Correlative fluorescence and atomic force microscopy to advance the bio-physical characterisation of co-culture of living cells.

An understanding of the cell mechanical properties involved in numerous cellular processes including cell division, cell migration/invasion, and cell morphology, is crucial in developing and informing cell physiology and function. Atomic force microscopy (AFM) offers a powerful biophysical technique that facilitates the imaging of living cells under physiological buffer conditions. However, AFM in isolation cannot discriminate between different cell types within heterogeneous samples for example in a solid biopsy. The current studies demonstrate the potential of AFM in combination with correlative fluorescence optical sectioning microscopy for live cell imaging. Furthermore, this work establishes the advantage of fluorescence-AFM imaging to distinguish and analyse single-cell bio-physical properties in mixed human cell populations, in real-time. Critically, our results show that correlative fluorescence-AFM imaging allows the simultaneous co-localised detection of fluorescence coupled with nano-mechanical mapping. The findings from this work contribute to the promotion and dissemination of correlative multimodal imaging in life sciences, providing a platform for further investigations in biological and pre-clinical research.

Live-imaging of Bioengineered Cartilage Tissue using Multimodal Non-linear Molecular Imaging.

Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.

Raman spectroscopy and coherent anti-Stokes Raman scattering imaging: prospective tools for monitoring skeletal cells and skeletal regeneration.

The use of skeletal stem cells (SSCs) for cell-based therapies is currently one of the most promising areas for skeletal disease treatment and skeletal tissue repair. The ability for controlled modification of SSCs could provide significant therapeutic potential in regenerative medicine, with the prospect to permanently repopulate a host with stem cells and their progeny. Currently, SSC differentiation into the stromal lineages of bone, fat and cartilage is assessed using different approaches that typically require cell fixation or lysis, which are invasive or even destructive. Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy present an exciting alternative for studying biological systems in their natural state, without any perturbation. Here we review the applications of Raman spectroscopy and CARS imaging in stem-cell research, and discuss the potential of these two techniques for evaluating SSCs, skeletal tissues and skeletal regeneration as an exemplar.

Solid Lipid Nanoparticles: A Potential Multifunctional Approach towards Rheumatoid Arthritis Theranostics.

Rheumatoid arthritis (RA) is the most common joint-related autoimmune disease and one of the most severe. Despite intensive investigation, the RA inflammatory process remains largely unknown and finding effective and long lasting therapies that specifically target RA is a challenging task. This study proposes a different approach for RA therapy, taking advantage of the new emerging field of nanomedicine to develop a targeted theranostic system for intravenous administration, using solid lipid nanoparticles (SLN), a biocompatible and biodegradable colloidal delivery system, surface-functionalized with an anti-CD64 antibody that specifically targets macrophages in RA. Methotrexate (MTX) and superparamagnetic iron oxide nanoparticles (SPIONs) were co-encapsulated inside the SLNs to be used as therapeutic and imaging agents, respectively. All the formulations presented sizes under 250 nm and zeta potential values lower than -16 mV, suitable characteristics for intravenous administration. Transmission electron microscopy (TEM) photographs indicated that the SPIONs were encapsulated inside the SLN matrix and MTX association efficiency values were higher than 98%. In vitro studies, using THP-1 cells, demonstrated that all formulations presented low cytotoxicity at concentrations lower than 500 µg/mL. It was proven that the proposed NPs were not cytotoxic, that both a therapeutic and imaging agent could be co-encapsulated and that the SLN could be functionalized for a potential future application such as anti-body specific targeting. The proposed formulations are, therefore, promising candidates for future theranostic applications.

Tracking adipogenic differentiation of skeletal stem cells by label-free chemically selective imaging.

