Publications by authors named "Margaret Mullin"

29 Publications

  • Page 1 of 1

Oligodendrocytes are susceptible to Zika virus infection in a mouse model of perinatal exposure: Implications for CNS complications.

Glia 2021 May 4. Epub 2021 May 4.

College of Medical, Veterinary, and Life Sciences, Institute of Infection, Immunity and Inflammation, Glasgow, UK.

Some children with proven intrauterine Zika virus (ZIKV) infection who were born asymptomatic subsequently manifested neurodevelopmental delays, pointing to impairment of development perinatally and postnatally. To model this, we infected postnatal day (P) 5-6 (equivalent to the perinatal period in humans) susceptible mice with a mammalian cell-propagated ZIKV clinical isolate from the Brazilian outbreak in 2015. All infected mice appeared normal up to 4 days post-intraperitoneal inoculation (dpi), but rapidly developed severe clinical signs at 5-6 dpi. All nervous tissue examined at 5/6 dpi appeared grossly normal. However, anti-ZIKV positive cells were observed in the optic nerve, brain, and spinal cord; predominantly in white matter. Co-labeling with cell type specific markers demonstrated oligodendrocytes and astrocytes support productive infection. Rarely, ZIKV positive neurons were observed. In spinal cord white matter, which we examined in detail, apoptotic cells were evident; the density of oligodendrocytes was significantly reduced; and there was localized microglial reactivity including expression of the NLRP3 inflammasome. Together, our observations demonstrate that a clinically relevant ZIKV isolate can directly impact oligodendrocytes. As primary oligodendrocyte cell death can lead later to secondary autoimmune demyelination, our observations may help explain neurodevelopmental delays in infants appearing asymptomatic at birth and commend lifetime surveillance.
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http://dx.doi.org/10.1002/glia.24010DOI Listing
May 2021

Defective apoptotic cell contractility provokes sterile inflammation, leading to liver damage and tumour suppression.

Elife 2021 Apr 19;10. Epub 2021 Apr 19.

Department of Chemistry and Biology, Ryerson University, Toronto, Canada.

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage-associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour-suppressive mechanism.
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http://dx.doi.org/10.7554/eLife.61983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087448PMC
April 2021

Regression of Melanoma Following Intravenous Injection of Plumbagin Entrapped in Transferrin-Conjugated, Lipid-Polymer Hybrid Nanoparticles.

Int J Nanomedicine 2021 6;16:2615-2631. Epub 2021 Apr 6.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G4 0RE, UK.

Background: Plumbagin, a naphthoquinone extracted from the officinal leadwort presenting promising anti-cancer properties, has its therapeutic potential limited by its inability to reach tumors in a specific way at a therapeutic concentration following systemic injection. The purpose of this study is to assess whether a novel tumor-targeted, lipid-polymer hybrid nanoparticle formulation of plumbagin would suppress the growth of B16-F10 melanoma in vitro and in vivo.

Methods: Novel lipid-polymer hybrid nanoparticles entrapping plumbagin and conjugated with transferrin, whose receptors are present in abundance on many cancer cells, have been developed. Their cellular uptake, anti-proliferative and apoptosis efficacy were assessed on various cancer cell lines in vitro. Their therapeutic efficacy was evaluated in vivo after tail vein injection to mice bearing B16-F10 melanoma tumors.

Results: The transferrin-bearing lipid-polymer hybrid nanoparticles loaded with plumbagin resulted in the disappearance of 40% of B16-F10 tumors and regression of 10% of the tumors following intravenous administration. They were well tolerated by the mice.

Conclusion: These therapeutic effects, therefore, make transferrin-bearing lipid-polymer hybrid nanoparticles entrapping plumbagin a highly promising anti-cancer nanomedicine.
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http://dx.doi.org/10.2147/IJN.S293480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8039437PMC
May 2021

Octadecyl chain-bearing PEGylated poly(propyleneimine)-based dendrimersomes: physicochemical studies, redox-responsiveness, DNA condensation, cytotoxicity and gene delivery to cancer cells.

Biomater Sci 2021 Feb 6;9(4):1431-1448. Epub 2021 Jan 6.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK.

