Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

Microneedle-based devices for point-of-care infectious disease diagnostics.

Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis.

Poly(lactic acid)/Carbon Nanotube Composite Microneedle Arrays for Dermal Biosensing.

Minimally invasive, reliable and low-cost in vivo biosensors that enable real-time detection and monitoring of clinically relevant molecules and biomarkers can significantly improve patient health care. Microneedle array (MNA)-based electrochemical sensors offer exciting prospects in this respect, as they can sample directly from the skin. However, their acceptability is dependent on developing a highly scalable and cost-effective fabrication strategy. In this work, we evaluated the potential for poly(lactic acid)/carboxyl-multiwalled carbon nanotube (PLA/ f-MWCNT) composites to be developed into MNAs and their effectiveness for dermal biosensing. Our results show that MNAs are easily made from solvent-cast nanocomposite films by micromolding. A maximum carbon nanotube (CNT) loading of 6 wt % was attained with the current fabrication method. The MNAs were mechanically robust, being able to withstand axial forces up to 4 times higher than necessary for skin insertion. Electrochemical characterization of these MNAs by differential pulse voltammetry (DPV) produced a linear current response toward ascorbic acid, with a limit of detection of 180 µM. In situ electrochemical performance was assessed by DPV measurements in ex vivo porcine skin. This showed active changes characterized by two oxidative peaks at 0.23 and 0.69 V, as a result of the diffusion of phosphate-buffered saline. The diagnostic potential of this waveform was further evaluated through a burn wound model. This showed an attenuated oxidative response at 0.69 V. Importantly, the impact of the burn could be measured at progressive distances from the burn site. Overall, alongside the scalable fabrication strategy, the DPV results promise efficient electrochemical biosensors based on CNT nanocomposite MNAs.

A central role for dityrosine crosslinking of Amyloid-ß in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the deposition of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta (Aß) fibrils. Copper has been implicated to play a role in Alzheimer's disease. Dimers of Aß have been isolated from AD brain and have been shown to be neurotoxic. RESULTS: We have investigated the formation of dityrosine cross-links in Aß42 formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress with elevated copper and shown that dityrosine can be formed in vitro in Aß oligomers and fibrils and that these links further stabilize the fibrils. Dityrosine crosslinking was present in internalized Aß in cell cultures treated with oligomeric Aß42 using a specific antibody for dityrosine by immunogold labeling transmission electron microscopy. Results also revealed the prevalence of dityrosine crosslinks in amyloid plaques in brain tissue and in cerebrospinal fluid from AD patients. CONCLUSIONS: Aß dimers may be stabilized by dityrosine crosslinking. These results indicate that dityrosine cross-links may play an important role in the pathogenesis of Alzheimer's disease and can be generated by reactive oxygen species catalyzed by Cu2+ ions. The observation of increased Aß and dityrosine in CSF from AD patients suggests that this could be used as a potential biomarker of oxidative stress in AD.