Translating ultrasound-mediated drug delivery technologies for CNS applications.

Ultrasound enhances drug delivery into the central nervous system (CNS) by opening barriers between the blood and CNS and by triggering release of drugs from carriers. A key challenge in translating setups from in vitro to in vivo settings is achieving equivalent acoustic energy delivery. Multiple devices have now been demonstrated to focus ultrasound to the brain, with concepts emerging to also target the spinal cord. Clinical trials to date have used ultrasound to facilitate the opening of the blood-brain barrier. While most have focused on feasibility and safety considerations, therapeutic benefits are beginning to emerge. To advance translation of these technologies for CNS applications, researchers should standardise exposure protocol and fine-tune ultrasound parameters. Computational modelling should be increasingly used as a core component to develop both in vitro and in vivo setups for delivering accurate and reproducible ultrasound to the CNS. This field holds promise for transformative advancements in the management and pharmacological treatment of complex and challenging CNS disorders.

Encapsulation of the growth factor neurotrophin-3 in heparinised poloxamer hydrogel stabilises bioactivity and provides sustained release.

Poloxamer-based hydrogels show promise to stabilise and sustain the delivery of growth factors in tissue engineering applications, such as following spinal cord injury. Typically, growth factors such as neurotrophin-3 (NT-3) degrade rapidly in solution. Similarly, poloxamer hydrogels also degrade readily and are, therefore, only capable of sustaining the release of a payload over a small number of days. In this study, we focused on optimising a hydrogel formulation, incorporating both poloxamer 188 and 407, for the sustained delivery of bioactive NT-3. Hyaluronic acid blended into the hydrogels significantly reduced the degradation of the gel. We identified an optimal hydrogel composition consisting of 20 % w/w poloxamer 407, 5 % w/w poloxamer 188, 0.6 % w/w NaCl, and 1.5 % w/w hyaluronic acid. Heparin was chemically bound to the poloxamer chains to enhance interactions between the hydrogel and the growth factor. The unmodified and heparin-modified hydrogels exhibited sustained release of NT-3 for 28 days while preserving the bioactivity of NT-3. Moreover, these hydrogels demonstrated excellent cytocompatibility and had properties suitable for injection into the intrathecal space, underscoring their suitability as a growth factor delivery system. The findings presented here contribute valuable insights to the development of effective delivery strategies for therapeutic growth factors for tissue engineering approaches, including the treatment of spinal cord injury.

Generation of direct current electrical fields as regenerative therapy for spinal cord injury: A review.

Electrical stimulation (ES) shows promise as a therapy to promote recovery and regeneration after spinal cord injury. ES therapy establishes beneficial electric fields (EFs) and has been investigated in numerous studies, which date back nearly a century. In this review, we discuss the various engineering approaches available to generate regenerative EFs through direct current electrical stimulation and very low frequency electrical stimulation. We highlight the electrode-tissue interface, which is important for the appropriate choice of electrode material and stimulator circuitry. We discuss how to best estimate and control the generated field, which is an important measure for comparability of studies. Finally, we assess the methods used in these studies to measure functional recovery after the injury and treatment. This work reviews studies in the field of ES therapy with the goal of supporting decisions regarding best stimulation strategy and recovery assessment for future work.

Investigating the influence of ultrasound parameters on ibuprofen drug release from hydrogels.

Hydrogels are promising ultrasound-responsive drug delivery systems. In this study, we investigated how different ultrasound parameters affected drug release and structural integrity of self-healing hydrogels composed of alginate or poloxamers. The effects of amplitude and duty cycle at low frequency (24 kHz) ultrasound stimulation were first investigated using alginate hydrogels at 2% w/v and 2.5% w/v. Increasing ultrasound amplitude increased drug release from these gels, although high amplitudes caused large variations in release and damaged the gel structure. Increasing duty cycle also increased drug release, although a threshold was observed with the lower pulsed 50% duty cycle achieving similar levels of drug release to a continuous 100% duty cycle. Poloxamer-based hydrogels were also responsive to the optimised parameters at low frequency (24 kHz, 20% amplitude, 50% duty cycle for 30 s) and showed similar drug release results to a 2.5% w/v alginate hydrogel. Weight loss studies demonstrated that the 2% w/v alginate hydrogel underwent significant erosion following ultrasound application, whereas the 2.5% w/v alginate and the poloxamer gels were unaffected by application of the same parameters (24 kHz, 20% amplitude, 50% duty cycle for 30 s). The rheological properties of the hydrogels were also unaffected and the FTIR spectra remained unchanged after low frequency ultrasound stimulation (24 kHz, 20% amplitude, 50% duty cycle for 30 s). Finally, high-frequency ultrasound stimulation (1 MHz, 3 W.cm-2, 50% duty cycle) was also trialled; the alginate gels were less responsive to this frequency, while no statistically significant impact on drug release was observed from the poloxamer gels. This study demonstrates the importance of ultrasound parameters and polymer selection in designing ultrasound-responsive hydrogels.