Stimuli-responsive nanocarriers have become increasingly important for nucleic acid and drug delivery in cancer therapy. Here, we report the synthesis, characterization and evaluation of disulphide-linked, octadecyl (C18 alkyl) chain-bearing PEGylated generation 3-diaminobutyric polypropylenimine dendrimer-based vesicles (or dendrimersomes) for gene delivery. The lipid-bearing PEGylated dendrimer was successfully synthesized through in situ two-step reaction. It was able to spontaneously self-assemble into stable, cationic, nanosized vesicles, with low critical aggregation concentration value, and also showed redox-responsiveness in presence of a glutathione concentration similar to that of the cytosolic reducing environment. In addition, it was able to condense more than 70% of DNA at dendrimer: DNA weight ratios of 5 : 1 and higher. This dendriplex resulted in an enhanced cellular uptake of DNA at dendrimer: DNA weight ratios of 10 : 1 and 20 : 1, by up to 16-fold and by up to 28-fold compared with naked DNA in PC-3 and DU145 prostate cancer cell lines respectively. At a dendrimer: DNA weight ratio of 20 : 1, it led to an increase in gene expression in PC-3 and DU145 cells, compared with DAB dendriplex. These octadecyl chain-bearing, PEGylated dendrimer-based vesicles are therefore promising redox-sensitive drug and gene delivery systems for potential applications in combination cancer therapy.
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http://dx.doi.org/10.1039/d0bm01441aDOI Listing
February 2021

Venetoclax causes metabolic reprogramming independent of BCL-2 inhibition.

Cell Death Dis 2020 08 13;11(8):616. Epub 2020 Aug 13.

Cancer Research UK Beatson Institute, University of Glasgow, Garscube Estate, Switchback Road, Glasgow, G61 1QH, UK.

BH3-mimetics are a new class of anti-cancer drugs that inhibit anti-apoptotic Bcl-2 proteins. In doing so, BH3-mimetics sensitise to cell death. Venetoclax is a potent, BCL-2 selective BH3-mimetic that is clinically approved for use in chronic lymphocytic leukaemia. Venetoclax has also been shown to inhibit mitochondrial metabolism, this is consistent with a proposed role for BCL-2 in metabolic regulation. We used venetoclax to understand BCL-2 metabolic function. Similar to others, we found that venetoclax inhibited mitochondrial respiration. In addition, we also found that venetoclax impairs TCA cycle activity leading to activation of reductive carboxylation. Importantly, the metabolic effects of venetoclax were independent of cell death because they were also observed in apoptosis-resistant BAX/BAK-deficient cells. However, unlike venetoclax treatment, inhibiting BCL-2 expression had no effect on mitochondrial respiration. Unexpectedly, we found that venetoclax also inhibited mitochondrial respiration and the TCA cycle in BCL-2 deficient cells and in cells lacking all anti-apoptotic BCL-2 family members. Investigating the basis of this off-target effect, we found that venetoclax-induced metabolic reprogramming was dependent upon the integrated stress response and ATF4 transcription factor. These data demonstrate that venetoclax affects cellular metabolism independent of BCL-2 inhibition. This off-target metabolic effect has potential to modulate venetoclax cytotoxicity.
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http://dx.doi.org/10.1038/s41419-020-02867-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426836PMC
August 2020

Anti-Tumor Activity of Intravenously Administered Plumbagin Entrapped in Targeted Nanoparticles.

J Biomed Nanotechnol 2020 Jan;16(1):85-100

Plumbagin, a natural naphthoquinone from the officinal leadwort, has recently been shown to exert promising anti-cancer effects. However, its therapeutic use is hampered by its failure to specifically reach tumors after intravenous administration, without secondary effects on normal tissues. Its poor solubility in water and rapid elimination following administration further limit its potential use. We hypothesize that the entrapment of plumbagin within PEGylated PLGA nanoparticles conjugated with transferrin, whose receptors are overexpressed on many types of cancer cells, could lead to a selective delivery of the drug to tumors following intravenous administration and enhance its chemotherapeutic effects. The objectives of this study were therefore to prepare and characterize transferrin-conjugated, PEGylated PLGA nanoparticles entrapping plumbagin, and to assess their anti-cancer efficacy as well as in tumor-bearing mice. The intravenous administration of transferrin-conjugated PEGylated PLGA nanoparticles resulted in the complete suppression of 10% of B16-F10 tumors and regression of 30% of the tumors, with improvement of the animal survival compared to controls. The treatment was well tolerated by the animals. Transferrin-bearing PEGylated PLGA nanoparticles entrapping plumbagin are therefore highly promising therapeutic systems, able to lead to tumor regression and even suppression after intravenous administration without visible toxicity.
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http://dx.doi.org/10.1166/jbn.2020.2874DOI Listing
January 2020

Camptothecin-based dendrimersomes for gene delivery and redox-responsive drug delivery to cancer cells.

