Less is more: Investigating the influence of cellular nanoparticle load on transfection outcomes in neural cells

Genetic engineering of cell transplant populations offers potential for delivery of neurotherapeutic factors to modify the regenerative microenvironment of the injured spinal cord. The use of magnetic nanoparticle (MNP) based vectors has reduced the traditional reliance on viral methods and their associated obstacles in terms of scale up and safety. Studies utilising magnetic assistive platforms for MNP‐mediated gene delivery have found transfection efficiency in astrocytes (a major transplant and homeostatic neural cell type) to be both frequency and amplitude‐dependent. It is widely assumed that increased intracellular particle load will enhance transfection efficiency in a cell population. Therefore, we tested repeat delivery of MNP:plasmid complexes in conjunction with oscillating magnetic field parameters‐ a process termed ‘magneto‐multifection’‐ in astrocytes of primary origin in an attempt to enhance transfection levels. We show i) levels of transfection using magneto‐multifection equal that seen with viral methods; ii) reporter protein expression using two reporter plasmids shows a diverse profile of single/dual transfected cells with implications for delivery of a ‘cocktail’ of neurotherapeutic proteins and, iii) contrary to expectation, an inverse relationship exists between particle load and reporter protein expression

Development of a nanomaterial bio-screening platform for neurological applications

Nanoparticle platforms are being intensively investigated for neurological applications. Current biological models used to identify clinically relevant materials have major limitations, e.g. technical/ethical issues with live animal experimentation, failure to replicate neural cell diversity, limited control over cellular stoichiometries and poor reproducibility. High-throughput neuro-mimetic screening systems are required to address these challenges. We describe an advanced multicellular neural model comprising the major non-neuronal/glial cells of the central nervous system (CNS), shown to account for ~99.5% of CNS nanoparticle uptake. This model offers critical advantages for neuro-nanomaterials testing while reducing animal use: one primary source and culture medium for all cell types, standardized biomolecular corona formation and defined/reproducible cellular stoichiometry. Using dynamic time-lapse imaging, we demonstrate in real-time that microglia (neural immune cells) dramatically limit particle uptake in other neural subtypes (paralleling post-mortem observations after nanoparticle injection in vivo), highlighting the utility of the system in predicting neural handling of biomaterials

Nanoengineering Neural Stem Cells on Biomimetic Substrates Using Magnetofection Technology

Tissue engineering studies are witnessing a major paradigm shift to cell culture on biomimetic materials that replicate native tissue features from which the cells are derived. Few studies have been performed in this regard for neural cells, particularly in nanomedicine. For example, platforms such as magnetic nanoparticles (MNPs) have proven efficient as multifunctional tools for cell tracking and genetic engineering of neural transplant populations. However, as far as we are aware, all current studies have been conducted using neural cells propagated on non-neuromimetic substrates that fail to represent the mechano-elastic properties of brain and spinal cord microenvironments. Accordingly, it can be predicted that such data is of less translational and physiological relevance than that derived from cells grown in neuromimetic environments. Therefore, we have performed the first test of magnetofection technology (enhancing MNP delivery using applied magnetic fields with significant potential for therapeutic application) and its utility in genetically engineering neural stem cells (NSCs; a population of high clinical relevance) propagated in biomimetic hydrogels. We demonstrate magnetic field application safely enhances MNP mediated transfection of NSCs grown as 3D spheroid structures in collagen which more closely replicates the intrinsic mechanical and structural properties of neural tissue than routinely used hard substrates. Further, as it is well known that MNP uptake is mediated by endocytosis we also investigated NSC membrane activity grown on both soft and hard substrates. Using high resolution scanning electron microscopy we were able to prove that NSCs display lower levels of membrane activity on soft substrates compared to hard, a finding which could have particular impact on MNP mediated engineering strategies of cells propagated in physiologically relevant systems

Electrophysiological Assessment of Primary Cortical Neurons Genetically Engineered using Iron Oxide Nanoparticles

The development of safe technologies to genetically modify neurons is of great interest in regenerative neurology, for both translational and basic science applications. Such approaches have conventionally been heavily reliant on viral transduction methods, which have safety and production limitations. Magnetofection (magnet-assisted gene transfer using iron oxide nanoparticles as vectors) has emerged as a highly promising non-viral alternative for safe and reproducible genetic modification of neurons. Despite the high potential of this technology, there is an important gap in our knowledge of the safety of this approach, namely, whether it alters neuronal function in adverse ways, such as by altering neuronal excitability and signaling. We have investigated the effects of magnetofection in primary cortical neurons by examining neuronal excitability using the whole cell patch clamp technique. We found no evidence that magnetofection alters the voltage-dependent sodium and potassium ionic currents that underpin excitability. Our study provides important new data supporting magnetofection as a safe technology for bioengineering of neuronal cell populations

Deploying clinical grade magnetic nanoparticles with magnetic fields to magnetolabel neural stem cells in adherent versus suspension cultures