Patternable Gelatin Methacrylate/PEDOT/Polystyrene Sulfonate Microelectrode Coatings for Neuronal Recording.

This manuscript addresses the need for new soft biomaterials that can be fabricated on the surface of microelectrodes to reduce the mechanical mismatch between biological tissues and electrodes and improve the performance at the neural interface. By electrochemical polymerization of poly(3,4-dioxythiophene) (PEDOT)/polystyrene sulfonate (PSS) through a gelatin methacrylate (GelMA) hydrogel, we demonstrate the synthesis of a conducting polymer hydrogel (CPH) to meet the performance criteria of bioelectrodes. The hybrid material can be photolithographically patterned and covalently attached to gold microelectrodes, forming an interpenetrating network, as confirmed by infrared spectroscopy. The GelMA/PEDOT/PSS coatings were found to be reversibly electroactive by cyclic voltammetry and had low impedance compared to bare gold and GelMA-coated microelectrodes. The CPH coatings showed impedance at levels similar to conventional PEDOT/PSS coatings at a frequency of 1000 Hz. CPH exhibited electrochemical stability over 1000 CV cycles, and its performance was maintained over 14 days. Biocompatibility of the CPH coatings was confirmed by primary hippocampal neuronal cultures via a neuronal viability assay. The CPH-coated microelectrode arrays (MEAs) successfully recorded neuronal activity from primary hippocampal neuronal cells. The CPH GelMA/PEDOT/PSS is a highly promising coating material to enhance microelectrode performance at the neural interface.

Development of agarose-gelatin bioinks for extrusion-based bioprinting and cell encapsulation.

Three-dimensional bioprinting continues to advance as an attractive biofabrication technique to employ cell-laden hydrogel scaffolds in the creation of precise, user-defined constructs that can recapitulate the native tissue environment. Development and characterisation of new bioinks to expand the existing library helps to open avenues that can support a diversity of tissue engineering purposes and fulfil requirements in terms of both printability and supporting cell attachment. In this paper, we report the development and characterisation of agarose-gelatin (AG-Gel) hydrogel blends as a bioink for extrusion-based bioprinting. Four different AG-Gel hydrogel blend formulations with varying gelatin concentration were systematically characterised to evaluate suitability as a potential bioink for extrusion-based bioprinting. Additionally, autoclave and filter sterilisation methods were compared to evaluate their effect on bioink properties. Finally, the ability of the AG-Gel bioink to support cell viability and culture after printing was evaluated using SH-SY5Y cells encapsulated in bioprinted droplets of the AG-Gel. All bioink formulations demonstrate rheological, mechanical and swelling properties suitable for bioprinting and cell encapsulation. Autoclave sterilisation significantly affected the rheological properties of the AG-Gel bioinks compared to filter sterilisation. SH-SY5Y cells printed and differentiated into neuronal-like cells using the developed AG-Gel bioinks demonstrated high viability (>90%) after 23 d in culture. This study demonstrates the properties of AG-Gel as a printable and biocompatible material applicable for use as a bioink.

Conducting polymer hydrogels with electrically-tuneable mechanical properties as dynamic cell culture substrates.

Hydrogels are a popular substrate for cell culture due to their mechanical properties closely resembling natural tissue. Stimuli-responsive hydrogels are a good platform for studying cell response to dynamic stimuli. Poly(N-isopropylacrylamide) (pNIPAM) is a thermo-responsive polymer that undergoes a volume-phase transition when heated to 32 °C. Conducting polymers can be incorporated into hydrogels to introduce electrically responsive properties. The conducting polymer, polypyrrole (PPy), has been widely studied as electrochemical actuators due to its electrochemical stability, fast actuation and high strains. We determine the volume-phase transition temperature of pNIPAM hydrogels with PPy electropolymerised with different salts as a film within the hydrogel network. We also investigate the electro-mechanical properties at the transition temperature (32 °C) and physiological temperature (37 °C). We show statistically significant differences in the Young's modulus of the hybrid hydrogel at elevated temperatures upon electrochemical stimulation, with a 5 kPa difference at the transition temperature. Furthermore, we show a three-fold increase in actuation at transition temperature compared to room temperature and physiological temperature, attributed to the movement of ions in/out of the PPy film that induce the volume-phase transition of the pNIPAM hydrogel. Furthermore, cell adhesion to the hybrid hydrogel was demonstrated with mouse articular chondrocytes.

A Subdural Bioelectronic Implant to Record Electrical Activity from the Spinal Cord in Freely Moving Rats.