Nanoscale 2019 Nov 15;11(42):20058-20071. Epub 2019 Oct 15.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK.

Combination therapy involving chemotherapeutic drugs and genes is emerging as a promising strategy to provide a synergistic therapeutic effect, to overcome drug resistance while reducing the severe side effects associated with conventional chemotherapeutic drugs. However, the lack of nanomedicines able to simultaneously carry anti-cancer drugs and nucleic acids limits the application of this therapeutic strategy. To overcome this issue, we proposed to synthesize a pro-drug dendrimer by conjugating the PEGylated, positively charged generation 3-diaminobutyric polypropylenimine dendrimer to the anti-cancer drug camptothecin with a redox-sensitive disulphide linkage, and evaluate its efficacy to co-deliver the complexed DNA and camptothecin to cancer cells. This PEGylated pro-drug dendrimer was found to spontaneously self-assemble into cationic (∼3-5 mV) vesicles at pH 7.4, at a critical aggregation concentration of about 200 μg mL. These vesicles (dendrimersomes) became smaller (150-200 nm) with increasing dendrimer concentration and remained stable over 7 days. They were able to release about 70% of the conjugated camptothecin in presence of 50 mM glutathione (equivalent to the intracellular environment of tumor tissue). They could also condense more than 85% of the DNA at dendrimer : DNA weight ratios of 5 : 1 and higher. DNA condensation occurred instantly and was found to be stable for at least 24 h. This led to an enhanced cellular uptake of DNA (by up to 1.6-fold) and increased gene transfection (by up to 2.4-fold) in prostate cancer cells in comparison with the unmodified dendrimer. These novel dendrimersomes are therefore promising for single carrier-based combination cancer therapy.
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http://dx.doi.org/10.1039/c9nr07254cDOI Listing
November 2019

Microfluidic manufacturing of different niosomes nanoparticles for curcumin encapsulation: Physical characteristics, encapsulation efficacy, and drug release.

Beilstein J Nanotechnol 2019 5;10:1826-1832. Epub 2019 Sep 5.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, G4 0RE Glasgow, United Kingdom.

Curcumin, a natural chemical compound found in that has been used in antitumor and anti-inflammation applications, exhibits very limited water solubility and rapid in vivo degradation, which limits its clinical application. To overcome these limitations, niosome nanoparticles were prepared by microfluidic mixing for curcumin encapsulation. Niosome nanoparticles are lipid-based, and composed of non-ionic surfactants with cholesterol orientated into a membrane bilayer structure. Two different non-ionic surfactants were used and the mixing parameters were varied to optimize the characteristics of the prepared niosomes. The prepared niosomes had an average particle size of 70-230 nm depending on the type of non-ionic surfactant used and the mixing parameter. Moreover, all prepared niosomes were monodisperse with an average polydispersity index ranging from 0.07 to 0.3. All prepared niosomes were spherical as demonstrated by transmission electron microscopy. Curcumin was encapsulated with a maximum encapsulation efficiency of around 60% using Tween 85 as the non-ionic surfactant. Niosomes prepared by microfluidic mixing provided a controlled release of curcumin, as indicated by the release profile of curcumin, improving its therapeutic capability. These results demonstrate that niosomes prepared by microfluidic mixing to encapsulate curcumin are a promising delivery system to reach target cells.
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http://dx.doi.org/10.3762/bjnano.10.177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753676PMC
September 2019

Transferrin-bearing liposomes entrapping plumbagin for targeted cancer therapy.

J Interdiscip Nanomed 2019 Jun 26;4(2):54-71. Epub 2019 Jun 26.

Strathclyde Institute of Pharmacy and Biomedical Sciences University of Strathclyde 161 Cathedral Street Glasgow G4 0RE UK.