Neural stem cells (NSCs) have a high therapeutic potential for patients with neurological disease/injury given their neuroregenerative and immunomodulatory capabilities. In recent years, magnetic nanoparticles (MNPs) have been used as contrast agents in translational studies, to track transplanted NSCs using non-invasive magnetic resonance imaging (MRI). However, NSC uptake of MNPs is inherently low in the absence of chemical/biological uptake enhancing strategies such as cell targeting peptides and transfection agents – approaches which may be cytotoxic and alter cellular physiology. By contrast, physical delivery strategies relying on magnetic assistive methods can safely enhance MNP uptake into multiple neural cell types. The utility of this approach has been demonstrated for gene delivery grade particles but their application in enhancing ‘magnetolabelling’ with clinical grade contrast agents has never been evaluated. Here, we show that applied oscillating magnetic fields can safely enhance the uptake of a clinical grade MNP (Lumirem/Ferumoxsil) into NSCs propagated as neurospheres (suspension cultures, the preferred format for transplantation) but offer limited benefit for monolayer (adherent) cultures. This physical delivery method therefore has potential to facilitate cell labelling for clinical therapies

Differences in magnetic particle uptake by CNS neuroglial subclasses: implications for neural tissue engineering

AIM: To analyze magnetic particle uptake and intracellular processing by the four main non-neuronal subclasses of the CNS: oligodendrocyte precursor cells; oligodendrocytes; astrocytes; and microglia. MATERIALS & METHODS: Magnetic particle uptake and processing were studied in rat oligodendrocyte precursor cells and oligodendrocytes using fluorescence and transmission electron microscopy, and the results collated with previous data from rat microglia and astrocyte studies. All cells were derived from primary mixed glial cultures. RESULTS: Significant intercellular differences were observed between glial subtypes: microglia demonstrate the most rapid/extensive particle uptake, followed by astrocytes, with oligodendrocyte precursor cells and oligodendrocytes showing significantly lower uptake. Ultrastructural analyses suggest that magnetic particles are extensively degraded in microglia, but relatively stable in other cells. CONCLUSION: Intercellular differences in particle uptake and handling exist between the major neuroglial subtypes. This has important implications for the utility of the magnetic particle platform for neurobiological applications including genetic modification, transplant cell labeling and biomolecule delivery to mixed CNS cell populations

Identifying the cellular targets of drug action in the central nervous system following corticosteroid therapy

Corticosteroid (CS) therapy is used widely in the treatment of a range of pathologies, but can delay production of myelin, the insulating sheath around central nervous system nerve fibers. The cellular targets of CS action are not fully understood, that is, "direct" action on cells involved in myelin genesis [oligodendrocytes and their progenitors the oligodendrocyte precursor cells (OPCs)] versus "indirect" action on other neural cells. We evaluated the effects of the widely used CS dexamethasone (DEX) on purified OPCs and oligodendrocytes, employing complementary histological and transcriptional analyses. Histological assessments showed no DEX effects on OPC proliferation or oligodendrocyte genesis/maturation (key processes underpinning myelin genesis). Immunostaining and RT-PCR analyses show that both cell types express glucocorticoid receptor (GR; the target for DEX action), ruling out receptor expression as a causal factor in the lack of DEX-responsiveness. GRs function as ligand-activated transcription factors, so we simultaneously analyzed DEX-induced transcriptional responses using microarray analyses; these substantiated the histological findings, with limited gene expression changes in DEX-treated OPCs and oligodendrocytes. With identical treatment, microglial cells showed profound and global changes post-DEX addition; an unexpected finding was the identification of the transcription factor Olig1, a master regulator of myelination, as a DEX responsive gene in microglia. Our data indicate that CS-induced myelination delays are unlikely to be due to direct drug action on OPCs or oligodendrocytes, and may occur secondary to alterations in other neural cells, such as the immune component. To the best of our knowledge, this is the first comparative molecular and cellular analysis of CS effects in glial cells, to investigate the targets of this major class of anti-inflammatory drugs as a basis for myelination deficits

Developing A New Strategy for Delivery of Neural Transplant Populations using Precursor Cell Sprays and Specialised Cell Media

Neural precursor/stem cell transplantation therapies promote regeneration in neurological injuries, but current cell delivery methods have drawbacks. These include risks with surgical microinjection (e.g., hemorrhage, embolism) and high cell loss with systemic delivery/passage through fine gauge needles. Aerosolized cell delivery offers significant benefits including rapid and minimally invasive cell delivery, and ease of delivery to end users. To develop this approach, it is necessary to prove that 1) aerosolization does not have detrimental effects on transplant cells and 2) suitable media can be identified to support cell delivery. To achieve these aims, cells are sprayed using a commercial spray device or stored in Hibernate-A, a CO2-independent nutrient solution. Histological assessments consist of cell viability analysis, immunocytochemistry, and EdU labeling. It is shown that a major neural precursor transplant population-oligodendrocyte precursor cells (OPCs)-survive following aerosolized delivery and retain their capacity for proliferation and differentiation (key to their repair function). Hibernate-A can support OPCs' survival without specialized maintenance conditions, with no detrimental impact on cell fate. It is considered that this data supports the concept of a novel class of advanced medical spray devices to facilitate transport and delivery of transplant populations in neural cell therapy