Coherent anti-Stokes Raman scattering (CARS) is a chemically selective label-free imaging technique which is rapidly emerging as a powerful alternative to conventional microscopy in biomedicine. The strength of this imaging approach is the provision of rapid insight into chemical distribution especially of small biomolecules such as lipids. The label-free, non-destructive and non-invasive nature of CARS lends itself for use with stem cells, as labelling or staining will render them otherwise unsuitable for therapy. Isolation, enrichment and characterisation of skeletal stem cells (SSCs) and their progeny is of tremendous significance in regenerative medicine. However, SSCs differentiation into bone, cartilage or fat cell types, is currently assessed using several invasive and, typically, destructive methodologies. Thus, CARS presents an exciting alternative to interrogate the differentiation of SSCs in their natural state. In the current study, we have examined the adipogenic differentiation of SSCs over time using CARS imaging and verified the observed differentiation using molecular analysis of gene expression as well as compared the results to conventional Oil Red O lipid staining. We find that the CARS analysis provides an enhanced resolution and definition of lipid droplets, detectable as early as 24 hours and 72 hours after adipogenic induction. Quantification of the CARS image data sets also showed a change in lipid droplet size distribution during the course of adipogenesis over 14 days. Furthermore, CARS provided a superior and facile approach to monitor changes in SSCs as a result of chemical modulation of adipogenic differentiation. The current studies pave the way for the use of CARS as a powerful chemical imaging tool in therapeutics, regenerative medicine and skeletal stem cell biology.

A new topical formulation for psoriasis: development of methotrexate-loaded nanostructured lipid carriers.

The aim of the present work was to develop and assess the potential of nanostructured lipid carriers (NLCs) loaded with methotrexate as a new approach for topical therapy of psoriasis. Methotrexate-loaded NLCs were prepared via a modified hot homogenization combined with ultrasonication techniques using either polysorbate 60 (P60) or 80 (P80) as surfactants. The produced NLCs were within the nanosized range (274-298 nm) with relatively low polydispersity index (<0.25) and zeta potential values around -40 mV. NLCs demonstrated storage stability at 25°C up to 28 days. The entrapment efficiency of methotrexate in NLC-P60 and -P80 was ∼65%. Cryo-SEM images showed the spherical shape of the empty and methotrexate-loaded NLCs. FT-IR confirmed methotrexate presence within the NLCs. The in vitro release of methotrexate from the NLCs followed a fast release pattern reaching ∼70% in 2h. In vitro skin penetration study demonstrated that methotrexate-loaded NLCs-P60 had higher skin penetration when compared to free methotrexate, suggesting a significant role of drug-nanocarriers on topical administration. Methotrexate-loaded NLC-P60 provided drug fluxes of 0.88 µg/cm(2)/h, higher (P<0.001) than with the free drug (control, 0.59 µg/cm(2)/h). The results indicate the potential of NLCs for the delivery of methotrexate to topical therapy of psoriasis.

Co-association of methotrexate and SPIONs into anti-CD64 antibody-conjugated PLGA nanoparticles for theranostic application.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease with severe consequences for the quality of life of sufferers. Regrettably, the inflammatory process involved remains unclear, and finding successful therapies as well as new means for its early diagnosis have proved to be daunting tasks. As macrophages are strongly associated with RA inflammation, effective diagnosis and therapy may encompass the ability to target these cells. In this work, a new approach for targeted therapy and imaging of RA was developed based on the use of multifunctional polymeric nanoparticles. METHODS: Poly(lactic-co-glycolic acid) nanoparticles were prepared using a single emulsion-evaporation method and comprisaed the co-association of superparamagnetic iron oxide nanoparticles (SPIONs) and methotrexate. The nanoparticles were further functionalized with an antibody against the macrophage-specific receptor, CD64, which is overexpressed at sites of RA. The devised nanoparticles were characterized for mean particle size, polydispersity index, zeta potential, and morphology, as well as the association of SPIONs, methotrexate, and the anti-CD64 antibody. Lastly, the cytotoxicity of the developed nanoparticles was assessed in RAW 264.7 cells using standard MTT and LDH assays. RESULTS: The nanoparticles had a mean diameter in the range of 130-200 nm and zeta potential values ranging from -32 mV to -16 mV. Association with either methotrexate or SPIONs did not significantly affect the properties of the nanoparticles. Conjugation with the anti-CD64 antibody, in turn, caused a slight increase in size and surface charge. Transmission electron microscopy confirmed the association of SPIONs within the poly(lactic-co-glycolic acid) matrix. Both anti-CD64 and methotrexate association were confirmed by Fourier transform infrared spectroscopy, and quantified yielding values as high as 36% and 79%, respectively. In vitro toxicity studies confirmed the methotrexate-loaded nanosystem to be more effective than the free drug. CONCLUSION: Multifunctional anti-CD64-conjugated poly(lactic-co-glycolic acid) nanoparticles for the combined delivery of methotrexate and SPIONs were successfully prepared and characterized. This nanosystem has the potential to provide a new theranostic approach for the management of RA.