The therapeutic potential of plumbagin, a naphthoquinone extracted from the officinal leadwort with anticancer properties, is hampered by its failure to specifically reach tumours at a therapeutic concentration after intravenous administration, without secondary effects on normal tissues. Its use in clinic is further limited by its poor aqueous solubility, its spontaneous sublimation, and its rapid elimination . We hypothesize that the entrapment of plumbagin within liposomes grafted with transferrin, whose receptors are overexpressed on many cancer cells, could result in a selective delivery to tumours after intravenous administration. The objectives of this study were therefore to prepare and characterize transferrin-targeted liposomes entrapping plumbagin and to evaluate their therapeutic efficacy and . The entrapment of plumbagin in transferrin-bearing liposomes led to an increase in plumbagin uptake by cancer cells and improved antiproliferative efficacy and apoptosis activity in B16-F10, A431, and T98G cell lines compared with that observed with the drug solution. the intravenous injection of transferrin-bearing liposomes entrapping plumbagin led to tumour suppression for 10% of B16-F10 tumours and tumour regression for a further 10% of the tumours. By contrast, all the tumours treated with plumbagin solution or left untreated were progressive. The animals did not show any signs of toxicity. Transferrin-bearing liposomes entrapping plumbagin are therefore highly promising therapeutic systems that should be further optimized as a therapeutic tool for cancer treatment.
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http://dx.doi.org/10.1002/jin2.56DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619241PMC
June 2019

Migration through physical constraints is enabled by MAPK-induced cell softening via actin cytoskeleton re-organization.

J Cell Sci 2019 05 31;132(11). Epub 2019 May 31.

Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.224071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589089PMC
May 2019

Development of transferrin-bearing vesicles encapsulating aspirin for cancer therapy.

J Liposome Res 2020 Jun 5;30(2):174-181. Epub 2019 Jun 5.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK.

Originally developed for the treatment of inflammatory disorders, the non-steroidal anti-inflammatory drug aspirin was shown to have a preventive effect against cancer in the past decades. Most importantly, recent studies suggested that it might also provide a therapeutic benefit in the treatment of cancer . However, this drug failed to specifically reach tumors at a therapeutic concentration following intravenous administration, thus resulting in lack of efficacy on tumors. In this work, we demonstrated that aspirin could be formulated in transferrin-bearing vesicles and that this tumor-targeted formulation could lead to an increase in the anti-proliferative efficacy of the drug in three cancer cell lines . The therapeutic efficacy of aspirin was significantly improved when formulated in transferrin-bearing vesicles, by about 2-fold compared to that of drug solution. These results are promising and support the optimization of this delivery system to further improve its potential as a therapeutic tool in combination with other anti-cancer therapies.
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http://dx.doi.org/10.1080/08982104.2019.1614054DOI Listing
June 2020

Environmental control programs the emergence of distinct functional ensembles from unconstrained chemical reactions.

Proc Natl Acad Sci U S A 2019 03 6;116(12):5387-5392. Epub 2019 Mar 6.

WestCHEM, School of Chemistry, University of Glasgow, Glasgow, United Kingdom G12 8QQ;

Many approaches to the origin of life focus on how the molecules found in biology might be made in the absence of biological processes, from the simplest plausible starting materials. Another approach could be to view the emergence of the chemistry of biology as process whereby the environment effectively directs "primordial soups" toward structure, function, and genetic systems over time. This does not require the molecules found in biology today to be made initially, and leads to the hypothesis that environment can direct chemical soups toward order, and eventually living systems. Herein, we show how unconstrained condensation reactions can be steered by changes in the reaction environment, such as order of reactant addition, and addition of salts or minerals. Using omics techniques to survey the resulting chemical ensembles we demonstrate there are distinct, significant, and reproducible differences between the product mixtures. Furthermore, we observe that these differences in composition have consequences, manifested in clearly different structural and functional properties. We demonstrate that simple variations in environmental parameters lead to differentiation of distinct chemical ensembles from both amino acid mixtures and a primordial soup model. We show that the synthetic complexity emerging from such unconstrained reactions is not as intractable as often suggested, when viewed through a chemically agnostic lens. An open approach to complexity can generate compositional, structural, and functional diversity from fixed sets of simple starting materials, suggesting that differentiation of chemical ensembles can occur in the wider environment without the need for biological machinery.
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http://dx.doi.org/10.1073/pnas.1813987116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431231PMC
March 2019

Redox-sensitive, cholesterol-bearing PEGylated poly(propylene imine)-based dendrimersomes for drug and gene delivery to cancer cells.

