Advancements in the co-formulation of biologic therapeutics

Biologic therapeutics are the medicines of the future and are destined to transform the approaches by which the causes and symptoms of diseases are cured and alleviated. These approaches will be accelerated through the development of novel strategies that target multiple pharmacologically active sites using a combination of different biologics, or mixtures of biologics and small molecule therapeutics. However, for this potential to be realised, advancements in co-formulation strategies for biologic therapeutics must be established. This review describes the current and emerging developments within this field and highlights the challenges and potential solutions, that will pave-the-way towards their clinical translation

Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of D-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements

New generation of bioreactors that advance extracellular matrix modelling and tissue engineering

Bioreactors hold a lot of promise for tissue engineering and regenerative medicine applications. They have multiple uses including cell cultivation for therapeutic production and for in vitro organ modelling to provide a more physiologically relevant environment for cultures compared to conventional static conditions. Bioreactors are often used in combination with scaffolds as the nutrient flow can enhance oxygen and diffusion throughout the 3D constructs to prevent the formation of necrotic cores. A variety of scaffolds have been fabricated to achieve a structural architecture that mimic native extracellular matrix. Future developments of in vitro models will incorporate the ability to non-invasively monitor the cellular microenvironment to enhance the understanding of in vitro conditions. This review details current advancements in bioreactor and scaffold systems and provides insight on how in vitro models can be augmented for future biomedical applications

Facile approach to generating polymeric nanoarrays containing populations of nanoparticles

The production of nanoarrays containing a population of entrapped, heterogeneous nanoparticles is reported. The nanoarray consists of a nanoporous film with pores of diameter 60-180 nm, formed from the phase separation of two immiscible polymers: polystyrene (PS) and polymethylmethacrylate coated onto glass or silicon wafer. Nanoparticles of PS (120 nm) and silica (90 nm) were deposited into the nanoporous films to generate the nanoarray containing a mixed population of nanoparticles

Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds

Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment

Enhanced uptake of nanoparticle drug carriers via a thermoresponsive shell enhances cytotoxicity in a cancer cell line

Polymer particles consisting of a biodegradable poly[lactide-co-glycolide] (PLGA) core and a thermoresponsive shell have been formulated to encapsulate the dye rhodamine 6G and the potent cytotoxic drug paclitaxel. Cellular uptake of these particles is significantly enhanced above the thermal transition temperature (TTT) of the polymer shells in the human breast carcinoma cell line MCF-7 as determined by flow cytometry and fluorescence microscopy. Paclitaxel-loaded particles display reduced and enhanced cytotoxicity below and above the TTT respectively compared to unencapsulated drug. The data suggests a potential route to enhanced anti-cancer efficacy through temperature-mediated cell targeting.© The Royal Society of Chemistry 2013

Improving effect of metal and oxide nanoparticles encapsulated in porous silica on fermentative biohydrogen production by Clostridium butyricum.

peer reviewedaudience: researcher, professional, student, popularizationThis paper investigated the enhancement effect of nanometre-sized metallic (Pd, Ag and Cu) or metallic oxide (Fe(x)O(y)) nanoparticles on fermentative hydrogen production from glucose by a Clostridium butyricum strain. These nanoparticles (NP) of about 2-3nm were encapsulated in porous silica (SiO(2)) and were added at very low concentration (10(-6)molL(-1)) in batch hydrogen production test. The cultures containing iron oxide NP produced 38% more hydrogen with a higher maximum H(2) production rate (HPR) of 58% than those without NP or with silica particles only. The iron oxide NP were used in a 2.5L sequencing-batch reactor and showed no significant effect on the yields (established at 2.2mol(hydrogen)mol(glucose)(-1)) but an improvement of the HPR (+113%, reaching a maximum HPR of 86mL(hydrogen)L(-1)h(-1)). These results suggest an improvement of the electron transfers trough some combinations between enzymatic activity and inorganic materials.Etude de la production d'hydrogène par les bactéries anaérobies chimiotrophes (dark-fermentation

Fluorescent nanosensors reveal dynamic pH gradients during biofilm formation

Understanding the dynamic environmental microniches of biofilms will permit us to detect, manage and exploit these communities. The components and architecture of biofilms have been interrogated in depth; however, little is known about the environmental microniches present. This is primarily because of the absence of tools with the required measurement sensitivity and resolution to detect these changes. We describe the application of ratiometric fluorescent pH-sensitive nanosensors, as a tool, to observe physiological pH changes in biofilms in real time. Nanosensors comprised two pH-sensitive fluorophores covalently encapsulated with a reference pH-insensitive fluorophore in an inert polyacrylamide nanoparticle matrix. The nanosensors were used to analyse the real-time three-dimensional pH variation for two model biofilm formers: (i) opportunistic pathogen Pseudomonas aeruginosa and (ii) oral pathogen Streptococcus mutans. The detection of sugar metabolism in real time by nanosensors provides a potential application to identify therapeutic solutions to improve oral health

Optically excited nanoscale ultrasonic transducers

In order to work at higher ultrasonic frequencies, for instance, to increase the resolution, it is necessary to fabricate smaller and higher frequency transducers. This paper presents an ultrasonic transducer capable of being made at a very small size and operated at GHz frequencies. The transducers are activated and read optically using pulsed lasers and without physical contact between the instrumentation and the transducer. This removes some of the practical impediments of traditional piezoelectric architectures (such as wiring) and allows the devices to be placed immediately on or within samples, reducing the significant effect of attenuation which is very strong at frequencies above 1 GHz. The transducers presented in this paper exploit simultaneous optical and mechanical resonances to couple the optical input into ultrasonic waves and vice versa. This paper discusses the mechanical and optical design of the devices at a modest scale (a few lm) and explores the scaling of the transducers toward the sub-micron scale. Results are presented that show how the transducers response changes depending on its local environment and how the resonant frequency shifts when the transducer is loaded by a printed protein sample

Enhanced distance-dependent fluorescence quenching using size tuneable core shell silica nanoparticles

Silica nanoparticles (SNPs) have been used as favoured platforms for sensor, drug delivery and biological imaging applications, due to their ease of synthesis, size-control and bespoke physico-chemical properties. In this study, we have developed a protocol for the synthesis of size-tuneable SNPs, with diameters ranging from 20 nm to 500 nm, through the optimisation of experimental components required for nanoparticle synthesis. This protocol was also used to prepare fluorescent SNPs, via covalent linkages of fluorophores, to the nanoparticle matrix using 3-aminopropyltriethoxysilane (APTES). This enabled the fabrication of ratiometric, fluorescent, pH-sensitive nanosensors (75 nm diameter) composed SNPs covalently linked to two pH-sensitive fluorescent dyes Oregon Green (OG) and 5(6)-carboxyfluorescein (FAM) and a reference fluorescent dye 5-(6)-carboxytetramethylrhodamine (TAMRA), extending the dynamic range of measurement from pH 3.5 to 7.5. In addition, size-tuneable, core-shell SNPs, covalently linked to a fluorescent TAMRA core were synthesised to investigate distance-dependant fluorescence quenching between TAMRA and black hole quencher 2 (BHQ2®) using nanometre-sized silica shells as physical spacers. The results showed a significant fluorescence quenching could be observed over greater distances than that reported for the classical distance-dependent molecular fluorescence quenching techniques, e.g. the Förster (fluorescence) resonance energy transfer (FRET). The methods and protocols we have detailed in this manuscript will provide the basis for the reproducible production of size tunable SNPs, which will find broad utility in the development of sensors for biological applications