Investigating Internalization of Reporter-Protein-Functionalized Polyhedrin Particles by Brain Immune Cells.

Achieving sustained drug delivery to the central nervous system (CNS) is a major challenge for neurological injury and disease, and various delivery vehicles are being developed to achieve this. Self-assembling polyhedrin crystals (POlyhedrin Delivery System; PODS) are being exploited for the delivery of therapeutic protein cargo, with demonstrated efficacy in vivo. However, to establish the utility of PODS for neural applications, their handling by neural immune cells (microglia) must be documented, as these cells process and degrade many biomaterials, often preventing therapeutic efficacy. Here, primary mouse cortical microglia were cultured with a GFP-functionalized PODS for 24 h. Cell counts, cell morphology and Iba1 expression were all unaltered in treated cultures, indicating a lack of acute toxicity or microglial activation. Microglia exhibited internalisation of the PODS, with both cytosolic and perinuclear localisation. No evidence of adverse effects on cellular morphology was observed. Overall, 20-40% of microglia exhibited uptake of the PODS, but extracellular/non-internalised PODS were routinely present after 24 h, suggesting that extracellular drug delivery may persist for at least 24 h.

In vitro model of traumatic brain injury to screen neuro-regenerative biomaterials.

Penetrating traumatic brain injury (pTBI) causes serious neurological deficits with no clinical regenerative therapies currently available. Tissue engineering strategies using biomaterial-based 'structural bridges' offer high potential to promote neural regeneration post-injury. This includes surgical grade materials which can be repurposed as biological scaffolds to overcome challenges associated with long approval processes and scaleup for human application. However, high throughput, pathomimetic models of pTBI are lacking for the developmental testing of such neuro-materials, representing a bottleneck in this rapidly emergent field. We have established a high throughput and facile culture model containing the major neural cell types which govern biomaterial handling in the central nervous system. We show that induction of traumatic injuries was feasible in the model, with post-injury implantation of a surgical grade biomaterial. Cellular imaging in lesions was achievable using standard epifluorescence microscopy methods. Key pathological features of pTBI were evident in vitro namely immune cell infiltration of lesions/biomaterial, with responses characteristic of cell scarring, namely hypertrophic astrocytes with GFAP upregulation. Based on our observations, we consider the high-throughput, inexpensive and facile pTBI model can be used to study biomaterial 'implantation' and evaluate neural cell-biomaterial responses. The model is highly versatile to test a range of laboratory and clinical grade materials for neural regeneration.

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.