Nanoscale 2018 Dec 29;10(48):22830-22847. Epub 2018 Nov 29.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK.

Stimuli-responsive nanocarriers have attracted increased attention as materials that can facilitate drug and gene delivery in cancer therapy. The present study reports the development of redox-sensitive dendrimersomes comprising disulfide-linked cholesterol-bearing PEGylated dendrimers, which can be used as drug and gene delivery systems. Two disulfide-linked cholesterol-bearing PEGylated generation 3 diaminobutyric polypropylenimine dendrimers have been successfully synthesized via an in situ two-step reaction. They were able to spontaneously self-assemble into stable, cationic, nanosized vesicles (or dendrimersomes) with lower critical aggregation concentration values for high-cholesterol-bearing vesicles. These dendrimersomes were able to entrap both hydrophilic and hydrophobic dyes, and they also showed a redox-responsive sustained release of the entrapped guests in the presence of a glutathione concentration similar to that of a cytosolic reducing environment. The high-cholesterol-bearing dendrimersomes were found to have a higher melting enthalpy, increased adsorption tendency on mica surface, entrapping ability for a larger amount of hydrophobic drugs, and increased resistance to redox-responsive environments in comparison with their low-cholesterol counterpart. In addition, both dendrimersomes were able to condense more than 85% of the DNA at all the tested ratios for the low-cholesterol vesicles, and at dendrimer : DNA weight ratios of 1 : 1 and higher for the high-cholesterol vesicles. These vesicles resulted in an enhanced cellular uptake of DNA, by up to 15-fold when compared with naked DNA with low-cholesterol vesicles. As a result, they increased the gene transfection on the PC-3 prostate cancer cell line, with the highest transfection being obtained with low-cholesterol vesicle complexes at a dendrimer : DNA weight ratio of 5 : 1 and high-cholesterol vesicle complexes at a dendrimer : DNA weight ratio of 10 : 1. These transfection levels were about 5-fold higher than those observed when treated with naked DNA. These cholesterol-bearing PEGylated dendrimer-based vesicles are, therefore, promising as redox-sensitive drugs and gene delivery systems for potential applications in combination cancer therapies.
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http://dx.doi.org/10.1039/c8nr08141gDOI Listing
December 2018

The sphingosine 1-phosphate receptor 2 is shed in exosomes from breast cancer cells and is N-terminally processed to a short constitutively active form that promotes extracellular signal regulated kinase activation and DNA synthesis in fibroblasts.

Oncotarget 2018 Jun 29;9(50):29453-29467. Epub 2018 Jun 29.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK.

We demonstrate here that the G protein-coupled receptor (GPCR), sphingosine 1-phosphate receptor 2 (S1P, Mr = 40 kDa) is shed in hsp70 and CD63 containing exosomes from MDA-MB-231 breast cancer cells. The receptor is taken up by fibroblasts, where it is N-terminally processed to a shorter form (Mr = 36 kDa) that appears to be constitutively active and able to stimulate the extracellular signal regulated kinase-1/2 (ERK-1/2) pathway and DNA synthesis. An N-terminally truncated construct of S1P, which may correspond to the processed form of the receptor generated in fibroblasts, was found to be constitutively active when over-expressed in HEK293 cells. Analysis based on the available crystal structure of the homologous S1P receptor suggests that, in the inactive-state, the N-terminus of S1P may tension TM1 so as to maintain a compressive action on TM7. This in turn may stabilise a closed basal state interface between the intracellular ends of TM7 and TM6. Cleavage and removal of the S1P N-terminal peptide is postulated to facilitate relaxation of TM1 and accompanying separation of TM6 and TM7. The latter transition is one of the key elements of G protein engagement and is required to open the intracellular coupling interface beneath the GPCR helix bundle. Therefore, removal at the N-terminus of S1P is likely to enhance G protein coupling. These findings provide the first evidence that S1P is released from breast cancer cells in exosomes and is processed by fibroblasts to promote ERK signaling and proliferation of these cells.
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http://dx.doi.org/10.18632/oncotarget.25658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047680PMC
June 2018

Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells.

PLoS One 2018 14;13(2):e0192562. Epub 2018 Feb 14.

Centre for Cell Engineering, University of Glasgow, Glasgow, Lanarkshire, United Kingdom.