Bioelectronic devices have found use at the interface with neural tissue to investigate and treat nervous system disorders. Here, the development and characterization of a very thin flexible bioelectronic implant inserted along the thoracic spinal cord in rats directly in contact with and conformable to the dorsal surface of the spinal cord are presented. There is no negative impact on hind-limb functionality nor any change in the volume or shape of the spinal cord. The bioelectronic implant is maintained in rats for a period of 12 weeks. The first subdural recordings of spinal cord activity in freely moving animals are presented; rats are plugged in via a recording cable and allowed to freely behave and move around on a raised platform. Recordings contained multiple distinct voltage waveforms spatially localize to individual electrodes. This device has great potential to monitor electrical signaling in the spinal cord after an injury and in the future, this implant will facilitate the identification of biomarkers in spinal cord injury and recovery, while enabling the delivery of localized electroceutical and chemical treatments.

An interpenetrating and patternable conducting polymer hydrogel for electrically stimulated release of glutamate.

Recent advances in drug delivery have made it possible to release bioactive agents from neural implants specifically to local tissues. Conducting polymer coatings have been explored as a delivery platform in bioelectronics, however, their utility is restricted by their limited loading capacity and stability. This study presents the fabrication of a stable conducting polymer hydrogel (CPH), comprising the hydrogel gelatin methacrylate (GelMA), and conducting polymer polypyrrole (PPy) for the electrically controlled delivery of glutamate (Glu). The hybrid GelMA/PPy/Glu can be photolithographically patterned and covalently bonded to an electrode. Fourier-transform infrared (FTIR) analysis confirmed the interpenetrating nature of PPy through the GelMA hydrogels. Electrochemical polymerisation of PPy/Glu through the GelMA hydrogels resulted in a significant increase in the charge storage capacity as determined by cyclic voltammetry (CV). Long-term electrochemical and mechanical stability was demonstrated over 1000 CV cycles and extracts of the materials were cytocompatible with SH-SY5Y neuroblastoma cell lines. Release of Glu from the CPH was responsive to electrical stimulation with almost five times the amount of Glu released upon constant reduction (-0.6 V) compared to when no stimulus was applied. Notably, GelMA/PPy/Glu was able to deliver almost 14 times higher amounts of Glu compared to conventional PPy/Glu films. The described CPH coatings are well suited in implantable drug delivery applications and compared to conducting polymer films can deliver higher quantities of drug in response to mild electrical stimulus. STATEMENT OF SIGNIFICANCE: Conducting polymer hydrogels (CPH) have been explored for the electrically controlled release of bioactives from implantable devices. Typically, the conducting polymer component does not fully penetrate the hydrogel. We report, for the first time, a completely interpenetrating CPH allowing for the full benefits of the composite material to be realised, the hydrogels provide a reservoir for drug delivery, and conducting polymer renders the material responsive to electrical stimulation for drug release. We report a CPH for the electrically controlled delivery of glutamate (excitatory neurotransmitter) where several-fold more glutamate can be delivered compared to conducting polymer films. The described CPH coatings are well suited for use in bioelectronic devices to deliver large quantities of drug in response to mild electrical stimulus.

Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays.

Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe techniques to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 h improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of our techniques by optimising it further for neurite outgrowth analysis. The presented methods achieve homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.

Stretchable microchannel-on-a-chip: A simple model for evaluating the effects of uniaxial strain on neuronal injury.

BACKGROUND: Axonal injury is a major component of traumatic spinal cord injury (SCI), associated with rapid deformation of spinal tissue and axonal projections. In vitro models enable us to examine these effects and screen potential therapies in a controlled, reproducible manner. NEW METHOD: A customized, stretchable microchannel system was developed using polydimethylsiloxane microchannels. Cortical and spinal embryonic rat neurons were cultured within the microchannel structures, allowing a uniaxial strain to be applied to isolated axonal processes. Global strains of up to 52% were applied to the stretchable microchannel-on-a-chip platform leading to local strains of up to 12% being experienced by axons isolated in the microchannels. RESULTS: Individual axons exposed to local strains between 3.2% and 8.7% developed beading within 30-minutes of injury. At higher local strains of 9.8% and 12% individual axons ruptured within 30-minutes of injury. Axon bundles, or fascicles, were more resistant to rupture at each strain level, compared to individual axons. At lower local strain of 3.2%, axon bundles inside microchannels and neuronal cells near entrances of them progressively swelled and degenerated over a period of 7 days after injury. COMPARISON WITH EXISTING METHOD(S): This method is simple, reliable and reproducible with good control and measurement of injury tolerance and morphological deformations using standard laboratory equipment. By measuring local strains, we observed that axonal injuries occur at a lower strain magnitude and a lower strain rate than previous methods reporting global strains, which may not accurately reflect the true axonal strain. CONCLUSIONS: We describe a novel stretchable microchannel-on-a-chip platform to study the effect of varying local strain on morphological characteristics of neuronal injury.

