse and human origin without any adverse effects on cell viability whilst providing substantial iron uptake (>5 pg Fe/cell) that was retained for 7 days in vitro. SPION labeling caused no significant reduction in phagocytic activity and a shift toward a reversible M1-like phenotype in bone marrow-derived macrophages (BMDMs). Finally, we show that SPION-labeled BMDMs delivered via the hepatic portal vein to mice are localized in the hepatic parenchyma resulting in a 50% drop in T2* in the liver. Engraftment of exogenous cells was confirmed via immunohistochemistry up to 3 weeks posttransplantation. DISCUSSION:A positively charged dextran-coated SPION is a promising tool to noninvasively track hepatic macrophage localization for therapeutic monitoring.

Perylene bisimides (PBIs) are one example of useful π-conjugated molecules that can be used in optoelectronic devices as n-type materials with strong visible light absorption. PBIs can self-assemble into a range of structures, but it is rare to be able to control the packing such that the same PBI can form either H-type or J-type aggregates. This is important because the conductivity pathways and optoelectronic properties are directly affected by this packing. Here, we show that we can control the packing of a single PBI functionalized with an amino acid by a subtle change in pH. Under one set of conditions, H-type aggregates form a gel when the pH is decreased. At a slightly different set of starting conditions, J-type aggregates are formed, but they cannot form a gel when the pH is lowered. We show that films formed from the self-assembled structures have very different photoconductive properties.

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used as contrast agents for stem cell tracking using magnetic resonance imaging (MRI). The total mass of iron oxide that can be internalised into cells without altering their viability or phenotype is an important criterion for the generation of contrast, with SPIONs designed for efficient labelling of stem cells allowing for an increased sensitivity of detection. Although changes in the ratio of polymer and iron salts in co‐precipitation reactions are known to affect the physicochemical properties of SPIONs, particularly core size, the effects of these synthesis conditions on stem cell labelling and magnetic resonance (MR) contrast have not been established. Here, we synthesised a series of cationic SPIONs with very similar hydrodynamic diameters and surface charges, but different polymer content. We have investigated how the amount of polymer in the co‐precipitation reaction affects core size and modulates not only the magnetic properties of the SPIONs but also their uptake into stem cells. SPIONs with the largest core size and lowest polymer content presented the highest magnetisation and relaxivity. These particles also had the greatest uptake efficiency without any deleterious effect on either the viability or function of the stem cells. However, for all particles internalised in cells, the T2 and T2* relaxivity was independent of the SPION's core size. Our results indicate that the relative mass of iron taken up by cells is the major determinant of MR contrast generation and suggest that the extent of SPION uptake can be regulated by the amount of polymer used in co‐precipitation reactions. Copyright © 2016 John Wiley & Sons, Ltd.

Aims: Despite advances in the surgical management and adjuvant chemotherapy, pancreatic cancer still has a very poor prognosis. Nanotechnology provides a novel approach for targeted drug delivery, possibly reducing the offset effects of systemic chemotherapy. Superparamagnetic iron oxide nanoparticles (SPIONs) are inorganic particles that can be functionalised as targeted drug vehicles with a stealth polymer coating and a specific antibody tag to recognise appropriate antigen expressing cells. Our aim is to develop and investigate the effects of SPIONs capable of targeted cell death of pancreatic cells by release of its chemotherapy payload. Methods: Micellar SPIONs, incorporating a gemcitabine pro-drug, conjugated to isotype and anti-CA19.9 antibodies (NP:CA19.9) were manufactured using self assembly methodology. Cellular uptake was assessed using transmission electron and co-localisation fluorescent microscopy. Antigen expression of cell lines was determined using indirect immunofluorescence. Antibody targeting was assessed using EZ4U cytotoxicity assay in BxPC-3 (Ca-19-9 +ve) and MiaPaCa-2 (Ca-19-9 -ve) cell lines. Results: Endocytosis of the SPIONs was demonstrated by their presence in endosomes and corroborated by colocalisation analysis (Manders coefficient = 0.92). The IC50 of gemcitabine was significantly improved using a nanohybrid attached with specific antibody targeting in BxPC-3 cells (10.245μM vs 5.143μM) [p < 0.001] but absent in MiaPaCa-2 cells (2.322μM vs 2.486μM) [n = 3]. Conclusions: We have developed a novel nanohybrid to target antigen expressing pancreatic cancer cells using a specific antibody tag. When loaded with modified gemcitabine, these SPIONs act as pH-triggered delivery vehicles capable of intracellular drug release. This could reduce off target effects leading to increased chemotherapy agent efficacy and offer the prospect for new treatments in pancreatic cancer.

Tracking stem cells in vivo using non-invasive techniques is critical to evaluate the efficacy and safety of stem cell therapies. Superparamagnetic iron oxide nanoparticles (SPIONs) enable cells to be tracked using magnetic resonance imaging (MRI), but to obtain detectable signal cells need to be labelled with a sufficient amount of iron oxide. For the majority of SPIONs, this can only be obtained with the use of transfection agents, which can adversely affect cell health. Here, we have synthesised a library of dextran-based polymer coated SPIONs with varying surface charge from -1.5 mV to +18.2 mV via a co-precipitation approach and investigated their ability to be directly internalised by stem cells without the need for transfection agents. The SPIONs were colloidally stable in physiological solutions. The crystalline phase of the particles was confirmed with powder X-ray diffraction and their magnetic properties were characterised using SQUID magnetometry and magnetic resonance. Increased surface charge led to six-fold increase in uptake of particles into stem cells and higher MRI contrast, with negligible change in cell viability. Cell tracking velocimetry was shown to be a more accurate method for predicting MRI contrast of stem cells compared to measuring iron oxide uptake through conventional bulk iron quantification.