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-tagged nanoparticles offer as novel therapeutics in regenerative medicine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192562PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812622PMC
April 2018

RIPK3 promotes adenovirus type 5 activity.

Cell Death Dis 2017 12 13;8(12):3206. Epub 2017 Dec 13.

Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, G61 1QH, UK.

Oncolytic adenoviral mutants infect human malignant cells and replicate selectively within them. This induces direct cytotoxicity that can also trigger profound innate and adaptive immune responses. However, the mechanism by which adenoviruses produce cell death remains uncertain. We previously suggested that type 5 adenoviruses, including the E1A CR2 deletion mutant dl922-947, might induce a novel form of programmed death resembling necroptosis. Here we have investigated the roles of core necrosis proteins RIPK1, RIPK3 and MLKL in the cytotoxicity of dl922-947 and other adenovirus serotypes. By electron microscopy, we show that dl922-947 induces similar necrotic morphology as TSZ treatment (TNF-α, Smac mimetic, zVAD.fmk). However, dl922-947-mediated death is independent of TNF-α signalling, does not require RIPK1 and does not rely upon the presence of MLKL. However, inhibition of caspases, specifically caspase-8, induces necroptosis that is RIPK3 dependent and significantly enhances dl922-947 cytotoxicity. Moreover, using CRISPR/Cas9 gene editing, we demonstrate that the increase in cytotoxicity seen upon caspase inhibition is also MLKL dependent. Even in the absence of caspase inhibition, RIPK3 expression promotes dl922-947 and wild-type adenovirus type 5 efficacy both in vitro and in vivo. Together, these results suggest that adenovirus induces a form of programmed necrosis that differs from classical TSZ necroptosis.
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http://dx.doi.org/10.1038/s41419-017-0110-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870599PMC
December 2017

Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence.

J Tissue Eng 2017 Jan-Dec;8:2041731417704428. Epub 2017 Apr 24.

Centre for Cell Engineering, Institute of Molecular, Cell and Systems Biology (IMCSB), The University of Glasgow, Glasgow, UK.

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies.
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http://dx.doi.org/10.1177/2041731417704428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460809PMC
April 2017

ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth.

EMBO Mol Med 2017 02;9(2):198-218

Cancer Research UK Beatson Institute, Glasgow, UK

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a Kras/p53 mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three-dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK-induced genes that facilitate extracellular matrix remodeling, with greatest fold-changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13 MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three-dimensional contexts. Treatment of Kras/p53 PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor-associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth.
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http://dx.doi.org/10.15252/emmm.201606743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286371PMC
February 2017

Tumor regression after intravenous administration of targeted vesicles entrapping the vitamin E α-tocotrienol.

J Control Release 2017 01 18;246:79-87. Epub 2016 Dec 18.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, United Kingdom. Electronic address:

The therapeutic potential of tocotrienol, a member of the vitamin E family of compounds with potent in vitro anti-cancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study is to determine whether a novel tumor-targeted vesicular formulation of tocotrienol would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo. In this work, we demonstrated that novel transferrin-bearing multilamellar vesicles entrapping α-T3 resulted in a dramatically improved (by at least 52-fold) therapeutic efficacy in vitro on A431 cell line, compared to the free drug. In addition, the intravenous administration of tocotrienol entrapped in transferrin-bearing vesicles resulted in tumor suppression for 30% of A431 and 60% of B16-F10 tumors, without visible toxicity. Mouse survival was enhanced by >13days compared to controls administered with the drug solution only. This tumor-targeted, tocotrienol-based nanomedicine therefore significantly improved the therapeutic response in cancer treatment.
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http://dx.doi.org/10.1016/j.jconrel.2016.12.014DOI Listing
January 2017

A Quiescent, Regeneration-Responsive Tissue Engineered Mesenchymal Stem Cell Bone Marrow Niche Model via Magnetic Levitation.

ACS Nano 2016 09 15;10(9):8346-54. Epub 2016 Sep 15.

School of Science, University of the West of Scotland , Paisley PA1 2BE, United Kingdom.