A Macroscopic Diffusion-Based Gradient Generator to Establish Concentration Gradients of Soluble Molecules Within Hydrogel Scaffolds for Cell Culture.

Concentration gradients of soluble molecules are ubiquitous within the living body and known to govern a number of key biological processes. This has motivated the development of numerous in vitro gradient-generators allowing researchers to study cellular response in a precise, controlled environment. Despite this, there remains a current paucity of simplistic, convenient devices capable of generating biologically relevant concentration gradients for cell culture assays. Here, we present the design and fabrication of a compartmentalized polydimethylsiloxane diffusion-based gradient generator capable of sustaining concentration gradients of soluble molecules within thick (5 mm) and thin (2 mm) agarose and agarose-collagen co-gel matrices. The presence of collagen within the agarose-collagen co-gel increased the mechanical properties of the gel. Our model molecules sodium fluorescein (376 Da) and FITC-Dextran (10 kDa) quickly established a concentration gradient that was maintained out to 96 h, with 24 hourly replenishment of the source and sink reservoirs. FITC-Dextran (40 kDa) took longer to establish in all hydrogel setups. The steepness of gradients generated are within appropriate range to elicit response in certain cell types. The compatibility of our platform with cell culture was demonstrated using a LIVE/DEAD® assay on terminally differentiated SH-SY5Y neurons. We believe this device presents as a convenient and useful tool that can be easily adopted for study of cellular response in gradient-based assays.

Evaluation of parylene derivatives for use as biomaterials for human astrocyte cell patterning.

Cell patterning is becoming increasingly popular in neuroscience because it allows for the control in the location and connectivity of cells. A recently developed cell patterning technology uses patterns of an organic polymer, parylene-C, on a background of SiO2. When cells are cultured on the parylene-C/SiO2 substrate they conform to the underlying parylene-C geometry. Parylene-C is, however, just one member of a family of parylene polymers that have varying chemical and physical properties. In this work, we investigate whether two commercially available mainstream parylene derivatives, parylene-D, parylene-N and a more recent parylene derivative, parylene-HT to determine if they enable higher fidelity hNT astrocyte cell patterning compared to parylene-C. We demonstrate that all parylene derivatives are compatible with the existing laser fabrication method. We then demonstrate that parylene-HT, parylene-D and parylene-N are suitable for use as an hNT astrocyte cell attractive substrate and result in an equal quality of patterning compared to parylene-C. This work supports the use of alternative parylene derivatives for applications where their different physical and chemical properties are more suitable.

Investigating parylene-HT as a substrate for human cell patterning.

We demonstrate, for the first time, how parylene-HT on SiO2 substrates can be used as a human cell patterning platform. We demonstrate this platform with hNT astrocytes, derived from the human NTera2.D1 cell line. We show how hNT astrocytes are attracted to Parylene-HT and repelled by the SiO2 and are shown to adopt a similar morphology as that attained on standard tissue culture polystyrene. Furthermore, parylene-HT was capable of patterning the astrocytes achieving a ratio of 8:1 for cells on parylene compared to SiO2. Thus, as parylene-HT has similar physical properties to parylene-C with the addition of UV and thermal resistance, parylene-HT represents a desirable alternative substrate for human cell patterning.

Investigation of the Ca2+ response of human hNT astrocytes to laser removal of cellular processes.

We demonstrate, for the first time, UV laser ablative microsurgery as a method for pruning astrocytic processes from live hNT astrocytic networks in vitro. Calcium fluorescence imaging was used to evaluate the cellular response to process ablation. The results showed that ablation of astrocyte processes induced an immediate increase in intracellular calcium level which propagated through the cells cytoplasm as a wave originating from the ablation site. The increased intracellular calcium dissipated from the body of the cell but remained high in the vicinity of the ablation site. Cell viability post ablation was confirmed by observing the integrity of the cell membrane. Ablation of astrocytic processes did not compromise cell viability whereas ablation of the cytoplasm using the same laser energy resulted in cell lysis.

Low cost, patterning of human hNT brain cells on parylene-C with UV & IR laser machining.

This paper describes the use of 800nm femtosecond infrared (IR) and 248nm nanosecond ultraviolet (UV) laser radiation in performing ablative micromachining of parylene-C on SiO2 substrates for the patterning of human hNT astrocytes. Results are presented that support the validity of using IR laser ablative micromachining for patterning human hNT astrocytes cells while UV laser radiation produces photo-oxidation of the parylene-C and destroys cell patterning. The findings demonstrate how IR laser ablative micromachining of parylene-C on SiO2 substrates can offer a low cost, accessible alternative for rapid prototyping, high yield cell patterning.