Nerve Growth Factor Sensitisation of TRPA1 on Sensory Neurones

Background: Sensory neurones from the trigeminal nerve innervate the oro-facial region and teeth. Transient receptor potential channels (TRPs) expressed by these neurones are responsible for relaying sensory information such as changes in ambient temperature, mechanical sensations and pain. Study of TRP channel expression and regulation in human sensory neurones therefore merits investigation to improve our understanding of allodynia and hyperalgesia. Objective: The objective of this study was to differentiate human dental pulp stem cells (hDPSCs) towards a neuronal phenotype (peripheral neuronal equivalents; PNEs) and employ this model to study TRP channel sensitisation. Method: hDPSCs were enriched by preferential adhesion to fibronectin, plated on coverslips (thickness 0) coated with poly-l-ornithine and laminin and then differentiated for 7 days in neurobasal A medium with additional supplementation. A whole cell patch clamp technique was used to investigate whether TRP channels on PNE membranes were modulated in the presence of nerve growth factor (NGF). PNEs were treated with NGF for 20 minutes immediately before experimentation and then stimulated for TRPA1 activity using cinnamaldehyde. Peak currents were read at 80 mV and -80 mV and compared to peak currents recorded in untreated PNEs. Data were analysed and plotted using Clampfit9 software (Molecular Devices, Sunnyvale, California, USA). Result: Results showed for the first time that pre-treatment of PNEs by NGF produced significantly larger inward and outward currents demonstrating that TRPA1 channels on PNE membranes were capable of becoming sensitised following treatment with NGF. Conclusion: Sensitisation of TRPA1 by NGF provides evidence of a mechanism for rapid neuronal sensitisation that is independent of TRPA1 gene expression

An in vitro model of nociceptors in human trigeminal nerves

Background: The oro-facial region is densely innervated by the trigeminal nerve, which when stimulated can induce noxious pain sensation and contribute to neurogenic inflammation in local tissues. Recent research on the expression of specialised ion channels on the trigeminal nerve has highlighted the need to undertake more extensive studies on ion channel expression/functionality with the aim of elucidating their role in pain sensations. A major family of such ion channels is the transient receptor potential (TRP) channels which are activated by a wide variety of thermal, mechanical or chemical stimuli and merit investigation as possible druggable targets for future analgesics.
Objective: Study of TRP channel expression and regulation in oro-facial tissues is hindered by the fact that the cell bodies of neurons innervating these tissues are located in the trigeminal ganglion. Using dental pulp stem cells differentiated towards peripheral neuronal equivalents (PNEs), we sought to determine TRP channel expression, functionality and potential modulation by cytokines in this novel model.
Method: Dental pulp stem cells (DPSCs) were grown on substrate-coated tissue culture plates and differentiated towards a neuronal phenotype using neuronal induction media. Quantitative polymerase chain reaction (qPCR) was performed on PNEs +/-cytokine treatment. Ion channel functionality was investigated using whole cell patch clamping.
Result: qPCR analysis showed that PNEs expressed the TRP channels TRPA1, TRPV1, TRPV4 and TRPM8. TRPA1 was the most abundantly expressed TRP channel studied whereas TRPM8 was lowly expressed. TRP channel expression was shown to be regulated by treatment with inflammatory cytokines. Patch clamp studies using specific agonists and antagonists for TRPA1 and TRPV1 showed these channels were functional.
Conclusion: PNEs differentiated from DPSCs provide a suitable model for TRP channel expression, regulation, and sensistisation in oro-facial tissues. This human neuronal model has potential for use in pre-clinical studies of novel analgesics.

Microneedle-mediated delivery of donepezil: Potential for improved treatment options in Alzheimer's disease

Transdermal drug delivery is an attractive route of drug administration, however there are relatively few marketed transdermal products. To increase delivery across the skin, strategies to enhance skin permeability are widely investigated, with microneedles demonstrating particular promise. Hydrogel-forming microneedles are inserted into the skin, and following dissolution of a drug loaded reservoir and movement of the drug through the created channels, the microneedle array is removed intact, and can then be readily and safely discarded. This study presents the formulation and evaluation of an integrated microneedle patch containing the Alzheimer's drug, donepezil hydrochloride. The integrated patch consisted of hydrogel-forming microneedles in combination with a donepezil hydrochloride containing film. Formulation and characterisation of plasticised films, prepared from poly(vinylpyrrolidone) or poly (methyl vinyl ether co-maleic anhydride/acid) (Gantrez(®)) polymers, is presented. Furthermore, in vitro permeation of donepezil hydrochloride across neonatal porcine skin from the patches was investigated, with 854.71 μg ± 122.71 μg donepezil hydrochloride delivered after 24 h, using the optimum patch formulation. Following administration of the patch to an animal model, plasma concentrations of 51.8 ± 17.6 ng/mL were obtained, demonstrating the success of this delivery platform for donepezil hydrochloride.

Transcending epithelial and intracellular biological barriers; a prototype DNA delivery device

Microneedle technology provides the opportunity for the delivery of DNA therapeutics by a non-invasive, patient acceptable route. To deliver DNA successfully requires consideration of both extra and intracellular biological barriers. In this study we present a novel two tier platform; i) a peptide delivery system, termed RALA, that is able to wrap the DNA into nanoparticles, protect the DNA from degradation, enter cells, disrupt endosomes and deliver the DNA to the nucleus of cells ii) a microneedle (MN) patch that will house the nanoparticles within the polymer matrix, breach the skin's stratum corneum barrier and dissolve upon contact with skin interstitial fluid thus releasing the nanoparticles into the skin. Our data demonstrates that the RALA is essential for preventing DNA degradation within the poly(vinylpyrrolidone) (PVP) polymer matrix. In fact the RALA/DNA nanoparticles (NPs) retained functionality when in the MN arrays after 28days and over a range of temperatures. Furthermore the physical strength and structure of the MNs was not compromised when loaded with the NPs. Finally we demonstrated the effectiveness of our MN-NP platform in vitro and in vivo, with systemic gene expression in highly vascularised regions. Taken together this 'smart-system' technology could be applied to a wide range of genetic therapies.