The bone marrow niche represents a specialized environment that regulates mesenchymal stem cell quiescence and self-renewal, yet fosters stem cell migration and differentiation upon demand. An in vitro model that embodies these features would open up the ability to perform detailed study of stem cell behavior. In this paper we present a simple bone marrow-like niche model, which comprises of nanomagnetically levitated stem cells cultured as multicellular spheroids within a type I collagen gel. The stem cells maintained are nestin positive and remain quiescent until regenerative demand is placed upon them. In response to coculture wounding, they migrate and appropriately differentiate upon engraftment. This tissue engineered regeneration-responsive bone marrow-like niche model will allow for greater understanding of stem cell response to injury and also facilitate as a testing platform for drug candidates in a multiwell plate format.
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http://dx.doi.org/10.1021/acsnano.6b02841DOI Listing
September 2016

Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish.

Cell Biochem Funct 2016 Aug 29;34(6):429-40. Epub 2016 Jul 29.

Department of Life Sciences, Glasgow Caledonian University, Glasgow, UK.

Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport.
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http://dx.doi.org/10.1002/cbf.3205DOI Listing
August 2016

Gold nanoparticle-siRNA mediated oncogene knockdown at RNA and protein level, with associated gene effects.

Nanomedicine (Lond) 2015 24;10(16):2513-25. Epub 2015 Aug 24.

Centre for Cell Engineering, Joseph Black Building, Glasgow University, G12 8QQ, UK.

Aims: RNAi is a powerful tool for gene silencing that can be used to reduce undesirable overexpression of oncogenes as a novel form of cancer treatment. However, when using RNAi as a therapeutic tool there is potential for associated gene effects. This study aimed to utilize gold nanoparticles to deliver siRNA into HeLa cells.

Results: Knockdown of the c-myc oncogene by RNAi, at the RNA, protein and cell proliferation level was achieved, while also identifying associated gene responses.

Discussion: The gold nanoparticles used in this study present an excellent delivery platform for siRNA, but do note associated gene changes.

Conclusion: The study highlights the need to more widely assess the cell physiological response to RNAi treatment, rather than focus on the immediate RNA levels.
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http://dx.doi.org/10.2217/NNM.15.95DOI Listing
June 2016

Tumor regression following intravenous administration of lactoferrin- and lactoferricin-bearing dendriplexes.

Nanomedicine 2015 Aug 29;11(6):1445-54. Epub 2015 Apr 29.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom. Electronic address:

Unlabelled: The possibility of using gene therapy for the treatment of cancer is limited by the lack of safe, intravenously administered delivery systems able to selectively deliver therapeutic genes to tumors. In this study, we investigated if the conjugation of the polypropylenimine dendrimer to lactoferrin and lactoferricin, whose receptors are overexpressed on cancer cells, could result in a selective gene delivery to tumors and a subsequently enhanced therapeutic efficacy. The conjugation of lactoferrin and lactoferricin to the dendrimer significantly increased the gene expression in the tumor while decreasing the non-specific gene expression in the liver. Consequently, the intravenous administration of the targeted dendriplexes encoding TNFα led to the complete suppression of 60% of A431 tumors and up to 50% of B16-F10 tumors over one month. The treatment was well tolerated by the animals. These results suggest that these novel lactoferrin- and lactoferricin-bearing dendrimers are promising gene delivery systems for cancer therapy.

From The Clinical Editor: Specific targeting of cancer cells should enhance the delivery of chemotherapeutic agents. This is especially true for gene delivery. In this article, the authors utilized a dendrimer-based system and conjugated this with lactoferrin and lactoferricin to deliver anti-tumor genes. The positive findings in animal studies should provide the basis for further clinical studies.
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http://dx.doi.org/10.1016/j.nano.2015.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4509555PMC
August 2015

The influence of particle size and static magnetic fields on the uptake of magnetic nanoparticles into three dimensional cell-seeded collagen gel cultures.

J Biomed Mater Res B Appl Biomater 2015 Aug 31;103(6):1294-301. Epub 2014 Oct 31.

Centre for Cell Engineering, University of Glasgow, Glasgow, UK, G12 8QQ.

