The functional and structural effects of an amphipathic pH responsive biopolymer: A comprehensive study in osteosarcoma cells

The use of amphipathic pH-responsive polymers to transport sugars across cell membranes has been shown to improve the cryopreservation of mammalian cells. However, the effect of these polymers on cell viability and morphology has not yet been thoroughly analysed. In this study, the objective was to investigate the functional and structural effects of an amphipathic polymer, PP-50, upon an osteosarcoma cell line (SAOS-2). Cellular growth curves confirmed similar doubling times in PP-50 treated cells and untreated cells. PP-50 concentrations (10-2000 μg/ml) were well tolerated by cells after 2, 24 and 48 hours of incubation, as measured by mitochondrial enzyme activity and lactate dehydrogenase release. Analysis by flow cytometry demonstrated that PP-50 did not induce any additional apoptosis or necrosis in polymer treated cells compared to untreated cells. Phase contrast, confocal and transmission electron microscopy analysis of PP-50 treated cells revealed no signs of morphological changes, with cells maintaining their nucleus size and membrane integrity after treatment. PP-50 was shown to have no negative cellular effects, which is a critical characteristic of polymers towards use in cryopreservation and biomedical applications. In addition, the methodology and protocols established in this work provided a robust and comprehensive analysis of PP-50’s cellular effects, thus making them well suited for determining these effects in mammalian cells exposed to other polymers intended for biological applications.Mercado S. A. wishes to thank the Agency for Science and Technology Research, CONICYT (Chile), for the provision of a studentship during the tenure of which this work was conducted. The authors would like to thank Nigel Miller, Department of Pathology, Dr Jeremy Skepper, Department of Anatomy, Radu Lazar, Dr Hassan Rahmoune and Dr Krishna Mahbubani, Department of Chemical Engineering and Biotechnology, all from the University of Cambridge.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.eurpolymj.2015.11.02

Increased cryosurvival of osteosarcoma cells using an amphipathic pH-responsive polymer for trehalose uptake.

Amphipathic pH-responsive polymers have shown to increase the permeability of cell membranes to trehalose hence improving the cryopreservation of mammalian cells. However, the trafficking of both the polymer and trehalose across the cell membrane has not yet been thoroughly analysed. The objective of this study was to investigate the effect on cryopreservation of the trafficking of the disaccharide trehalose along PP-50, an amphipathic polymer, through an osteosarcoma cell line (SAOS-2). Confocal microscopy analysis confirmed the presence of intracellular labelled trehalose only when incubated in the presence of PP-50. Further analysis confirmed that both trehalose and PP-50 localised in the cytoplasm, accumulated mainly in the perinuclear area. Quantitative analysis of the colocalisation between trehalose and PP-50 showed Pearson and Manders coefficients of 0.862 ± 0.008 and 0.766 ± 0.033, respectively, suggesting a high degree of intracellular colocalisation between these molecules. Cryopreserved cells pre-incubated with trehalose and PP-50 showed increased cryosurvival when compared with cells pre-incubated in the absence of the polymer. PP-50 showed to be directly involved in the uptake of trehalose, a critical characteristic towards use in cryopreservation and biomedical applications.Agency for Science and Technology Research, CONICYT (Chile) (Studentship)This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.cryobiol.2016.08.00

Antioxidant modified amphiphilic polymer improves intracellular cryoprotectant delivery and alleviates oxidative stress in HeLa cells

The design and synthesis of a dual-function, cell permeating polymer with an antioxidative property is described and its use for the intracellular delivery of the cryoprotectant trehalose into HeLa cells is demonstrated. The polymer, PVitE-25, was created by grafting the water insoluble hydrophobic antioxidant (±)-α-Tocopherol (vitamin E) onto pendant carboxylate groups of a biocompatible cell permeating polymer, poly (L-lysine iso-phthalamide) (PLP). The modification increased the intracellular delivery efficiency of the polymer and also introduced an antioxidative effect that was able to reduce 85% of reactive oxygen species (ROS) in HeLa cells incubated with 1 mM hydrogen peroxide (H2O2), as determined by 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) probe. PVitE-25 was also used to load the cropreservative trehalose into HeLa cells prior to freezing such that the level of cell viability measured 48 hours after cell revival was comparable to that observed with a standard Me2SO-based cryopreservation protocol. This is the first report of a synthetic intracellular delivery system that facilitates the intracellular delivery of the cryoprotectant, trehalose, and mitigates oxidative stress during the freeze thaw cycle of cryopreservation

The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pH-responsive pseudo-peptides

pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly(l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL(−1), the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly(l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications

The intracellular fate of an amphipathic pH-responsive polymer: Key characteristics towards drug delivery.