Over recent decades there has been and continues to be major advances in the imaging, diagnosis and potential treatment of medical conditions, by the use of magnetic nanoparticles. However, to date the majority of cell delivery studies employ a traditional 2D monolayer culture. This article aims to determine the ability of various sized magnetic nanoparticles to penetrate and travel through a cell seeded collagen gel model, in the presence or absence of a magnetic field. Three different sized (100, 200, and 500 nm) nanoparticles were employed in the study. The results showed cell viability was unaffected by the presence of nanoparticles over a 24-h test period. The initial uptake of the 100 nm nanoparticle into the collagen gel structure was superior compared to the larger sized nanoparticles under the influence of a magnetic field and incubated for 24 h. Interestingly, it was the 200 nm nanoparticles, which proved to penetrate the gel furthest, under the influence of a magnetic field, during the initial culture stage after 1-h incubation.
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http://dx.doi.org/10.1002/jbm.b.33302DOI Listing
August 2015

Elucidating the function of penetratin and a static magnetic field in cellular uptake of magnetic nanoparticles.

Pharmaceuticals (Basel) 2013 Feb 6;6(2):204-22. Epub 2013 Feb 6.

Centre for Cell Engineering, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK.

Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrin- medicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake. Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake.
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http://dx.doi.org/10.3390/ph6020204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816682PMC
February 2013

Applications of β-limit dextrin as a matrix forming excipient for fast disintegrating buccal dosage formats.

J Pharm Pharm Sci 2012 ;15(5):669-79

Glycologic Limited, 70 Cowcaddens Road, Glasgow, UK.

Purpose: To compare the properties of buccal delivery matrices (wafers) made with dextrin, β-limit dextrin and pre-gelatinised starch.

Methods: The constituent α-glucans were tested for their mucoadhesive properties in solution plus their content of crystalline material (differential scanning calorimetry, DSC). Wafers were made by lyophilisation of aqueous solutions/dispersions of the α-glucans. Physical properties of the wafers were evaluated using texture analysis, dissolution coupled to photography and scanning electron microscopy (SEM).

Results: The results highlighted how the β-limit dextrins chemical and physical properties were ideally suited for the production of buccal delivery wafers. Dissolution testing confirmed the excellent hydration profile of the β-limit dextrin (within wafers) with time. Using SEM it was evident that the homogeneous "bee-hive" like structure of the β-limit dextrin wafers, unlike the other α-glucans, provided a rapidly hydratable strong porous matrix.

Conclusions: The β-limit dextrin α-glucan makes a superb (lyophilised) mucoadhesive delivery structure for the delivery of active agents to the buccal mucosa.
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http://dx.doi.org/10.18433/j3rc8mDOI Listing
July 2013

Influence of both a static magnetic field and penetratin on magnetic nanoparticle delivery into fibroblasts.

Nanomedicine (Lond) 2011 Dec 2;6(10):1719-31. Epub 2011 Aug 2.

Center for Cell Engineering, Joseph Black Building, Glasgow University, G12 8QQ, UK.

Aim: With regards to nanoparticles, all biomedical applications require cellular uptake, which to date remains a hurdle to further progress. This study aims to compare both the attractive force of a static magnetic field and the cell penetrating capability of penetratin; two techniques currently employed to enhance cell uptake.

Materials & Methods: Fluorescent magnetic nanoparticles were functionalized with penetratin and cells were challenged with or without the particles in the presence/absence of a static magnetic field (350 mT). Following analysis of the magnetic field applied, cellular uptake and behavior was assessed in terms of fluorescence microscopy, clathrin and caveolin levels, scanning electron microscopy and transmission electron microscopy.

Results: Modeling of the field applied demonstrated varying field patterns across the cell culture area, reflected by higher particle uptake at higher field strengths. Both penetratin and the magnetic field increased cell uptake with penetratin proving more efficient. Interestingly, the magnetic field stimulated clathrin-mediated endocytosis and subsequent particle uptake.
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http://dx.doi.org/10.2217/nnm.11.65DOI Listing
December 2011

Campylobacter jejuni 81-176 forms distinct microcolonies on in vitro-infected human small intestinal tissue prior to biofilm formation.

Microbiology (Reading) 2010 Oct 8;156(Pt 10):3079-3084. Epub 2010 Jul 8.

Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow, UK.

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2 h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4 h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-176 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man.
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http://dx.doi.org/10.1099/mic.0.039867-0DOI Listing
October 2010

The effect of static magnetic fields and tat peptides on cellular and nuclear uptake of magnetic nanoparticles.

Biomaterials 2010 May 26;31(15):4392-400. Epub 2010 Feb 26.

Centre for Cell Engineering, Glasgow University, Glasgow, UK.

Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field.
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http://dx.doi.org/10.1016/j.biomaterials.2010.01.096DOI Listing
May 2010