Biopolymers have become important drug delivery systems for therapeutic molecules by enhancing their accessibility and efficacy intracellularly. However, the transport of these drugs across the cell membrane and their release into the cytosol remain a challenge. The trafficking of poly (l-lysine iso-phthalamide) grafted with phenylalanine (PP-50) was investigated using an osteosarcoma cell line (SAOS-2). Colocalisation of this amphipathic biopolymer with endocytosis tracers, such as transferrin and lactosylceramide, suggested that PP-50 is partially internalised by both clathrin and caveolin-mediated endocytosis. Macropinocytosis was also investigated, but a smaller correlation was found between this mechanism and PP-50 transport. A significant decrease in polymer-mediated calcein uptake was found when cells were pre-incubated with endocytosis inhibitors, suggesting also the use of a combination of mechanisms for cell internalisation. In addition, PP-50 colocalisation with endosome and lysosome pathway markers showed that the polymer was able to escape the endolysosomal compartment before maturation. This is a critical characteristic of a biopolymer towards use as drug delivery systems and biomedical applications.Agency for Science and Technology Research, CONICYT (Chile) (Studentship)This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.msec.2016.08.00

Mimicking the impact of infant tongue peristalsis on behaviour of solid oral dosage forms administered during breastfeeding

An in vitro simulation system was developed to study the effect of an infant’s peristaltic tongue motion during breastfeeding on oral rapidly disintegrating tablets in the mouth, for use in rapid product candidate screening. These tablets are being designed for use inside a modified nipple shield worn by a mother during breastfeeding, a proposed novel platform technology to administer drugs and nutrients to breastfeeding infants. In this study, the release of a model compound, sulforhodamine B, from tablet formulations was studied under physiologically relevant forces induced by compression and rotation of a tongue mimic. The release profiles of the sulforhodamine B in flowing deionised water were found to be statistically different using 2-way ANOVA with matching, when tongue mimic rotation was introduced for two compression levels representing two tongue strengths (P=0.0013 and P<0.0001 for the lower and higher compression settings, respectively). Compression level was found to be a significant factor for increasing model compound release at rotational rates representing non-nutritive breastfeeding (P=0.0162). This novel apparatus is the first to simulate the motion and pressures applied by the tongue, and could be used in future infant oral product development

The development of a weak anion micro-capillary film for protein chromatography.

In this study, the surface of a microporous walled micro-capillary film (MMCF) was modified into a weak anion exchanger by coupling cyanuric chloride and 2-diethylaminoethylamine (DEAE) to the ethylene-vinyl alcohol (EVOH) matrix. Fourier transform infrared spectroscopy (FTIR) measurements of modified and unmodified MMCFs confirmed the addition of a triazine ring and DEAE onto the membrane. Binding of bovine serum albumin (BSA) at pH 7.2 was found to follow a Langmuir isotherm with a maximum equilibrium binding of 12.4mg BSA/mL adsorbent and 8.2mg BSA/mL adsorbent under static and flow conditions, respectively. The ion exchange capacity, determined by Mohr's titration of chlorine atoms displaced from the functionalised surface, was found to be 195±21μmol Cl-/mL of adsorber, comparable to commercial ion exchangers. BSA adsorption onto the ion exchanger was strongly pH-dependant, with an observed reduction in binding above pH 8.2. Frontal experiments of a BSA (5mg/mL) and lysozyme (5mg/mL) mixture demonstrated successful separation of BSA from lysozyme at more than 97% purity as verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separation between similarly charged anionic molecules was also achieved using BSA (5mg/mL) and herring sperm DNA (0.25mg/mL). BSA was extracted at 100% purity, demonstrating the ability of MMCF-DEAE to remove significant DNA contamination from a protein solution. These experiments highlight the potential for MMCFs to be used for fast protein purification in preparative chromatography application.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.chroma.2016.09.00

The effect of Me2_{2}SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me$_{2}$SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me$_{2}$SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me$_{2}$SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me$_{2}$SO before, and after, cryopreservation. Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.The authors would like to acknowledge the Engineering and Physical Sciences Research Council (EPSRC; UK, EP/F500491/1) and Bioprocessing Research Industry Club (BBSRC/BRIC; UK, BB/I017602/1) for their support and funding

Bioprocess modelling of biohydrogen production by Rhodopseudomonas palustris: Model development and effects of operating conditions on hydrogen yield and glycerol conversion efficiency

This research explores the photofermentation of glycerol to hydrogen by Rhodopseudomonas palus- tris, with the objective to maximise hydrogen production. Two piecewise models are designed to simulate the entire growth phase of R. palustris; a challenge that few dynamic models can accomplish. The parameters in both models were fitted by the present batch experiments through the solution of the underlying optimal control problems by means of stable and accurate discretisation techniques. It was found that an initial glutamate to glycerol ratio of 0.25 was optimal, and was independent of the initial biomass concentration. The glycerol conversion efficiency was found to depend on initial biomass concentration and its computational peak is 64.4%. By optimising a 30-day industrially relevant batch process, the hydrogen productivity was improved to be 37.7 mL·g biomass-1·hr-1 and the glycerol conversion efficiency was maintained at 58%. The models can then be applied as the connection to transfer biohydrogen production from laboratory scale into industrial scale.Authors N. Xiao and Dr. K. T. Mahbubani are funded through the KACST-Cambridge Center for Advanced Material Manufacture, the author E. A. del Rio-Chanona is found by CONACyT scholarship No. 522530 from the Secretariat of Public Education and the Mexican government.This is the accepted manuscript. The final version is available from Elsevier at http://www.sciencedirect.com/science/article/pii/S0009250915001815